Journal Cover Articles
Selected Publications
- [34] A transgene-free, human peri-gastrulation embryo model with trilaminar embryonic disc-, amnion- and yolk sac-like structures
Shiyu Sun, Yi Zheng, Norio Kobayashi, Yung Su Kim, Xuefeng Xue, Yue Liu, Yanhong Xu, Jinglei Zhai, Hongmei Wang, and Jianping Fu
bioRxiv, 2024.
DOI: 10.1101/2024.08.05.606556.
Abstract | PDF | Supplemental Materials | PubMed
The ultimate outcome of the gastrulation in mammalian development is a recognizable trilaminar disc structure containing organized cell lineages with spatially defined identities in an emerging coordinate system. Despite its importance in human development, gastrulation remains difficult to study. Stem cell-based embryo models, including those that recapitulate different aspects of pre- and peri-gastrulation human development, are emerging as promising tools for studying human embryogenesis. However, it remains unclear whether existing human embryo models are capable of modeling the development of the trilaminar embryonic disc structure, a hallmark of human gastrulation. Here we report a transgene-free human embryo model derived solely from primed human pluripotent stem cells (hPSCs), which recapitulates various aspects of peri-gastrulation human development, including formation of trilaminar embryonic layers situated between dorsal amnion and ventral definitive yolk sac and primary hematopoiesis. We term this model the peri-gastrulation trilaminar embryonic disc (PTED) embryoid. The development of PTED embryoid does not follow natural developmental sequences of cell lineage diversification or spatial organization. Instead, it exploits both extrinsic control of tissue boundaries and intrinsic self-organizing properties and embryonic plasticity of the diverse peri-gastrulation-stage cell lineages, leading to the emergence of in vivo-like tissue organization and function at a global scale. Our lineage tracing study reveals that in PTED embryoids, embryonic and extraembryonic mesoderm cells, as well as embryonic and extraembryonic endoderm cells, share common progenitors emerging during peri-gastrulation development. Active hematopoiesis and blood cell generation are evident in the yolk sac-like structure of PTED embryoids. Together, PTED embryoids provide a promising and ethically less challenging model for studying self-organizing properties of peri-gastrulation human development. - [33] Criteria for standardization of stem-cell-based embryo models
Alfonso Martinez Arias, Nicolas Rivron, Naomi Moris, Patrick Tam, Cantas Alev, Jianping Fu, Anna-Katerina Hadjantonakis, Jacob H. Hanna, Gabriella Minchiotti, Olivier Pourquie, Guojun Sheng, Liliana Solnica Krezel, Jesse Veenvliet, and Aryeh Warmflash
Nature Cell Biology, in press, 2024.
DOI: 10.1038/s41556-024-01492-x.
Abstract | PDF | PubMed
Pluripotent Stem Cells (PSCs) are being used to generate models of early embryogenesis promising for discovery and translational research. To be useful these models require a critical consideration of their level of efficiency and fidelity to natural embryos. Herein, we propose criteria that define the utility of stem-cell-based embryo models of human embryogenesis. - [32] A comprehensive human embryo reference tool using single-cell RNA-sequencing data
Cheng Zhao, Alvaro Plaza Reyes, John Paul Schell, Jere Weltner, Nicolás M. Ortega, Yi Zheng, Åsa K. Björklund, Joonas Sokka, Ras Torokovic, Brian Cox, Janet Rossant, Jianping Fu, Sophie Petropoulos, and Fredrik Lanner
Nature Methods, in press, 2024.
Abstract | PDF | PubMed
Stem cell-based embryo models offer unprecedented experimental tools for studying early human development. The usefulness of embryo models hinges on their molecular, cellular and structural fidelities to their in vivo counterparts. To authenticate human embryo models, single-cell RNA-sequencing has been utilised for unbiased transcriptional profiling. However, a well-organised and integrated human single-cell RNA-sequencing dataset, serving as a universal reference for benchmarking human embryo models, remains unavailable. Herein, we developed such a reference, through integration of six publicly available human datasets covering developmental stages from the zygote to the gastrula. Lineage annotations are contrasted and validated with available human and non-human primate datasets. Using stabilised UMAP, we further constructed a web tool, using which query datasets can be projected on the reference and annotated with predicted cell identities. Using this reference tool, we examined several recently published human embryo models. Our analysis reveals the risk of mis-annotation of cell lineages in embryo models when relevant human embryo references, such as the one developed in this work, were not utilized for benchmarking and authentication. - [31] Bioengineering embryo models
Xufeng Xue, Yue Liu, and Jianping Fu
Nature Reviews Bioengineering, in press, 2024.
DOI: 10.1038/s44222-024-00241-x.
Abstract | PDF | PubMed
Stem cell-based embryo models, which recapitulate symmetry breaking, pattern formation and tissue morphogenesis during early development, provide promising experimental tools to study development of mammalian species, including humans. Despite considerable progress in embryo modeling using cultured stem cells, it remains challenging to construct embryo models with high fidelity, efficiency, controllability, and in vivo-like cellular organization and tissue architecture. This is largely due to intrinsic variabilities in self-organization and differentiation of mammalian stem cells in uncontrolled culture environments utilized in current embryo modeling. In this review, we argue that bioengineering tools, which are powerful for controlling topological boundaries and dynamic chemical and mechanical signals and thus are efficient in guiding symmetry breaking, pattern formation, tissue morphogenesis and tissue-tissue interactions, should be utilized for constructing high-fidelity, high-efficiency embryo models. In this review, we first discuss pattern formation and morphogenesis during embryonic development, and selectively examine different embryo models to highlight the importance of bioengineering controls in developing these models. We then explore how different bioengineering approaches useful for guiding pattern formation, morphogenesis, cell fate decisions and cell-cell interfaces can be utilized in stem cell-based embryo modeling to promote their efficiency, reproducibility, controllability, complexity, and in vivo relevance. - [30] Towards developing human organs via embryo models and chimeras
Jun Wu and Jianping Fu
Cell, vol. 187, pp. 3194-3219, 2024.
DOI: 10.1016/j.cell.2024.05.027.
Abstract | PDF | PubMed
Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting and organoids, only offer partial solutions. This Perspective focuses on two emerging approaches promising for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development, as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed, before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outline future prospects for advancing both fields towards the generation of human tissues and organs for basic research and translational applications. - [29] A human pluripotent stem cell-based somitogenesis model using microfluidics
Yue Liu, Yung Su Kim, Xufeng Xue, Yuchuan Miao, Norio Kobayashi, Shiyu Sun, Robin Zhexuan Yan, Qiong Yang, Olivier Pourquié, and Jianping Fu
Cell Stem Cell, vol. 31, pp. 1113-1126, 2024.
DOI: 10.1101/2023.10.29.564399.
Abstract | PDF | Supplemental Materials | PubMed
Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation. - [28] A patterned human neural tube model using microfluidic gradients
Xufeng Xue, Yung Su Kim, Alfredo-Isaac Ponce-Arias, Richard O'laughlin, Robin Zhexuan Yan, Norio Kobayashi, Rami Yair Tshuva, Yu-Hwai Tsai, Shiyu Sun, Yi Zheng, Yue Liu, Frederick C.K. Wong, Azim Surani, Jason R. Spence, Hongjun Song, Guo-Li Ming, Orly Reiner, and Jianping Fu
Nature, vol. 628, pp. 391-399, 2024.
DOI: 10.1038/s41586-024-07204-7.
Abstract | PDF | Supplemental Materials | PubMed
Human nervous system is arguably the most complex but highly organized organ. Foundation of its complexity and organization is laid down during regional patterning of neural tube (NT), the embryonic precursor to human nervous system. Historically, studies of NT patterning have relied on animal models to uncover underlying principles. Recently, human pluripotent stem cell (hPSC)-based models of neurodevelopment, including neural organoids1-5 and bioengineered NT development models6-10, are emerging. However, existing hPSC-based models fail to recapitulate neural patterning along both rostral-caudal (R-C) and dorsal-ventral (D-V) axes in a three-dimensional (3D) tubular geometry, a hallmark of NT development. Herein we report a hPSC-based, microfluidic NT-like structure (or µNTLS), whose development recapitulates some critical aspects of neural patterning in both brain and spinal cord (SC) regions and along both R-C and D-V axes. The µNTLS was utilized for studying neuronal lineage development, revealing pre-patterning of axial identities of neural crest (NC) progenitors and functional roles of neuromesodermal progenitors (NMPs) and caudal gene CDX2 in SC and trunk NC development. We further developed D-V patterned, microfluidic forebrain-like structures (µFBLS) with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic human forebrain pallium and subpallium developments, respectively. Together, both µNTLS and µFBLS offer 3D lumenal tissue architectures with an in vivo-like spatiotemporal cell differentiation and organization, promising for studying human neurodevelopment and disease. - [27] Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity
Hyung Chul Lee, Nidia M. M. Oliveira, Cato Hastings, Peter Baillie-Benson, Adam A. Moverley, Hui-Chun Lu, Yi Zheng, Elise L. Wilby, Timothy T. Weil, Karen M. Page, Jianping Fu, Naomi Moris, and Claudio D. Stern
Nature Communications, vol. 15, 1463, 2024.
DOI: 10.1038/s41467-024-45772-4.
Abstract | PDF | Supplemental Materials | PubMed
Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevents ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT, establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in human embryoids and Drosophila, suggesting an ancient evolutionary origin. - [26] Derivation of human primordial germ cell-like cells in an embryonic-like culture
Sajedeh Nasr Esfahani, Yi Zheng, Auriana Arabpour, Agnes M. Resto Irizarry, Norio Kobayashi, Xufeng Xue, Yue Shao, Cheng Zhao, Nicole L. Agranonik, Megan Sparrow, Timothy J. Hunt, Jared Faith, Mary Jasmine Lara, Qiu Ya Wu, Sherman Silber, Sophie Petropoulos, Ran Yang, Kenneth R. Chien, Amander T. Clark, and Jianping Fu
Nature Communications, vol. 15, 167, 2024.
DOI: 10.1038/s41467-023-43871-2.
Abstract | PDF | Supplemental Materials | PubMed
Featured in Nature Communications Editors’ Highlights
We report the induction of human primordial germ cell-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs) derived from hPSCs induce hPGCLC specification through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived induced pluripotent stem cell lines using this biomimetic platform, demonstrating its promise for screening applications. - [25] Morphogenesis beyond in vivo
Yue Liu, Xufeng Xue, Shiyu Sun, Norio Kobayashi, Yung Su Kim, and Jianping Fu
Nature Reviews Physics, vol. 6, pp. 28-44, 2024.
DOI: 10.1038/s42254-023-00669-x.
Abstract | PDF | PubMed
Morphogenetic events during development shape the body plan and establish structural foundations for tissue forms and functions. Driven by technical and ethical constraints in acquiring spatiotemporal information of development, especially for humans, both stem cell-based, in vitro development models and theoretical models have been constructed to recapitulate morphogenetic events during development. These in vitro experimental and theoretical models offer convenient accessibility, great efficiency, and favorable modulability. However, their physiological relevance often remains obscure due to their simplistic nature, obstructing their applicability as faithful and predictive models of natural development. In this review we examine existing in vitro experimental and theoretical models of various developmental events while comparing them with the current knowledge of natural development, with particular considerations of biomechanical driving forces and stereotypic morphogenetic features. We highlight state-of-the-art methodologies employed in constructing these in vitro models and emphasize the biomechanical and biophysical principles these models have helped unveil. Finally, challenges faced by the current in vitro experimental and theoretical models will be discussed, and we propose how theoretical modeling and in vitro experimental models should be combined with in vivo studies to advance fundamental understanding of development. - [24] Changing the public perception of human embryology
Nicolas C. Rivron, Alfonso Martinez-Arias, Karen Sermon, Christine Mummery, Hans Schöler, James Wells, Jenny Nichols, Anna-Katerina Hadjantonakis, Madeline A. Lancaster, Jianping Fu, Janet Rossant, and Kazuto Kato
Nature Cell Biology, vol. 25, pp. 1717-1719, 2023.
DOI: 10.1038/s41556-023-01289-4.
Abstract | PDF | PubMed
Human embryology is flourishing thanks to an impetus provided by embryo models formed from stem cells. These scientific advances require meticulous modelling, a refined ethical framework, but also sensible public communication. Securing public support is essential to laying the foundations needed to achieve societal impact. - [23] Why researchers should use human embryo models with caution
Janet Rossant and Jianping Fu
Nature, vol. 622, pp. 22-24, 2023.
DOI: 10.1038/d41586-023-03062-x.
Abstract | PDF | PubMed
Scientists should carefully consider whether embryo models based on human stem cells are essential to their work because of the associated practical and ethical challenges. Appropriately used, stem-cell-based models of human embryonic development could transform our understanding of how human life begins. We ask researchers to carefully consider whether there is a strong scientific rationale for replicating an entire human embryo from stem cells. Pushing ahead without careful deliberation risks a public backlash that could stall the progress in this exciting field. - [22] Mechanically enhanced biogenesis of gut spheroids with instability-driven morphomechanics
Feng Lin, Xia Li, Shiyu Sun, Zhongyi Li, Jianbo Bai, Lin Song, Bo Li, Jianping Fu, and Yue Shao
Nature Communications, vol. 14, 6016, 2023.
DOI: 10.1038/s41467-023-41760-2.
Abstract | PDF | Supplemental Materials | PubMed
Region-specific gut spheroids are precursors for gastrointestinal and pulmonary organoids that hold great promise for fundamental studies and translations. However, efficient production of gut spheroids remains challenging due to a lack of control and mechanistic understanding of gut spheroid morphogenesis. Here, we report an efficient biomaterial system, termed micropatterned gut spheroid generator (μGSG), to generate gut spheroids from human pluripotent stem cells through mechanically enhanced tissue morphogenesis. We show that μGSG enhances the biogenesis of gut spheroids independent of micropattern shape and size; instead, mechanically enforced cell multilayering and crowding is demonstrated as a general, geometry-insensitive mechanism that is necessary and sufficient for promoting spheroid formation. Combining experimental findings and an active-phase-field morphomechanics theory, our study further reveals an instability-driven mechanism and a mechanosensitive phase diagram governing spheroid pearling and fission in μGSG. This work unveils mechanobiological paradigms based on tissue architecture and surface tension for controlling tissue morphogenesis and advancing organoid technology. - [21] Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids
Zongyong Ai, Baohua Niu, Yu Yin, Lifeng Xiang, Gaohui Shi, Kui Duan, Sile Wang, Yingjie Hu, Chi Zhang, Chengting Zhang, Lujuan Rong, Ruize Kong, Tingwei Chen, Yixin Guo, Wanlu Liu, Nan Li, Shumei Zhao, Xiaoqing Zhu, Xuancheng Mai, Yonggang Li, Ze Wu, Yi Zheng, Jianping Fu, Weizhi Ji, and Tianqing Li
Cell Research, vol. 33, pp. 661-678, 2023.
DOI: 10.1038/s41422-023-00846-8.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Janet Rossant, Cell Research, vol. 33, pp. 1-2, 2023. [PDF]
Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage development as well as underlying mechanisms remains obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling pathways synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast development and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights into human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis. - [20] Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development
Yi Zheng, Robin Zhexuan Yan, Shiyu Sun, Mutsumi Kobayashi, Lifeng Xiang, Ran Yang, Alexander Goedel, Yu Kang, Xuefeng Xue, Sajedeh Nasr Esfahani, Yue Liu, Agnes M. Resto Irizarry, Weisheng Wu, Yunxiu Li, Weizhi Ji, Yuyu Niu, Kenneth R. Chien, Tianqing Li, Toshihiro Shioda, and Jianping Fu
Cell Stem Cell, vol. 29, pp. 1402-1419, 2022.
DOI: 10.1016/j.stem.2022.08.009.
Abstract | PDF | Supplemental Materials | PubMed
Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast towards the amniotic ectoderm, primitive streak / mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells. - [19] Engineering multiscale structural orders for high-fidelity embryoids and organoids
Yue Shao, and Jianping Fu
Cell Stem Cell, vol. 29, pp. 722-743, 2022.
DOI: 10.1016/j.stem.2022.04.003.
Abstract | PDF | PubMed
Embryoids and organoids hold great promise for human biology and medicine. Herein, we discuss conceptual and technological frameworks useful for developing high-fidelity embryoids and organoids that display tissue- and organ-level phenotypes and functions, which are critically needed for decoding developmental programs and improving translational applications. Through dissecting the layers of inputs controlling mammalian embryogenesis, we review recent progress in reconstructing multiscale structural orders in embryoids and organoids. Bioengineering tools useful for multiscale, multimodal structural engineering of tissue- and organ-level cellular organization and microenvironment are also discussed to present integrative, bioengineering-directed approaches to achieve next-generation, high-fidelity embryoids and organoids. - [18] Amnion signals are essential for mesoderm formation in primates
Ran Yang, Alexander Goedel, Yu Kang, Chengyang Si, Chu Chu, Yi Zheng, Zhenzhen Chen, Peter J. Gruber, Yao Xiao, Chikai Zhou, Chuen-Yan Leung, Yongchang Chen, Jianping Fu, Weizhi Ji, Fredrik Lanner, Yuyu Niu, and Kenneth Chien
Nature Communications, vol. 12, 5126, 2021.
DOI: 10.1101/2020.05.28.118703.
Abstract | PDF | Supplemental Materials | PubMed
The in vivo signaling network controlling primitive streak formation is well understood in mouse, but largely unexplored in primates. Herein, we generated a transcriptional atlas consisting of >100 000 cells spanning peri-implantation to early steak stages in non-human primates. We uncovered that ISL1, a gene with well-established role in mouse-cardiogenesis, is highly expressed in the primate amnion. Strikingly, ISL1 hypomorphic primate mutant embryos fail to induce primitive streak. We identified BMP4 as a key signaling pathway in this process and confirmed this in human embryonic stem cells, suggesting conserved function in humans. Notably, no viable ISL1 hypomorphic mutant embryo could be recovered after embryo transfer confirming the essential requirement of ISL1 and highlighting the importance of the amnion as a signaling center during primate embryogenesis. This study demonstrates the potential and importance of in vitro primate model systems to investigate the genetics of early human development including mesoderm specification. - [17] Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair
Dennis W. Zhou, Marc A. Fernández-Yagüe, Elijah N. Holland, Andrés F. García, Nicolas S. Castro, Eric B. O’Neill, Jeroen E.G. Eyckmans, Christopher S. Chen, Jianping Fu, David D. Schlaepfer, and Andrés J. García
Nature Communications, vol. 12, 2359, 2021.
DOI: 10.1038/s41467-021-22602-5.
Abstract | PDF | Supplemental Materials | PubMed
How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization and traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK binding interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair. - [16] Spatially resolved cell polarity proteomics of a human epiblast model
Sicong Wang, Chien-Wei Lin, Chari L. Cortez, Amber E. Carleton, Craig Johnson, Linnea E. Taniguchi, Ryan F. Townshend, Venkatesha Basrur, Alexey I. Nesvizhskii, Amy W. Hudson, Blake R. Hill, Peng Zou, Jianping Fu, Deborah L. Gumucio, Mara C. Duncan, and Kenichiro Taniguchi
Science Advances, vol. 7, eabd8407, 2021.
DOI: 10.1126/sciadv.abd8407.
Abstract | PDF | Supplemental Materials | PubMed
The pro-amniotic cavity of the implanting human embryo forms from an unpolarized mass of epithelial pluripotent epiblast cells. This process requires apico-basal polarization and radial organization of epiblast cells, followed by central lumen expansion. To gain global mechanistic insights into these critical but poorly understood early steps, we developed a spatially targeted approach to compare the proteomes of apical vs. basolateral membrane territories in a three-dimensional (3D) human pluripotent stem cell-derived epiblast-like model. We employed APEX2-based proximity biotinylation with quantitative mass spectrometry and revealed a variety of proteins without previous annotation to specific membrane sub-domains. Functional experiments validated the requirement for several apically enriched proteins in epiblast-like morphogenesis. In particular, we identified a previously unappreciated role for the AP-1 clathrin adaptor complex-dependent apical trafficking in lumen expansion. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models. - [15] First complete model of the human embryo
Yi Zheng, and Jianping Fu
Nature, vol. 591, pp. 531-532, 2021.
DOI: 10.1038/d41586-021-00581-3.
Abstract | PDF | PubMed
Early in development, human embryos form a structure called the blastocyst. Two research groups have now generated human blastocyst-like structures from cells in a dish, providing a valuable model for advancing human embryology. - [14] Stem-cell-based embryo models for fundamental research and translation
Jianping Fu, Aryeh Warmflash, and Lutolf P. Matthias
Nature Materials, vol. 20, pp. 132-144, 2021.
DOI: 10.1038/s41563-020-00829-9. PMCID: PMC7855549.
Abstract | PDF | PubMed
Despite its importance, understanding the early phases of human development has been significantly limited by availability of human samples. The recent emergence of stem cell-derived embryo models, a new field aiming to use stem cells to construct in vitro models to recapitulate snapshots of the development of the mammalian conceptus, opens up exciting opportunities to promote fundamental understanding of human development and advance reproductive and regenerative medicine. This review provides a summary of the current knowledge of early mammalian development, using mouse and human conceptuses as models, and emphasizes their similarities and critical differences. We then highlight existing embryo models that mimic different aspects of mouse and human development. We further discuss bioengineering tools used for controlling multicellular interactions and self-organization critical for the development of these models. We conclude with a discussion of the important next steps and exciting future opportunities of stem cell-derived embryo models for fundamental discovery and translation. - [13] Mechanical tension promotes formation of gastrulation-like nodes and patterns mesoderm specification in human embryonic stem cells
Jonathon M. Muncie, Nadia M.E. Ayad, Johnathon N. Lakins, Xufeng Xue, Jianping Fu, and Valerie M. Weaver
Developmental Cell, vol. 55, pp. 679-694, 2020.
DOI: 10.1101/2020.02.10.943076. PMCID: PubMed - in process.
Abstract | PDF | Supplemental Materials | PubMed
Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization that motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that enhance cell-cell interactions to direct the self-organization of cultured hESCs into “gastrulation-like” nodes. Tissue geometries that generated local nodes of high cell-adhesion tension and induced these self-organized tissue nodes drove BMP4-dependent gastrulation by enhancing phosphorylation and junctional release of β-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development. - [12] Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche
Yuanyuan Zheng, Xufeng Xue, Agnes M. Resto Irizarry, Zida Li, Yue Shao, Yi Zheng, Gang Zhao, and Jianping Fu
Science Advances, vol. 5, eaax5933, 2019.
DOI: 10.1126/sciadv.aax5933. PMCID: PubMed - in process.
Abstract | PDF | Supplemental Materials | PubMed
Despite its importance in the central nervous system development, the development of the human neural tube (NT) remains poorly understood, given interspecies divergence and challenges of studying human embryo specimens. Here we report a human NT development model, in which NT-like tissues, termed neuroepithelial (NE) cysts, are generated in a bioengineered neurogenic environment through self-organization of human pluripotent stem cells (hPSCs). NE cysts correspond to the neural plate in the dorsal ectoderm layer and possess a default dorsal identity. Dorsal-ventral (DV) patterning of NE cysts is achieved using retinoic acid and/or Sonic hedgehog, featuring sequential emergence of the ventral floor plate, P3 and pMN domains in discrete, adjacent regions and an dorsal territory progressively restricted to the opposite dorsal pole. Together, this study reports the development of a hPSC-based, DV patterned NE cyst system for modeling human NT development, useful for understanding the self-organizing principles that guide NT patterning and hence to study neural development and disease. - [11] Controlled modeling of human epiblast and amnion development using stem cells
Yi Zheng, Xufeng Xue, Yue Shao, Sicong Wang, Sajedeh Nasr Esfahani, Zida Li, Jonathon M. Muncie, Johnathon N. Lakins, Valerie M. Weaver, Deborah L. Gumucio, and Jianping Fu
Nature, vol. 573, pp. 421-425, 2019.
DOI: 10.1038/s41586-019-1535-2. PMCID: PubMed - in process.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Amander T. Clark, Nature, vol. 573, pp. 350-351, 2019. [PDF]
Highlighted by Nature News, "Embryo-like structures created from human stem cells". (link)
Highlighted by Nature Podcast, "Modelling early embryos". (link)
Highlighted by National Public Radio (NPR) News, "Scientists create a device that can mass-produce human embryoids". (link)
Highlighted by BBC News, "Embryoids from stem cells". (link)
Highlighted by MIT Technology Review, "Meet the 'artificial embryos' being called uncanny and spectacular". (link)
Highlighted by Chemical & Engineering News (C&EN), "Microfluidic device brews human embryo-like structures". (link)
Early human embryonic development involves extensive lineage diversification, cell fate specification and tissue patterning. Despite its basic and clinical importance, early human embryonic development remains mysterious due to interspecies divergence and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast (EPI) and amniotic ectoderm (AM) parts of the conceptus, including lumenogenesis and expansion of a pro-amniotic cavity, formation of a bipolar embryonic sac, specification of primordial germ cells (PGCs) and of primitive streak (PS) cells. We further show that AM-like cells (AMLCs) function as a signaling center to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model might assist in the rational design of differentiation protocols of hPSCs for disease modeling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects. - [10] Debate ethics of embryo models from stem cells
Nicolas Rivron, Martin Pera, Janet Rossant, Alfonso Martinez Arias, Magdalena Zernicka-Goetz, Jianping Fu, Suzanne van den Brink, Annelien Bredenoord, Wybo Dondorp, Guido de Wert, Insoo Hyun, Megan Munsie, and Rosario Isasi
Nature, vol. 564, pp. 183-185, 2018.
DOI: 10.1038/d41586-018-07663-9. PMCID: PubMed - in process.
Abstract | PDF | PubMed
Take a look at what has been achieved in the emerging field of stem cell models of embryonic development, along with the ethics of where research may go in the future. - [9] Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells
Xufeng Xue, Yubing Sun, Agnes M. Resto-Irizarry, Ye Yuan, Koh Meng Aw Yong, Yi Zheng, Shinuo Weng, Yue Shao, Yimin Chai, Lorenz Studer, and Jianping Fu
Nature Materials, vol. 17, pp. 633-641, 2018.
DOI: 10.1038/s41563-018-0082-9.
Abstract | PDF | Supplemental Materials | PubMed
Recommended in F1000Prime by David Schaffer and Rocio Sampayo. [PDF]
Commentary by Mukul Tewary and Peter W. Zandstra, Nature Materials, vol. 17, pp. 571-572, 2017. [PDF]
Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals - cell shape and cytoskeletal contractile force - instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease. - [8] A pluripotent stem cell-based model for post-implantation human amniotic sac development
Yue Shao, Kenichiro Taniguchi, Ryan F. Townshend, Toshio Miki, Deborah L. Gumucio, and Jianping Fu
Nature Communications, vol. 8, 208, 2017.
DOI: 10.1038/s41467-017-00236-w. PMCID: PMC5547056.
Abstract | PDF | Supplemental Materials | PubMed
Recommended in F1000Prime by Magdalena Zernicka-Goetz and Marta Shahbazi. [PDF]
Highlighted by MIT Technology Review, "Artificial human embryos are coming, and no one knows how to handle them". (link)
Development of the asymmetric amniotic sac—with the embryonic disc and amniotic ectoderm occupying opposite poles—is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology. - [7] Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche
Yue Shao, Kenichiro Taniguchi, Katherine Gurdziel, Ryan F. Townshend, Xufeng Xue, Koh Meng Aw Yong, Jianming Sang, Jason R. Spence, Deborah L. Gumucio, and Jianping Fu
Nature Materials, vol. 16, pp. 419-425, 2017.
DOI: 10.1038/nmat4829. PMCID: PMC5374007.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Martin Pera, Nature Materials, vol. 16, pp. 394-395, 2017. [PDF]
Amniogenesis - the development of amnion - is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP–SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine. - [6] Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis
Shinuo Weng, Yue Shao, Weiqiang Chen, and Jianping Fu
Nature Materials, vol. 15, pp. 961-967, 2016.
DOI: 10.1038/nmat4654. PMCID: PMC4996707.
Abstract | PDF | Supplemental Materials | PubMed
Mechanical homeostasis - a fundamental process by which cells maintain stable states under environmental perturbations - is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths via either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modeling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviors in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight previously underappreciated physical nature of the mechanical homeostasis of cells. - [5] Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells
Yubing Sun, Koh Meng Aw Yong, Luis G. Villa-Diaz, Xiaoli Zhang, Weiqiang Chen, Renee Philson, Shinuo Weng, Haoxing Xu, Paul H. Krebsbach, and Jianping Fu
Nature Materials, vol. 13, pp. 599-604, 2014.
DOI: 10.1038/nmat3945. PMCID: PMC4051885.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Emily Rhodes Lowry & Christopher E. Henderson, Nature Materials, vol. 13, pp. 543-544, 2014. [PDF]
Commentary by Ning Wang, Cell Stem Cell, vol. 14, pp. 701-703, 2014. [PDF]Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive1. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs. - [4] Adhesive signature-based, label-free isolation of human pluripotent stem cells
Ankur Singh, Shalu Suri, Ted T. Lee, Jamie M. Chilton, Marissa T. Cooke, Weiqiang Chen, Jianping Fu, Steven L. Stice, Hang Lu, Todd C. McDevitt, and Andrés J. García
Nature Methods, vol. 10, pp. 438-444, 2013.
DOI: 10.1038/nmeth.2437. PMCID: PMC3641175.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Oscar J. Abilez & Joseph C. Wu, Nature Materials, vol. 12, pp. 474-476, 2013; web link.
The ability to efficiently isolate undifferentiated human pluripotent stem cells (hPSCs) as colonies from contaminating non-pluripotent cells is a crucial step in the stem cell field to maintain survival efficiency and karyotype stability. Currently, this isolation relies primarily on time-intensive manual isolation or single cell sorting. Here we demonstrate significant differences in focal adhesion assembly and adhesion strength among undifferentiated hPSCs (hiPSC, hESC) and parental/feeder layer cells, partially reprogrammed cells, spontaneously differentiated and directly differentiated progenitor cells. This distinct ‘adhesive signature’ of hPSCs is exploited to rapidly (< 10 min) and efficiently isolate fully reprogrammed undifferentiated hiPSCs as intact colonies from partially reprogrammed cells, parental cells, feeder layer cells, spontaneously differentiated cells, and terminally differentiated cardiomyocytes using microfluidics technology termed µSHEAR (micro Stem cell High-Efficiency Adhesion-based Recovery). hPSCs, irrespective of source, passage number, and feeder-free matrix, are isolated in a label-free fashion and enriched to > 95-99% purity and survival without adversely affecting the transcriptional profile, differentiation potential or karyotype of the pluripotent cells. This low-cost, rapid, label-free, high-throughput strategy is applicable to isolate undifferentiated hiPSCs from heterogeneous reprogramming cultures and differentiating cell populations during routine hPSC culture and can be expanded to purify stem cells of specific lineages, such as neurons and cardiomyocytes. - [3] Mechanical regulation of cell function with geometrically modulated elastomeric substrates
Jianping Fu, Yang-Kao Wang, Michael T. Yang, Ravi A. Desai, Xiang Yu, Zhijun Liu, and Christopher S. Chen
Nature Methods, vol. 7, pp. 733-736, 2010.
DOI: 10.1038/nmeth.1487. PMCID: PMC3069358.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Amnon Buxboim & Dennis Discher, Nature Methods, vol. 7, pp. 695-697, 2010. [PDF]We report here the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We have demonstrated that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility can predict later stem cell fate decisions in single cells. - [2] A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and protein
Jianping Fu, Reto B. Schoch, Anna L. Stevens, Steven R. Tannenbaum, and Jongyoon Han
Nature Nanotechnology, vol. 2, pp. 121-128, 2007.
DOI: 10.1038/nnano.2006.206. PMCID: PMC2621439.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Robert Austin, Nature Nanotechnology, vol. 2, pp. 121-128, 2007. [PDF]We report here a microfabricated anisotropic sieving structure consisting of a two-dimensional periodic nanofluidic filter array. The designed structural anisotropy causes different-sized or -charged biomolecules to follow distinct trajectories, leading to efficient separation. Continuous-flow size-based separation of DNA and proteins, as well as electrostatic separation of proteins, have been achieved, demonstrating the potential use of this device as a generic molecular sieving structure for an integrated biomolecule sample preparation and analysis system. - [1] Molecular sieving in periodic free-energy landscapes created by patterned nanofilter arrays
Jianping Fu, Juhwan Yoo, and Jongyoon Han
Physical Review Letters, vol. 97, 018103, 2006.
DOI: 10.1103/PhysRevLett.97.018103. PMCID: PMC1752241.
Abstract | PDF | PubMedWe present an experimental study of Ogston-like sieving process of rod-like DNA in patterned periodic nanofluidic filter arrays. The electrophoretic motion of DNA through the array is described as a biased Brownian motion overcoming periodically modulated free-energy landscape. A kinetic model, constructed based on the equilibrium partitioning theory and the Kramers theory, explains the field-dependent mobility well.
Journal Articles and Book Chapters
2024
- [164] Substrate rigidity modulates segmentation clock dynamics in isolated presomitic mesoderm cells
Chun-Yen Sung, Usha Kadiyala, Owen Blanchard, Liam Yourston, Derek Walker, Linyuan Li, Jianping Fu, and Qiong Yang
bioRxiv, 2024.
DOI: 10.1101/2024.07.02.601712.
Abstract | PDF | PubMed
The segmentation clock, a genetic oscillator in the presomitic mesoderm (PSM), is known to be influenced by biochemical signals, yet its potential regulation by mechanical cues remains unclear. The complex PSM microenvironment has made it challenging to isolate the effects of mechanical perturbations on clock behavior. Here we investigated how mechanical stimuli affect clock oscillations by culturing zebrafish PSM cells on PDMS micropost arrays with tunable rigidities (0.6-1200 kPa). We observed an inverse sigmoidal relationship between surface rigidity and both the percentage of oscillating cells and the number of oscillation cycles, with a switching threshold between 3-6 kPa. The periods of oscillating cells showed a consistently broad distribution across rigidity changes. Moreover, these cells exhibited distinct biophysical properties, such as reduced motility, contractility, and sustained circularity. These findings highlight the crucial role of cell-substrate interactions in regulating segmentation clock behavior, providing insights into the mechanobiology of somitogenesis. - [163] A transgene-free, human peri-gastrulation embryo model with trilaminar embryonic disc-, amnion- and yolk sac-like structures
Shiyu Sun, Yi Zheng, Norio Kobayashi, Yung Su Kim, Xuefeng Xue, Yue Liu, Yanhong Xu, Jinglei Zhai, Hongmei Wang, and Jianping Fu
bioRxiv, 2024.
DOI: 10.1101/2024.08.05.606556.
Abstract | PDF | Supplemental Materials | PubMed
The ultimate outcome of the gastrulation in mammalian development is a recognizable trilaminar disc structure containing organized cell lineages with spatially defined identities in an emerging coordinate system. Despite its importance in human development, gastrulation remains difficult to study. Stem cell-based embryo models, including those that recapitulate different aspects of pre- and peri-gastrulation human development, are emerging as promising tools for studying human embryogenesis. However, it remains unclear whether existing human embryo models are capable of modeling the development of the trilaminar embryonic disc structure, a hallmark of human gastrulation. Here we report a transgene-free human embryo model derived solely from primed human pluripotent stem cells (hPSCs), which recapitulates various aspects of peri-gastrulation human development, including formation of trilaminar embryonic layers situated between dorsal amnion and ventral definitive yolk sac and primary hematopoiesis. We term this model the peri-gastrulation trilaminar embryonic disc (PTED) embryoid. The development of PTED embryoid does not follow natural developmental sequences of cell lineage diversification or spatial organization. Instead, it exploits both extrinsic control of tissue boundaries and intrinsic self-organizing properties and embryonic plasticity of the diverse peri-gastrulation-stage cell lineages, leading to the emergence of in vivo-like tissue organization and function at a global scale. Our lineage tracing study reveals that in PTED embryoids, embryonic and extraembryonic mesoderm cells, as well as embryonic and extraembryonic endoderm cells, share common progenitors emerging during peri-gastrulation development. Active hematopoiesis and blood cell generation are evident in the yolk sac-like structure of PTED embryoids. Together, PTED embryoids provide a promising and ethically less challenging model for studying self-organizing properties of peri-gastrulation human development. - [162] Small molecule valproic acid enhances ventral patterning of human neural tube organoids by regulating Wnt and Shh signaling
Yuanyuan Zheng, Fangrong Zhang, Haifeng Nie, Xinyu Li, Jaili Xun, Shengmin Xu, Jianping Fu, and Lijun Wu
Cell Proliferation, in press, 2024.
DOI: 10.1111/cpr.13737.
Abstract | PDF | Supplemental Materials | PubMed
WNT signaling is critical for dorsal-ventral (DV) patterning of embryonic neural tube, the process through which neuroepithelial precursors give rise to specific classes of neurons along the DV axis of the neural tube. Previous studies show that valproic acid (VPA), a teratogen and drug for treating seizures and bipolar disorder, could activate WNT signaling. However, little is known about the impact of VPA on DV patterning of human neural tube. Here, patterned human neural tube organoids (hNTOs) were utilized to investigate the impact of VPA on neural tube formation and subsequent DV patterning. Our data showed that even though VPA treatments had a negative impact on the formation of hNTOs, it significantly enhanced the efficiency of ventral patterned hNTOs, when VPA was added to hNTO culture during the entire differentiation process. RNA sequencing and RT-qPCR analysis demonstrated that VPA activated endogenous WNT signaling in VPA-treated hNTOs. Surprisingly, our transcriptome analysis also identified upregulation of genes for degradation of GLI2 and GLI3 proteins, whose truncated N-terminal fragment are transcriptional repressors of SHH signaling. Thus, VPA might enhance ventral patterning of hNTOs through both activating WNT signaling, which could antagonize SHH signaling by inducing GLI3 expression, and/or inhibiting SHH signaling by inducing GLI protein degradation. We further obtained results to show that VPA treatment could increase ventral patterning of hNTOs without affecting their formation when the initiation time of VPA treatment was delayed and its duration was reduced. Thus, this study demonstrates that VPA could enhance the generation of more reproducible hNTOs with ventral patterning, opening the avenues for the applications of hNTOs in neural development and disease modeling studies. - [161] Criteria for standardization of stem-cell-based embryo models
Alfonso Martinez Arias, Nicolas Rivron, Naomi Moris, Patrick Tam, Cantas Alev, Jianping Fu, Anna-Katerina Hadjantonakis, Jacob H. Hanna, Gabriella Minchiotti, Olivier Pourquie, Guojun Sheng, Liliana Solnica Krezel, Jesse Veenvliet, and Aryeh Warmflash
Nature Cell Biology, in press, 2024.
DOI: 10.1038/s41556-024-01492-x.
Abstract | PDF | PubMed
Pluripotent Stem Cells (PSCs) are being used to generate models of early embryogenesis promising for discovery and translational research. To be useful these models require a critical consideration of their level of efficiency and fidelity to natural embryos. Herein, we propose criteria that define the utility of stem-cell-based embryo models of human embryogenesis. - [160] A comprehensive human embryo reference tool using single-cell RNA-sequencing data
Cheng Zhao, Alvaro Plaza Reyes, John Paul Schell, Jere Weltner, Nicolás M. Ortega, Yi Zheng, Åsa K. Björklund, Joonas Sokka, Ras Torokovic, Brian Cox, Janet Rossant, Jianping Fu, Sophie Petropoulos, and Fredrik Lanner
Nature Methods, in press, 2024.
Abstract | PDF | PubMed
Stem cell-based embryo models offer unprecedented experimental tools for studying early human development. The usefulness of embryo models hinges on their molecular, cellular and structural fidelities to their in vivo counterparts. To authenticate human embryo models, single-cell RNA-sequencing has been utilised for unbiased transcriptional profiling. However, a well-organised and integrated human single-cell RNA-sequencing dataset, serving as a universal reference for benchmarking human embryo models, remains unavailable. Herein, we developed such a reference, through integration of six publicly available human datasets covering developmental stages from the zygote to the gastrula. Lineage annotations are contrasted and validated with available human and non-human primate datasets. Using stabilised UMAP, we further constructed a web tool, using which query datasets can be projected on the reference and annotated with predicted cell identities. Using this reference tool, we examined several recently published human embryo models. Our analysis reveals the risk of mis-annotation of cell lineages in embryo models when relevant human embryo references, such as the one developed in this work, were not utilized for benchmarking and authentication. - [159] Bioengineering embryo models
Xufeng Xue, Yue Liu, and Jianping Fu
Nature Reviews Bioengineering, in press, 2024.
DOI: 10.1038/s44222-024-00241-x.
Abstract | PDF | PubMed
Stem cell-based embryo models, which recapitulate symmetry breaking, pattern formation and tissue morphogenesis during early development, provide promising experimental tools to study development of mammalian species, including humans. Despite considerable progress in embryo modeling using cultured stem cells, it remains challenging to construct embryo models with high fidelity, efficiency, controllability, and in vivo-like cellular organization and tissue architecture. This is largely due to intrinsic variabilities in self-organization and differentiation of mammalian stem cells in uncontrolled culture environments utilized in current embryo modeling. In this review, we argue that bioengineering tools, which are powerful for controlling topological boundaries and dynamic chemical and mechanical signals and thus are efficient in guiding symmetry breaking, pattern formation, tissue morphogenesis and tissue-tissue interactions, should be utilized for constructing high-fidelity, high-efficiency embryo models. In this review, we first discuss pattern formation and morphogenesis during embryonic development, and selectively examine different embryo models to highlight the importance of bioengineering controls in developing these models. We then explore how different bioengineering approaches useful for guiding pattern formation, morphogenesis, cell fate decisions and cell-cell interfaces can be utilized in stem cell-based embryo modeling to promote their efficiency, reproducibility, controllability, complexity, and in vivo relevance. - [158] Towards developing human organs via embryo models and chimeras
Jun Wu and Jianping Fu
Cell, vol. 187, pp. 3194-3219, 2024.
DOI: 10.1016/j.cell.2024.05.027.
Abstract | PDF | PubMed
Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting and organoids, only offer partial solutions. This Perspective focuses on two emerging approaches promising for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development, as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed, before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outline future prospects for advancing both fields towards the generation of human tissues and organs for basic research and translational applications. - [157] Contributions of FAK, vinculin, and talin to YAP nuclear localization in adherent fibroblasts
Elijah N. Holland, Marc A. Fernández-Yagüe, Dennis W. Zhou, Eric B. O’Neill, Ayanna U. Woodfolk, Jianping Fu, David D. Schlaepfer, and Andrés J. García
Biomaterials, vol. 308, 122542, 2024.
DOI: 10.1016/j.biomaterials.2024.122542.
Abstract | PDF | Supplemental Materials | PubMed
Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity. - [156] Metabolic glycoengineering-enabled molecularly specific acoustic tweezing cytometry for targeted mechanical stimulation of cell surface sialoglycans
Weiping Li, Jiatong Guo, Eric E. Hobson, Xufeng Xue, Qingjiang Li, Jianping Fu, Cheri X. Deng, and Zhongwu Guo
Angewandte Chemie International Edition, vol. 63, e202401921, 2024.
DOI: 10.1002/anie.202401921.
Abstract | PDF | Supplemental Materials | PubMed
This study developed a new type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated via metabolic glycoengineering (MGE). This enabled application of mechanical forces to sialoglycans on hPSCs via molecularly specific acoustic tweezing cytometry (mATC), i.e., displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinct mechanoresponses during the early stages of hPSC differentiation. Taken together, the results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs than integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction. - [155] A human pluripotent stem cell-based somitogenesis model using microfluidics
Yue Liu, Yung Su Kim, Xufeng Xue, Yuchuan Miao, Norio Kobayashi, Shiyu Sun, Robin Zhexuan Yan, Qiong Yang, Olivier Pourquié, and Jianping Fu
Cell Stem Cell, vol. 31, pp. 1113-1126, 2024.
DOI: 10.1101/2023.10.29.564399.
Abstract | PDF | Supplemental Materials | PubMed
Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation. - [154] A patterned human neural tube model using microfluidic gradients
Xufeng Xue, Yung Su Kim, Alfredo-Isaac Ponce-Arias, Richard O'laughlin, Robin Zhexuan Yan, Norio Kobayashi, Rami Yair Tshuva, Yu-Hwai Tsai, Shiyu Sun, Yi Zheng, Yue Liu, Frederick C.K. Wong, Azim Surani, Jason R. Spence, Hongjun Song, Guo-Li Ming, Orly Reiner, and Jianping Fu
Nature, vol. 628, pp. 391-399, 2024.
DOI: 10.1038/s41586-024-07204-7.
Abstract | PDF | Supplemental Materials | PubMed
Human nervous system is arguably the most complex but highly organized organ. Foundation of its complexity and organization is laid down during regional patterning of neural tube (NT), the embryonic precursor to human nervous system. Historically, studies of NT patterning have relied on animal models to uncover underlying principles. Recently, human pluripotent stem cell (hPSC)-based models of neurodevelopment, including neural organoids1-5 and bioengineered NT development models6-10, are emerging. However, existing hPSC-based models fail to recapitulate neural patterning along both rostral-caudal (R-C) and dorsal-ventral (D-V) axes in a three-dimensional (3D) tubular geometry, a hallmark of NT development. Herein we report a hPSC-based, microfluidic NT-like structure (or µNTLS), whose development recapitulates some critical aspects of neural patterning in both brain and spinal cord (SC) regions and along both R-C and D-V axes. The µNTLS was utilized for studying neuronal lineage development, revealing pre-patterning of axial identities of neural crest (NC) progenitors and functional roles of neuromesodermal progenitors (NMPs) and caudal gene CDX2 in SC and trunk NC development. We further developed D-V patterned, microfluidic forebrain-like structures (µFBLS) with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic human forebrain pallium and subpallium developments, respectively. Together, both µNTLS and µFBLS offer 3D lumenal tissue architectures with an in vivo-like spatiotemporal cell differentiation and organization, promising for studying human neurodevelopment and disease. - [153] Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity
Hyung Chul Lee, Nidia M. M. Oliveira, Cato Hastings, Peter Baillie-Benson, Adam A. Moverley, Hui-Chun Lu, Yi Zheng, Elise L. Wilby, Timothy T. Weil, Karen M. Page, Jianping Fu, Naomi Moris, and Claudio D. Stern
Nature Communications, vol. 15, 1463, 2024.
DOI: 10.1038/s41467-024-45772-4.
Abstract | PDF | Supplemental Materials | PubMed
Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevents ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT, establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in human embryoids and Drosophila, suggesting an ancient evolutionary origin. - [152] Derivation of human primordial germ cell-like cells in an embryonic-like culture
Sajedeh Nasr Esfahani, Yi Zheng, Auriana Arabpour, Agnes M. Resto Irizarry, Norio Kobayashi, Xufeng Xue, Yue Shao, Cheng Zhao, Nicole L. Agranonik, Megan Sparrow, Timothy J. Hunt, Jared Faith, Mary Jasmine Lara, Qiu Ya Wu, Sherman Silber, Sophie Petropoulos, Ran Yang, Kenneth R. Chien, Amander T. Clark, and Jianping Fu
Nature Communications, vol. 15, 167, 2024.
DOI: 10.1038/s41467-023-43871-2.
Abstract | PDF | Supplemental Materials | PubMed
Featured in Nature Communications Editors’ Highlights
We report the induction of human primordial germ cell-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs) derived from hPSCs induce hPGCLC specification through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived induced pluripotent stem cell lines using this biomimetic platform, demonstrating its promise for screening applications. - [151] Morphogenesis beyond in vivo
Yue Liu, Xufeng Xue, Shiyu Sun, Norio Kobayashi, Yung Su Kim, and Jianping Fu
Nature Reviews Physics, vol. 6, pp. 28-44, 2024.
DOI: 10.1038/s42254-023-00669-x.
Abstract | PDF | PubMed
Morphogenetic events during development shape the body plan and establish structural foundations for tissue forms and functions. Driven by technical and ethical constraints in acquiring spatiotemporal information of development, especially for humans, both stem cell-based, in vitro development models and theoretical models have been constructed to recapitulate morphogenetic events during development. These in vitro experimental and theoretical models offer convenient accessibility, great efficiency, and favorable modulability. However, their physiological relevance often remains obscure due to their simplistic nature, obstructing their applicability as faithful and predictive models of natural development. In this review we examine existing in vitro experimental and theoretical models of various developmental events while comparing them with the current knowledge of natural development, with particular considerations of biomechanical driving forces and stereotypic morphogenetic features. We highlight state-of-the-art methodologies employed in constructing these in vitro models and emphasize the biomechanical and biophysical principles these models have helped unveil. Finally, challenges faced by the current in vitro experimental and theoretical models will be discussed, and we propose how theoretical modeling and in vitro experimental models should be combined with in vivo studies to advance fundamental understanding of development.
2023
- [150] Rapid responses of human pluripotent stem cells to cyclic mechanical strains applied to integrin by acoustic tweezing cytometry
Zhaoyi Xu, Shiying Liu, Xufeng Xue, Weiping Li, Jianping Fu, and Cheri X. Deng
Scientific Reports, vol. 13, 18030, 2023.
DOI: 10.1038/s41598-023-45397-5.
Abstract | PDF | Supplemental Materials | PubMed
Acoustic tweezing cytometry (ATC) is an ultrasound-based biophysical technique that has shown the capability to promote differentiation of human pluripotent stem cells (hPSCs). This study systematically examined how hPSCs would respond to cyclic mechanical strains applied by ATC via displacement of integrin-bound microbubbles using ultrasound pulses. Our data show downregulation of pluripotency marker Octamer-binding transcription factor 4 (OCT4) by at least 10% and reduced nuclear localization of Yes-associated protein (YAP) by 50% in hPSCs immediately after ATC applications for as short as 1 min. Analysis of the movements of integrin-anchored microbubbles under ATC stimulations reveals viscoelastic characteristics and deformation of the integrin-cytoskeleton (CSK) linkage that are influenced by ultrasound pulse duty cycles from 5% to 50% without detachment. Real-time tracking of multiple integrin-bound microbubbles during ATC applications demonstrates that microbubble displacements correlate with OCT4 downregulation in hPSCs. Together, our data showing rapid downregulation of OCT4 in hPSCs in respond to ATC stimulations highlight the unique mechanosensitivity of hPSCs to integrin-targeted cyclic force/strain that are dependent on duty cycle of ultrasound pulses, providing insights into the mechanism of ATC-induced accelerated differentiation of hPSCs. - [149] Changing the public perception of human embryology
Nicolas C. Rivron, Alfonso Martinez-Arias, Karen Sermon, Christine Mummery, Hans Schöler, James Wells, Jenny Nichols, Anna-Katerina Hadjantonakis, Madeline A. Lancaster, Jianping Fu, Janet Rossant, and Kazuto Kato
Nature Cell Biology, vol. 25, pp. 1717-1719, 2023.
DOI: 10.1038/s41556-023-01289-4.
Abstract | PDF | PubMed
Human embryology is flourishing thanks to an impetus provided by embryo models formed from stem cells. These scientific advances require meticulous modelling, a refined ethical framework, but also sensible public communication. Securing public support is essential to laying the foundations needed to achieve societal impact. - [148] Why researchers should use human embryo models with caution
Janet Rossant and Jianping Fu
Nature, vol. 622, pp. 22-24, 2023.
DOI: 10.1038/d41586-023-03062-x.
Abstract | PDF | PubMed
Scientists should carefully consider whether embryo models based on human stem cells are essential to their work because of the associated practical and ethical challenges. Appropriately used, stem-cell-based models of human embryonic development could transform our understanding of how human life begins. We ask researchers to carefully consider whether there is a strong scientific rationale for replicating an entire human embryo from stem cells. Pushing ahead without careful deliberation risks a public backlash that could stall the progress in this exciting field. - [147] Mechanically enhanced biogenesis of gut spheroids with instability-driven morphomechanics
Feng Lin, Xia Li, Shiyu Sun, Zhongyi Li, Jianbo Bai, Lin Song, Bo Li, Jianping Fu, and Yue Shao
Nature Communications, vol. 14, 6016, 2023.
DOI: 10.1038/s41467-023-41760-2.
Abstract | PDF | Supplemental Materials | PubMed
Region-specific gut spheroids are precursors for gastrointestinal and pulmonary organoids that hold great promise for fundamental studies and translations. However, efficient production of gut spheroids remains challenging due to a lack of control and mechanistic understanding of gut spheroid morphogenesis. Here, we report an efficient biomaterial system, termed micropatterned gut spheroid generator (μGSG), to generate gut spheroids from human pluripotent stem cells through mechanically enhanced tissue morphogenesis. We show that μGSG enhances the biogenesis of gut spheroids independent of micropattern shape and size; instead, mechanically enforced cell multilayering and crowding is demonstrated as a general, geometry-insensitive mechanism that is necessary and sufficient for promoting spheroid formation. Combining experimental findings and an active-phase-field morphomechanics theory, our study further reveals an instability-driven mechanism and a mechanosensitive phase diagram governing spheroid pearling and fission in μGSG. This work unveils mechanobiological paradigms based on tissue architecture and surface tension for controlling tissue morphogenesis and advancing organoid technology. - [146] Modeling development using microfluidics: Bridging gaps to foster fundamental and translational research
Shiyu Sun, Xufeng Xue, and Jianping Fu
Current Opinion in Genetics & Development, vol. 82, 102097, 2023.
DOI: 10.1016/j.gde.2023.102097.
Abstract | PDF | PubMed
In vitro stem cell-derived embryo and organ models, termed embryoids and organoids, respectively, provide promising experimental tools to study physiological and pathological processes in mammalian development and organ formation. Most of current embryoid and organoid systems are developed using conventional three-dimensional cultures that lack controls of spatiotemporal extracellular signals. Microfluidics, an established technology for quantitative controls and quantifications of dynamic chemical and physical environments, has recently been utilized for developing nextgeneration embryoids and organoids in a controllable and reproducible manner. In this review, we summarize recent progress in constructing microfluidics-based embryoids and organoids. Development of these models demonstrates the successful applications of microfluidics in establishing morphogen gradients, accelerating medium transport, exerting mechanical forces, facilitating tissue coculture studies, and improving assay throughput, thus supporting using microfluidics for building next-generation embryoids and organoids for fundamental and translational research. - [145] Stem cell-derived embryo models: A frontier of human embryology
Norio Kobayashi, and Jianping Fu
Medical Review, 2023.
DOI: 10.1515/mr-2023-0009.
Abstract | PDF | PubMed
Studying human development remains difficult due to limited accessibility to human embryonic tissues. Prompted by the availability of human stem cells that share molecular and cellular similarities with embryonic and extraembryonic cells in peri-implantation human embryos, researchers have now successfully developed stem cellbased human embryo models that are promising as experimental tools for studying early human development. In this Perspective, we discuss the current progress in mouse and human stem cell-derived embryo models and highlight their promising applications in advancing the fundamental understanding of mammalian development. - [144] Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids
Zongyong Ai, Baohua Niu, Yu Yin, Lifeng Xiang, Gaohui Shi, Kui Duan, Sile Wang, Yingjie Hu, Chi Zhang, Chengting Zhang, Lujuan Rong, Ruize Kong, Tingwei Chen, Yixin Guo, Wanlu Liu, Nan Li, Shumei Zhao, Xiaoqing Zhu, Xuancheng Mai, Yonggang Li, Ze Wu, Yi Zheng, Jianping Fu, Weizhi Ji, and Tianqing Li
Cell Research, vol. 33, pp. 661-678, 2023.
DOI: 10.1038/s41422-023-00846-8.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Janet Rossant, Cell Research, vol. 33, pp. 1-2, 2023. [PDF]
Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage development as well as underlying mechanisms remains obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling pathways synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast development and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights into human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis. - [143] Impact of adhesive area on cellular traction force and spread area
Elijah N. Holland, Deborah Lobaccaro, Jianping Fu, Andrés J. García
Journal of Biomedical Materials Research Part A, vol. 111, pp. 609-617, 2023.
DOI: 10.1002/jbm.a.37518.
Abstract | PDF | PubMed
Cells integrate endogenous and exogenous mechanical forces to sense and respond to environmental signals. In particular, cell-generated microscale traction forces regulate cellular functions and impact macroscale tissue function and development. Many groups have developed tools for measuring cellular traction forces, including microfabricated post array detectors (mPADs). mPADs are a powerful tool that provides direct traction force measurements through imaging post deflections and utilizing Bernoulli-Euler beam theory. In this technical note, we investigated how mPADs presenting two different top surface areas but similar effective stiffness influence cellular spread area and traction forces for murine embryonic fibroblasts and human mesenchymal stromal cells. When focal adhesion size was restricted via mPAD top surface area, we observed a decrease in both cell spread area and cell traction forces as the mPAD top surface area decreased, but the traction force-cell area linear relationship was maintained, which is indicative of cell contractility. We conclude that the mPAD top surface area is an important parameter to consider when utilizing mPADs to measure cellular traction forces. Furthermore, the slope of the traction force-cell area linear relationship provides a useful metric to characterize cell contractility on mPADs. - [142] Mechanosensitive channel-based membrane tension biosensor
Yen-Yu Hsu, Agnes M. Resto Irizarry, Jianping Fu, and Allen P. Liu
ACS Sensors, vol. 8, pp. 12-18, 2023.
DOI: 10.1021/acssensors.2c01921.
Abstract | PDF | Supplemental Materials | PubMed
Plasma membrane tension has been shown as a global physical organizer of cellular activities. Technical limitations of current membrane tension measurement techniques have hampered in-depth investigation of cellular membrane biophysics and its role in regulating cellular processes. Here, we develop an optical membrane tension biosensor by repurposing an E. coli mechanosensitive channel via insertion of circularly permuted GFP (cpGFP), which undergoes a large conformational rearrangement associated with channel activation and thus fluorescence intensity changes under increased membrane tension.
2022
- [141] In vitro germ cell induction from fertile and infertile monozygotic twin research participants.
Erica C. Pandolfi, Mark Duhon, Fei-Man Hsu, Yi Zheng, Jianping Fu, Sherman J. Silber, and Amander T. Clark
Cell Reports Medicine, vol. 3, 100782, 2022.
DOI: 10.1016/j.xcrm.2022.100782.
Abstract | PDF | PubMed
Primary ovarian insufficiency (POI) is a disease that affects around 1% of all women and involves depletion or dysfunction of germ cells. POI is also diagnosed in monozygotic (MZ) twins and in rare cases POI is discordant. In discordant POI, the etiology is hypothesized to originate during prenatal development due to unequal distribution of primordial germ cells (PGCs) amongst MZ twins who share an amniotic sac. To evaluate whether loss of germ cells can be reversed using reprogramming, we consented MZ twins with discordant POI to donate a skin punch biopsy and generated human induced pluripotent stem cells (hiPSCs). We discovered that germ cell loss can be reversed following reprogramming with germ cell induction occurring in hiPSC-derived amnion tissue, supporting a promising solution for treating POI in MZ twins. - [140] Stem cell-based models of early mammalian development
Aidan Terhune, Jeyoon Bok, Shiyu Sun, and Jianping Fu
Development, vol. 149, dev.201015, 2022.
DOI: 10.1242/dev.201015.
Abstract | PDF | PubMed
The complex process by which a single celled zygote develops into a viable embryo is nothing short of a miraculous wonder of the natural world. Elucidating how this process is orchestrated in humans has long eluded the grasp of scientists due to ethical and practical limitations. Thankfully, pluripotent stem cells that resemble early developmental cell types possess the ability to mimic certain embryonic development events. As such, murine and human stem cells have been leveraged by scientists to create in vitro models that aim to recapitulate different stages of early mammalian development. Here, we examine the wide variety of stem cell-based embryo models that have been developed to recapitulate and study embryonic events, from pre-implantation development through to early organogenesis. We discuss the applications of these embryo models, key considerations regarding their importance within the field, and how such models are expected to grow and evolve to achieve exciting new milestones in the future. - [139] Organoids as tools for fundamental discovery and translation - A Keystone Symposia report
Jennifer Cable, Matthias P. Lutolf, Jianping Fu, Sunghee Estelle Park, Athanasia Apostolou, Shuibing Chen, Cheng Jack Song, Jason R. Spence, Prisca Liberali, Madeline Lancaster, Anna B. Meier, Nicole Min Qian Pek, James M. Wells, Meghan M. Capeling, Ana Uzquiano, Samira Musah, Meritxell Huch, Mina Gouti, Pleun Hombrink, Georgio Quadrato
Annals of the New York Academy of Sciences, vol. 1518, pp. 196-208, 2022.
DOI: 10.1111/nyas.14874.
Abstract | PDF | PubMed
Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproducehumanbiology. Successfully addressing such limitations would increase the viability of organoids as models for drug development and preclinical testing. On April 3–6, 2022, experts in organoid development and biology convened at the Keystone Symposium “Organoids as tools for fundamental discovery and translation” to discuss recent advances and insights from this relatively new model system into human development and disease. - [138] Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development
Yi Zheng, Robin Zhexuan Yan, Shiyu Sun, Mutsumi Kobayashi, Lifeng Xiang, Ran Yang, Alexander Goedel, Yu Kang, Xuefeng Xue, Sajedeh Nasr Esfahani, Yue Liu, Agnes M. Resto Irizarry, Weisheng Wu, Yunxiu Li, Weizhi Ji, Yuyu Niu, Kenneth R. Chien, Tianqing Li, Toshihiro Shioda, and Jianping Fu
Cell Stem Cell, vol. 29, pp. 1402-1419, 2022.
DOI: 10.1016/j.stem.2022.08.009.
Abstract | PDF | Supplemental Materials | PubMed
Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast towards the amniotic ectoderm, primitive streak / mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells. - [137] Engineering multiscale structural orders for high-fidelity embryoids and organoids
Yue Shao, and Jianping Fu
Cell Stem Cell, vol. 29, pp. 722-743, 2022.
DOI: 10.1016/j.stem.2022.04.003.
Abstract | PDF | PubMed
Embryoids and organoids hold great promise for human biology and medicine. Herein, we discuss conceptual and technological frameworks useful for developing high-fidelity embryoids and organoids that display tissue- and organ-level phenotypes and functions, which are critically needed for decoding developmental programs and improving translational applications. Through dissecting the layers of inputs controlling mammalian embryogenesis, we review recent progress in reconstructing multiscale structural orders in embryoids and organoids. Bioengineering tools useful for multiscale, multimodal structural engineering of tissue- and organ-level cellular organization and microenvironment are also discussed to present integrative, bioengineering-directed approaches to achieve next-generation, high-fidelity embryoids and organoids. - [136] Elucidating the behavior of trophectoderm derivatives in mouse implantation
Aidan H. Terhune, and Jianping Fu
Developmental Cell, vol. 57, pp. 295-297, 2022.
DOI: 10.1016/j.devcel.2022.01.010.
Abstract | PDF | PubMed
Studying mammalian implantation in utero is difficult, but many in vitro models of peri-implantation development lack contributions from extra-embryonic tissues. Two recently published Developmental Cell papers present biomimetic systems for culturing peri-implantation mouse blastocysts ex vivo. These papers reveal dynamics and developmental impacts of two essential trophectoderm derivatives: extra-embryonic ectoderm and trophoblast.
2021
- [135] Micro/nanoengineered technologies for human pluripotent stem cell maintenance and differentiation
Sajedeh Nasr Esfahani, Agnes M. Resto Irizarry, Xufeng Xue, Samuel Byung-Deuk Lee, Yue Shao, and Jianping Fu
Nano Today, vol. 41, 101310, 2021.
DOI: 10.1016/j.nantod.2021.101310.
Abstract | PDF | PubMed
Human pluripotent stem cells (hPSCs) are a promising source of cells for cell replacement-based therapies as well as modeling human development and diseases in vitro. However, achieving fate control of hPSCs with a high yield and specificity remains challenging. The fate specification of hPSCs is regulated by biochemical and biomechanical cues in their environment. Driven by this knowledge, recent exciting advances in micro/nanoengineering have been leveraged to develop a broad range of tools for the generation of extracellular biomechanical and biochemical signals that determine the behavior of hPSCs. In this review, we summarize such micro/nanoengineered technologies for controlling hPSC fate and highlight the role of biochemical and biomechanical cues such as substrate rigidity, surface topography, and cellular confinement in hPSC-based technologies that are on the horizon. - [134] Machine learning-assisted imaging analysis of a human epiblast model
Agnes M. Resto Irizarry, Sajedeh Nasr Esfahani, Yi Zheng, Robin Zhexuan Yan, Patrick Kinnunen, and Jianping Fu
Integrative Biology, vol. 13, pp. 221-229, 2021.
DOI: 10.1093/intbio/zyab014.
Abstract | PDF | PubMed
Selected as the front cover story by Integrative Biology.
The human embryo is a complex structure that emerges and develops as a result of cell-level decisions guided by both intrinsic genetic programs and cell-cell interactions. Given limited accessibility and associated ethical constraints of human embryonic tissue samples, researchers have turned to the use of human stem cells to generate embryo models to study specific embryogenic developmental steps. However, to study complex self-organizing developmental events using embryo models, there is a need for computational and imaging tools for detailed characterization of cell-level dynamics in embryo models at the single cell level. In this work, we obtained live cell imaging data from a human pluripotent stem cell (hPSC)-based epiblast model that can recapitulate the lumenal epiblast cyst formation soon after implantation of the human blastocyst. By processing imaging data with a Python pipeline that incorporates both cell tracking and event recognition with the use of a CNN-LSTM machine learning model, we obtained detailed temporal information of changes in cell state and neighborhood during the dynamic growth and morphogenesis of lumenal hPSC cysts. The use of this tool combined with reporter lines for cell types of interest will drive future mechanistic studies of hESC fate specification in embryo models and will advance our understanding of how cell-level decisions lead to global organization and emergent phenomena. - [133] Human embryo research, stem cell-derived embryo models and in vitro gametogenesis: Considerations leading to the revised ISSCR guidelines
Amander T. Clark, Ali Brivanlou, Jianping Fu, Kazuto Kato, Debra Mathews, Kathy K. Niakan, Nicolas Rivron, Mitinori Saitou, Azim Surani, Fuchou Tang, and Janet Rossant
Stem Cell Reports, vol. 16, pp. 1-9, 2021.
DOI: 10.1016/j.stemcr.2021.05.008.
Abstract | PDF | PubMed
The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process. - [132] ISSCR Guidelines for Stem Cell Research and Clinical Translation: The 2021 update
Robin Lovell-Badge, Eric Anthony, Roger A. Barker, Tania Bubela, Ali H. Brivanlou, Melissa Carpenter, R. Alta Charo, Amander Clark, Ellen Clayton, Yali Cong, George Q. Daley, Jianping Fu, Misao Fujita, Andy Greenfield, Steve A. Goldman, Lori Hill, Insoo Hyun, Rosario Isasi, Jeffrey Kahn, Kazuto Kato, Jin-Soo Kim, Jonathan Kimmelman, Jürgen A. Knoblich, Debra Mathews, Nuria Montserrat, Jack Mosher, Megan Munsie, Hiromitsu Nakauchi, Luigi Naldini, Gail Naughton, Kathy Niakan, Ubaka Ogbogu, Roger Pedersen, Nicolas Rivron, Heather Rooke, Janet Rossant, Jeff Round, Mitinori Saitou, Douglas Sipp, Julie Steffann, Jeremy Sugarman, Azim Surani, Jun Takahashi, Fuchou Tang, Leigh Turner, Patricia J. Zettler, and Xiaomei Zhai
Stem Cell Reports, vol. 16, pp. 1-11, 2021.
DOI: 10.1016/j.stemcr.2021.05.012.
Abstract | PDF | PubMed
The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here. - [131] SnapShot: Embryo models
Nicolas Rivron, and Jianping Fu
Stem Cell Reports, vol. 16, pp. 1142, 2021.
DOI: 10.1016/j.stemcr.2021.04.012.
Abstract | PDF | PubMed
Stem Cell Report theme issue of stem cell-based embryo models. - [130] It takes a village to form embryo models
Nicolas Rivron, and Jianping Fu
Stem Cell Reports, vol. 16, pp. 1011-1013, 2021.
DOI: 10.1016/j.stemcr.2021.04.014.
Abstract | PDF | PubMed
Stem Cell Report Editorail on the theme issue of stem cell-based embryo models. - [129] Branching development of early post-implantation human embryonic-like tissues in 3D stem cell culture
Kejie Chen, Yi Zheng, Xufeng Xue, Yue Liu, Agnes M. Resto Irizarry, Huaijing Tang, and Jianping Fu
Biomaterials, vol. 275, 120898, 2021.
DOI: 10.1016/j.biomaterials.2021.120898.
Abstract | PDF | Supplemental Materials | PubMed
Human embryonic stem cells (hESCs) have the intrinsic capacity to self-organize and generate patterned tissues. In vitro models that coax hESCs to form embryonic-like structures by modulating physical environments and priming with chemical signals have become a powerful tool for dissecting the regulatory mechanisms underlying early human development. Here we present a 3D suspension culture system of hESCs that can generate post-implantation, pre-gastrulation embryonic-like tissues in a highly efficient and controllable manner. The efficiency of the development of asymmetric tissues, which mimic the post-implantation amniotic sac, was about 50% in the 3D suspension culture. Quantitative imaging profiling and unsupervised trajectory analysis revealed that hESC aggregates first entered into a transitional stage expressing Brachyury (or T), after which their development branched into different paths to develop into asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues, respectively. Moreover, the branching developmental trajectory of embryonic-like structures was affected by the initial cell seeding density or cluster size. A higher percentage of amniotic-like tissues was observed under a small initial cell seeding density. Conversely, a large initial cell seeding density promoted the development of mesodermal-like tissues. Our results suggest that hESCs have the intrinsic capability to sense the initial cell population size, which in turn regulates their differentiation and self-organization into different embryonic-like tissues. Our 3D suspension culture thus provides a promising experimental tool to study the interplay between tissue topology and self-organization and progressive embryonic development using in vitro hESC-based models. - [128] Reprogrammed iBlastoids contain amnion-like cells but not trophectoderm
Cheng Zhao, Alvaro Plaza Reyes, John Paul Schell, Jere Weltner, Nicolás Ortega, Yi Zheng, Åsa K. Björklund, Janet Rossant, Jianping Fu, Sophie Petropoulos, and Fredrik Lanner
bioRxiv, 2021.2005.2007.442980, 2021.
DOI: 10.1101/2021.05.07.442980.
Abstract | PDF | Supplemental Materials | PubMed
Two recent papers in Nature show that human blastocyst-like structures (or blastoids) can be generated from human pluripotent stem cells or through reprogramming of fibroblasts, respectively. Both papers perform extensive single cell transcriptional analysis and compare blastoid cells with the cells in preimplantation human embryos, leading to a conclusion that the blastoids contain cell lineages corresponding to the epiblast, primitive endoderm and trophectoderm in preimplantation human embryos. Transcriptional analysis is, however, critically dependent on having relevant reference samples, not only of targeted cell types but also of potential alternative cell lineages. For this reason, we have reevaluated the blastoid data with a more comprehensive cellular reference, including extended cultures of blastocysts, several stem cell-based embryo models and a gastrulation stage human specimen. From this reanalysis we resolve that reprogrammed blastoids by Liu et al. fail to generate cells with trophectoderm profiles. Instead, cells identified as trophectoderm lineages in reprogrammed blastoids possess a transcriptional profile more representative of amniotic cells in post-implantation human embryos. Our reanalysis also shows that stem cell-derived blastoids1 did contain trophectoderm-like cells, highlighting the potential of human blastoids to model blastocyst development.. - [127] Generation of fate patterns via intercellular forces
Hayden Nunley, Xufeng Xue, Jianping Fu, and David K. Lubensky
bioRxiv, 2021.2004.2030.442205, 2021.
Physical Review Letters, under review, 2021.
DOI: 10.1101/2021.04.30.442205.
Abstract | PDF | Supplemental Materials |PubMed
Studies of fate patterning often emphasize cell-cell communication via diffusible chemicals rather than via mechanical forces. Recent experiments on stem cell colonies demonstrated that without exogenous chemical gradients, fate patterning can be biased by mechanical stretching; these findings inspire a model of mechanical patterning: fate affects contractility, and in-plane pressure biases fate. Cells at the colony boundary, more contractile than cells at the center, generate a pattern via force transmission. In agreement with previous observations, our model implies that the outer domain's width depends weakly on colony diameter. Our model predicts that this same width depends nonmonotonically on substrate stiffness. With experimental confirmation of model predictions, we argue that mechanical pressure can act like a morphogen, effecting cell-cell communication. - [126] Amnion signals are essential for mesoderm formation in primates
Ran Yang, Alexander Goedel, Yu Kang, Chengyang Si, Chu Chu, Yi Zheng, Zhenzhen Chen, Peter J. Gruber, Yao Xiao, Chikai Zhou, Chuen-Yan Leung, Yongchang Chen, Jianping Fu, Weizhi Ji, Fredrik Lanner, Yuyu Niu, and Kenneth Chien
Nature Communications, vol. 12, 5126, 2021.
DOI: 10.1101/2020.05.28.118703.
Abstract | PDF | Supplemental Materials | PubMed
The in vivo signaling network controlling primitive streak formation is well understood in mouse, but largely unexplored in primates. Herein, we generated a transcriptional atlas consisting of >100 000 cells spanning peri-implantation to early steak stages in non-human primates. We uncovered that ISL1, a gene with well-established role in mouse-cardiogenesis, is highly expressed in the primate amnion. Strikingly, ISL1 hypomorphic primate mutant embryos fail to induce primitive streak. We identified BMP4 as a key signaling pathway in this process and confirmed this in human embryonic stem cells, suggesting conserved function in humans. Notably, no viable ISL1 hypomorphic mutant embryo could be recovered after embryo transfer confirming the essential requirement of ISL1 and highlighting the importance of the amnion as a signaling center during primate embryogenesis. This study demonstrates the potential and importance of in vitro primate model systems to investigate the genetics of early human development including mesoderm specification. - [125] Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair
Dennis W. Zhou, Marc A. Fernández-Yagüe, Elijah N. Holland, Andrés F. García, Nicolas S. Castro, Eric B. O’Neill, Jeroen E.G. Eyckmans, Christopher S. Chen, Jianping Fu, David D. Schlaepfer, and Andrés J. García
Nature Communications, vol. 12, 2359, 2021.
DOI: 10.1038/s41467-021-22602-5.
Abstract | PDF | Supplemental Materials | PubMed
How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization and traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK binding interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair. - [124] Spatially resolved cell polarity proteomics of a human epiblast model
Sicong Wang, Chien-Wei Lin, Chari L. Cortez, Amber E. Carleton, Craig Johnson, Linnea E. Taniguchi, Ryan F. Townshend, Venkatesha Basrur, Alexey I. Nesvizhskii, Amy W. Hudson, Blake R. Hill, Peng Zou, Jianping Fu, Deborah L. Gumucio, Mara C. Duncan, and Kenichiro Taniguchi
Science Advances, vol. 7, eabd8407, 2021.
DOI: 10.1126/sciadv.abd8407.
Abstract | PDF | Supplemental Materials | PubMed
The pro-amniotic cavity of the implanting human embryo forms from an unpolarized mass of epithelial pluripotent epiblast cells. This process requires apico-basal polarization and radial organization of epiblast cells, followed by central lumen expansion. To gain global mechanistic insights into these critical but poorly understood early steps, we developed a spatially targeted approach to compare the proteomes of apical vs. basolateral membrane territories in a three-dimensional (3D) human pluripotent stem cell-derived epiblast-like model. We employed APEX2-based proximity biotinylation with quantitative mass spectrometry and revealed a variety of proteins without previous annotation to specific membrane sub-domains. Functional experiments validated the requirement for several apically enriched proteins in epiblast-like morphogenesis. In particular, we identified a previously unappreciated role for the AP-1 clathrin adaptor complex-dependent apical trafficking in lumen expansion. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models. - [123] First complete model of the human embryo
Yi Zheng, and Jianping Fu
Nature, vol. 591, pp. 531-532, 2021.
DOI: 10.1038/d41586-021-00581-3.
Abstract | PDF | PubMed
Early in development, human embryos form a structure called the blastocyst. Two research groups have now generated human blastocyst-like structures from cells in a dish, providing a valuable model for advancing human embryology. - [122] A microfluidics-based stem cell model of early post-implantation human development
Yi Zheng, Yue Shao, and Jianping Fu
Nature Protocols, vol. 16, pp. 309-326, 2021.
DOI: 10.1038/s41596-020-00417-w.
Abstract | PDF | PubMed
Here we describe the design and implementation of a microfluidic device for the development of a stem cell model of human embryo development using human pluripotent stem cells (hPSCs). The microfluidic human embryo model exhibits molecular and cellular features resembling the progressive development of the post-implantation human embryo. The compartmentalized configuration of the microfluidic device allows the formation of spherical hPSC colonies in prescribed locations in the device. Implementing different culture protocols using the device, two opposite regions of each hPSC colony would be exposed to two different exogenous chemical environments, respectively. Under such asymmetrical chemical conditions, several early post-implantation human embryo development landmarks, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells, can be robustly recapitulated using the microfluidic device. The microfluidic human embryo model is compatible with live imaging, immunofluorescence staining, fluorescent in situ hybridization and single-cell sequencing. The superior controllability and scalability of the microfluidic human embryo model render it as an ideal tractable experimental system to study early post-implantation human development. The microfluidic human embryo model will be useful for advancing understanding of the previously inaccessible phases of early post-implantation human embryonic development. The reported protocol takes about 5 days to complete, including steps of microfluidic device fabrication (2 days), cell seeding (1 day), and progressive development of the microfluidic model up to gastrulation-like events (1-2 days). - [121] Stem-cell-based embryo models for fundamental research and translation
Jianping Fu, Aryeh Warmflash, and Lutolf P. Matthias
Nature Materials, vol. 20, pp. 132-144, 2021.
DOI: 10.1038/s41563-020-00829-9.
Abstract | PDF | PubMed
Despite its importance, understanding the early phases of human development has been significantly limited by availability of human samples. The recent emergence of stem cell-derived embryo models, a new field aiming to use stem cells to construct in vitro models to recapitulate snapshots of the development of the mammalian conceptus, opens up exciting opportunities to promote fundamental understanding of human development and advance reproductive and regenerative medicine. This review provides a summary of the current knowledge of early mammalian development, using mouse and human conceptuses as models, and emphasizes their similarities and critical differences. We then highlight existing embryo models that mimic different aspects of mouse and human development. We further discuss bioengineering tools used for controlling multicellular interactions and self-organization critical for the development of these models. We conclude with a discussion of the important next steps and exciting future opportunities of stem cell-derived embryo models for fundamental discovery and translation.
2020
- [120] Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation
Zhenzhen Fan, Xufeng Xue, Jianping Fu, and Cheri X. Deng
Scientific Reports, vol. 10, 18253, 2020.
DOI: 10.1038/s41598-020-75347-4.
Abstract | PDF | Supplemental Materials | PubMed
Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2+ diffusion in hESC colonies. We quantify cell-cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2+ waves in hESC colonies induced by sonoporation of single cells. - [119] Effect of cell spreading on rosette formation by human pluripotent stem cell-derived neural progenitor cells
Ryan F. Townshend, Yue Shao, Sicong Wang, Chari L. Cortez, Sajedeh Nasr Esfahani, Jason R. Spence, K. Sue O’Shea, Jianping Fu, Deborah L. Gumucio, and Kenichiro Taniguchi
Frontiers in Cell and Developmental Biology, vol. 8, 588941, 2020.
DOI: 10.3389/fcell.2020.588941.
Abstract | PDF | PubMed
Neural rosettes (NSC-rosettes) are radially arranged groups of cells containing a central apical lumen during in vitro culture of human pluripotent stem cell (hPSC)-derived neural stem cells (NSC). NSC-rosettes are thought to mimic the early neural tube and represent important developmental models for neural tube morphogenesis. However, current NSC-rosette assays are not synchronized or consistent among different hPSC lines, hindering comparative and quantitative mechanistic analyses. Here, we report a rapid and robust protocol to induce rosette formation within 6 hours after evenly-sized “colonies” of NSC are generated through physical cutting of uniformly polarized NESTIN+/PAX6+/PAX3+/DACH1+ NSC monolayers. These NSC rosettes show apically polarized lumens studded with primary cilia. Using this assay, we demonstrate reduced lumenal size in the absence of PODXL1, an important apical determinant recently identified as a potential causal gene for juvenile Parkinsonism. Interestingly, time lapse imaging reveals an unexpected dynamic aspect of NSC rosette formation: cut NSC colonies initiate rapid spreading prior to exhibiting signs of radial organization. At the colony leading edge, widespread formation of filopodia and lamellipodia is seen. Further, using chemical, genetic and biomechanical tools, we show that NSC-rosette morphogenesis requires this spreading activity that are tightly regulated by actin cytoskeleton. This robust and quantitative NSC-rosette platform provides a sensitive system for the further investigation of cellular and molecular mechanisms underlying NSC-rosette morphogenesis. - [118] Mechanical tension promotes formation of gastrulation-like nodes and patterns mesoderm specification in human embryonic stem cells
Jonathon M. Muncie, Nadia M.E. Ayad, Johnathon N. Lakins, Xufeng Xue, Jianping Fu, and Valerie M. Weaver
Developmental Cell, vol. 55, pp. 679-694, 2020.
DOI: 10.1101/2020.02.10.943076.
Abstract | PDF | Supplemental Materials | PubMed
Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization that motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that enhance cell-cell interactions to direct the self-organization of cultured hESCs into “gastrulation-like” nodes. Tissue geometries that generated local nodes of high cell-adhesion tension and induced these self-organized tissue nodes drove BMP4-dependent gastrulation by enhancing phosphorylation and junctional release of β-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development. - [117] How can microfluidic and microfabrication approaches make experiments more physiologically relevant?
Lydia L. Sohn, Petra Schwille, Andreas Hierlemann, Savas Tay, Josep Samitier, Jianping Fu, and Peter Loskill
Cell Systems, vol. 11, pp. 1-3, 2020.
DOI: 10.1016/j.cels.2020.07.003.
Abstract | PDF | PubMed
Microfabricated and microfluidic devices enable standardized handling, precise spatiotemporal manipulation of cells and liquids, and recapitulation of cellular environments, tissues, and organ-level biology. We asked researchers how these devices can make in vitro experiments more physiologically relevant. - [116] The future of biomedical engineering - Bioengineering of organoids and tissue development
George A. Truskey and Jianping Fu
Current Opinion in Biomedical Engineering, vol. 13, A1-A2, 2020.
DOI: 10.1016/j.cobme.2020.07.002.
Abstract | PDF
The future of bioengineering for multicellular organoids and tissue models has never been brighter. The articles in the special issue cover the latest bioengineering advances in human embryo models (or embryoids), kidney organoids, tumor organoids including glioblastoma multiforme (GBM) models, models of neurological disorders, and computational methods for organoid systems. This issue also includes reviews on the engineered extracellular matrix (ECM) for epithelial morphogenesis and engineering principles, challenges, and opportunities relevant to organoids and tissue models. - [115] Bioengineered pluripotent stem cell models: New approaches to explore early human embryo development
Agnes M. Resto Irizarry, Sajedeh Nasr Esfahani, and Jianping Fu
Current Opinion in Biotechnology, vol. 66, pp. 52-58, 2020.
DOI: 10.1016/j.copbio.2020.06.005.
Abstract | PDF | PubMed
Human development is a complex process in which environmental signals and factors encoded by the genome interact to engender cell fate changes and self-organization that drive the progressive formation of the human body. Herein, we discuss engineered biomimetic platforms with controllable environments that are being used to develop human pluripotent stem cell (hPSC)-based embryo models (or embryoids) that recapitulate a wide range of early human embryonic developmental events. Coupled with genome editing tools, single-cell analysis, and computational models, they can be used to parse the spatiotemporal dynamics that lead to differentiation, patterning, and growth in early human development. Furthermore, we discuss ongoing efforts in human extraembryonic lineage derivation and what can be learned from mouse embryoid models that have used both embryonic and extraembryonic stem cells. Finally, we discuss promising bioengineering tools for the generation of more controllable systems and the need for validation of findings from hPSC-based embryoid models. - [114] Multiplexed luminescence oxygen channeling immunoassay based on dual-functional barcodes with host-guest structure: A facile and robust suspension array platform
Qingsheng Guo, Yao Wang, Cang Chen, Dan Wei, Jianping Fu, Hong Xu, and Hongchen Gu
Small, vol. 16, pp. 1907521, 2020.
DOI: 10.1002/smll.201907521.
Abstract | PDF | PubMed
The development of a powerful immunoassay platform with capacities of both simplicity and high multiplexing is promising for disease diagnosis. To meet this urgent need, for the first time, a multiplexed luminescent oxygen channeling immunoassay (multi‐LOCI) platform by implementation of LOCI with suspension array technology is reported. As the microcarrier of the platform, a unique dual‐functional barcode with a host–guest structure composed of a quantum dot host bead (QDH) and LOCI acceptor beads (ABs) is designed, in which QDH provides function of high coding capacity while ABs facilitate the LOCI function. The analytes bridge QDH@ABs and LOCI donor beads (DBs) into a close proximity, forming a QDH@ABs–DBs “host–guest–satellite” superstructure that generates both barcode signal from QDH and LOCI signal induced by singlet oxygen channeling between ABs and DBs. Through imaging‐based decoding, different barcodes are automatically distinguished and colocalized with LOCI signals. Importantly, the assay achieves simultaneous detection of multiple analytes within one reaction, simply by following a “mix‐and‐measure” protocol without the need for tedious washing steps. Furthermore, the multi‐LOCI platform is validated for real sample measurements. With the advantages of robustness, simplicity, and high multiplexing, the platform holds great potential for the development of point‐of‐care diagnostics. - [113] Synthetic human embryology: Towards a quantitative future
Yue Shao, and Jianping Fu
Current Opinion in Genetics and Development, vo. 63, pp. 30-35, 2020.
DOI: 10.1016/j.gde.2020.02.013.
Abstract | PDF | PubMed
Study of early human embryo development is essential for advancing reproductive and regenerative medicine. Traditional human embryological studies rely on embryonic tissue specimens, which are difficult to acquire due to technical challenges and ethical restrictions. The availability of human stem cells with developmental potentials comparable to pre- and peri-implantation human embryonic and extraembryonic cells, together with properly engineered in vitro culture environments, allow for the first time researchers to generate self-organized multicellular structures in vitro that mimic the structural and molecular features of their in vivo counterparts. The development of these stem cell-based, synthetic human embryo models offer a paradigm-shifting experimental system for quantitative measurements and perturbations of multicellular development, critical for advancing human embryology and reproductive and regenerative medicine without using intact human embryos. - [112] Modeling of human neurulation using bioengineered pluripotent stem cell culture
Xufeng Xue, Ryan P. Wang, and Jianping Fu
Current Opinion in Biomedical Engineering, vol. 13, pp. 127-133, 2020.
DOI: 10.1016/j.cobme.2020.02.002.
Abstract | PDF | PubMed
Leveraging the developmental potential and self-organizing property of human pluripotent stem (hPS) cells, researchers have developed tractable models of peri- and post-implantation development of the human embryo. Owing to their compatibility to live imaging, genome editing, mechanical perturbation and measurement, and screening applications, these models offer promising quantitative experimental platforms to advance human embryology and reproductive and regenerative medicine. Herein, we provide a review of recent progress in using hPS cells to generate models of early human neural development or neurulation, including neural induction of the ectoderm and regional patterning of the neural tube. These models, even in their nascent developmental stages, have already revealed intricate cell-cell signaling and mechanoregulation mechanisms likely involved in tissue patterning during early neural development. We also discuss future opportunities in modeling early neural development by incorporating bioengineering tools to control precisely neural tissue morphology and architecture, morphogen dynamics, intracellular signaling events, and cell-cell interactions to further the development of this emerging field and expand its applications.
2019
- [111] Human primordial germ cells are specified from lineage-primed progenitors
Di Chen, Na Sun, Lei Hou, Rachel Kim, Jared Faith, Marianna Aslanyan, Yu Tao, Yi Zheng, Jianping Fu, Wanlu Liu, Manolis Kellis, and Amander Clark
Cell Reports, vol. 29, pp. 4568-4582, 2019.
DOI: 10.1016/j.celrep.2019.11.083.
Abstract | PDF | PubMed
In vitro gametogenesis is the process of making germline cells from human pluripotent stem cells. The foundation of this model is the quality of the first progenitors called primordial germ cells (PGCs), which in vivo are specified during the peri-implantation window of human development. Here, we show that human PGC (hPGC) specification begins at day 12 post-fertilization. Using single-cell RNA sequencing of hPGC-like cells (hPGCLCs) differentiated from pluripotent stem cells, we discovered that hPGCLC specification involves resetting pluripotency toward a transitional state with shared characteristics between naive and primed pluripotency, followed by differentiation into lineage-primed TFAP2A+ progenitors. Applying the germline trajectory to TFAP2C mutants reveals that TFAP2C functions in the TFAP2A+ progenitors upstream of PRDM1 to regulate the expression of SOX17. This serves to protect hPGCLCs from crossing the Weismann’s barrier to adopt somatic cell fates and, therefore, is an essential mechanism for successfully initiating in vitro gametogenesis. - [110] Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche
Yuanyuan Zheng, Xufeng Xue, Agnes M. Resto Irizarry, Zida Li, Yue Shao, Yi Zheng, Gang Zhao, and Jianping Fu
Science Advances, vol. 5, eaax5933, 2019.
DOI: 10.1126/sciadv.aax5933.
Abstract | PDF | Supplemental Materials | PubMed
Despite its importance in the central nervous system development, the development of the human neural tube (NT) remains poorly understood, given interspecies divergence and challenges of studying human embryo specimens. Here we report a human NT development model, in which NT-like tissues, termed neuroepithelial (NE) cysts, are generated in a bioengineered neurogenic environment through self-organization of human pluripotent stem cells (hPSCs). NE cysts correspond to the neural plate in the dorsal ectoderm layer and possess a default dorsal identity. Dorsal-ventral (DV) patterning of NE cysts is achieved using retinoic acid and/or Sonic hedgehog, featuring sequential emergence of the ventral floor plate, P3 and pMN domains in discrete, adjacent regions and an dorsal territory progressively restricted to the opposite dorsal pole. Together, this study reports the development of a hPSC-based, DV patterned NE cyst system for modeling human NT development, useful for understanding the self-organizing principles that guide NT patterning and hence to study neural development and disease. - [109] Controlled modeling of human epiblast and amnion development using stem cells
Yi Zheng, Xufeng Xue, Yue Shao, Sicong Wang, Sajedeh Nasr Esfahani, Zida Li, Jonathon M. Muncie, Johnathon N. Lakins, Valerie M. Weaver, Deborah L. Gumucio, and Jianping Fu
Nature, vol. 573, pp. 421-425, 2019.
DOI: 10.1038/s41586-019-1535-2.
Abstract | PDF | PubMed
Commentary by Amander T. Clark, Nature, vol. 573, pp. 350-351, 2019. [PDF]
Highlighted by Nature News, "Embryo-like structures created from human stem cells". (link)
Highlighted by Nature Podcast, "Modelling early embryos". (link)
Highlighted by National Public Radio (NPR) News, "Scientists create a device that can mass-produce human embryoids". (link)
Highlighted by BBC News, "Embryoids from stem cells". (link)
Highlighted by MIT Technology Review, "Meet the 'artificial embryos' being called uncanny and spectacular". (link)
Highlighted by Chemical & Engineering News (C&EN), "Microfluidic device brews human embryo-like structures". (link)
Early human embryonic development involves extensive lineage diversification, cell fate specification and tissue patterning. Despite its basic and clinical importance, early human embryonic development remains mysterious due to interspecies divergence and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast (EPI) and amniotic ectoderm (AM) parts of the conceptus, including lumenogenesis and expansion of a pro-amniotic cavity, formation of a bipolar embryonic sac, specification of primordial germ cells (PGCs) and of primitive streak (PS) cells. We further show that AM-like cells (AMLCs) function as a signaling center to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model might assist in the rational design of differentiation protocols of hPSCs for disease modeling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects. - [108] Mass-producible microporous silicon membranes for specific leukocyte subset isolation, immunophenotyping, and personalized immunomodulatory drug screening in vitro
Andrew Stephens, Robert Nidetz, Nicolas Mesyngier, Meng Ting Chung, Yujing Song, Jianping Fu, and Katsuo Kurabayashi
Lab on Chip, vol. 19, pp. 3065-3076, 2019.
DOI: 10.1039/c9lc00315k.
Abstract | PDF | Supplemental Materials | PubMed
Widespread commercial and clinical adaptation of biomedical microfluidic technology has been limited in large part due to the lack of mass producibility of polydimethylsiloxane (PDMS) and glass-based devices commonly as reported in the literature. Here, we present a batch-fabricated, robust, and mass-producible immunophenotyping microfluidic device using silicon micromachining processes. Our Si and glass-based microfluidic device, named the silicon microfluidic immunophenotyping assay (SiMIPA), consists of a highly porous (~40%) silicon membrane that can selectively separate microparticles below a certain size threshold. The device is capable of isolating and stimulating specific leukocyte populations, and allows for measuring their secretion of cell signaling proteins by means of a no-wash homogeneous chemiluminescence-based immunoassay. The high manufacturing throughput (~170 devices per wafer) makes a large quantity of SiMIPA chips readily available for clinically relevant applications, which normally require large dataset acquisitions for statistical accuracy. With 30 SiMIPA chips, we performed in vitro immunomodulatory drug screening on isolated leukocyte subsets, yielding 5 data points at 6 drug concentrations. Furthermore, the excellent structural integrity of the device allowed for samples and reagents to be loaded using a micropipette, greatly simplifying the experimental protocol. - [107] Microengineered human amniotic ectoderm tissue assay for high-content development phenotyping
Sajedeh Nasr Esfahani, Yue Shao, Agnes M. Resto Irizarry, Zida Li, Xufeng Xue, Deborah L. Gumucio, and Jianping Fu
Biomaterials, vol. 216, 119244, 2019.
DOI: 10.1016/j.biomaterials.2019.119244.
Abstract | PDF | Supplemental Materials | PubMed
During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study. - [106] Back-focal-plane interferometric detection of nanoparticles in spatially confined microfluidic channels
Abhay Kotnala, Yi Zheng, Jianping Fu, and Wei Cheng
Review of Scientific Instructments, vol. 90, 023107, 2019.
DOI: 10.1063/1.5074194.
Abstract | PDF | PubMed
Nanoparticles are important in several areas of modern biomedical research. However, detection and characterization of nanoparticles is challenging due to their small size. Back-focal-plane interferometry (BFPI) is a highly sensitive technique that has been used in laser tweezers for quantitative measurement of force and displacement. The utility of BFPI for detection and characterization of nanoparticles, however, has not yet been achieved. Here we show that BFPI can be used for rapid probing of a suspension of nanoparticles in a spatially confined microfluidic channel. We show that the Gaussian Root-mean-squared noise of the BFPI signal is highly sensitive to the nanoparticle size and can be used as a parameter for rapid detection of nanoparticles at a single-particle level and characterization of particle heterogeneities in a suspension. By precisely aligning the optical trap relative to the channel boundaries, individual polystyrene particles with a diameter as small as 63 nm can be detected using BFPI with a high signal-to-noise ratio. - [105] Biophysical phenotyping and modulation of ALDH+ inflammatory breast cancer stem-like cells
Weiqiang Chen, Steven G. Allen, Weiyi Qian, Zifeng Peng, Shuo Han, Xiang Li, Yubing Sun, Chelsea Fournier, Liwei Bao, Raymond H.W. Lam, Sofia D. Merajver, and Jianping Fu
Small, vol. 15, 1802891, 2019.
DOI: 10.1002/smll.201802891.
Abstract | PDF | Supplemental Materials | PubMed
Cancer stem cells (CSCs), as defined through specific marker expression methods, have been shown to initiate tumorigenesis, have the capacity to self-renew, and initiate cancer metastasis in many cancer types. Although identification of CSCs through marker expression helps separate and define the CSC compartment, it does not directly provide information on how or why this cancer cell subpopulation is more metastatic or tumorigenic. In this study, we comprehensively profiled the functional and biophysical characteristics of aggressive and lethal inflammatory breast cancer (IBC) CSCs at the single-cell level using multiple microengineered tools. We found distinct functional (cell migration, growth, adhesion, invasion, self-renewal) and biophysical (cell deformability, adhesion strength, and contractility) properties of IBC CSCs compared to their non-CSC counterpart, providing biophysical insights into why IBC CSCs has an enhanced propensity to metastasize compared to other IBC non-CSCs. We further show that the cellular biophysical phenotype can predict and determine IBC cells’ tumorigenic ability with high accuracy. IBC cells selected and modulated through biophysical features – adhesion and stiffness – showed characteristics of CSCs in vitro and enhanced tumorigenicity in in vivo murine models of primary tumor growth. Overall, our multiparametric cellular biophysical phenotyping and determination of IBC CSCs yields a new understanding of IBC’s metastatic properties and how they might develop and be targeted for therapeutic interventions. - [104] Biophysical phenotypes and determinants of anterior vs. posterior primitive streak cells derived from human pluripotent stem cells
Feng Lin, Yue Shao, Xufeng Xue, Yi Zheng, Zida Li, Chunyang Xiong, and Jianping Fu
Acta Biomaterialia, vol. 86, pp. 125-134, 2019.
DOI: 10.1016/j.actbio.2019.01.017.
Abstract | PDF | Supplemental Materials | PubMed
Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, our understanding of cellular mechanism(s) underlying cell fate diversification along the anterior-posterior axis of the PS remains incomplete. Furthermore, difference in biophysical phenotypes between anterior and posterior PS cells, which could affect their functions and regulate their fate decisions, remain uncharacterized. Herein, anterior and posterior PS cells were derived using human pluripotent stem cell (hPSC)-based in vitro culture systems. We observed that anterior and posterior PS cells displayed significantly different biophysical phenotypes, including cell morphology, migration, and traction force generation, which was further regulated by different levels of Activin A- and BMP4-mediated developmental signaling. Our data further suggested that intracellular cytoskeletal contraction could mediate anterior and posterior PS differentiation and phenotypic bifurcation through its effect on Activin A- and BMP4-mediated intracellular signaling events. Together, our data provide new information about biophysical phenotypes of anterior and posterior PS cells and reveal an important role of intracellular cytoskeletal contractility in regulating anterior and posterior PS differentiation of hPSCs.
2018
- [103] Debate ethics of embryo models from stem cells
Nicolas Rivron, Martin Pera, Janet Rossant, Alfonso Martinez Arias, Magdalena Zernicka-Goetz, Jianping Fu, Suzanne van den Brink, Annelien Bredenoord, Wybo Dondorp, Guido de Wert, Insoo Hyun, Megan Munsie, and Rosario Isasi
Nature, vol. 564, pp. 183-185, 2018.
DOI: 10.1038/d41586-018-07663-9.
Abstract | PDF | PubMed
Take a look at what has been achieved in the emerging field of stem cell models of embryonic development, along with the ethics of where research may go in the future. - [102] Acoustic actuation of integrin-bound microbubbles for mechanical phenotyping during differentiation and morphogenesis of human embryonic stem cells
Zhenzhen Fan, Xufeng Xue, Al Christopher De Leon, Agata Exner, Jianping Fu, and Cheri X. Deng
Small, vol. 14, 1803137, 2018.
DOI: 10.1002/smll.201803137.
Abstract | PDF | PubMed
Early human embryogenesis is a dynamic developmental process, involving continuous and concomitant changes in gene expression, structural reorganization, and cellular mechanics. However, lack of investigation methods has limited our understanding of how cellular mechanical property changes during early human embryogenesis. In this study, ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry, ATC) is employed for in situ measurement of cell stiffness during human embryonic stem cell (hESC) differentiation and morphogenesis. Cell stiffness, which is regulated by cytoskeleton structure, remains unchanged in undifferentiated hESCs, but significantly increases during neural differentiation. Further, using our recently established in vitro 3D embryogenesis models, ATC measurements reveals that cells continue to stiffen while maintaining pluripotency during epiblast cyst formation. In contrast, during amniotic cyst formation, cells first become stiffer during luminal cavity formation, but softens significantly when cells differentiate to form amniotic cysts. These results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change. ATC therefore provides a versatile platform for in situ measurement of cellular mechanical property, and cell stiffness may be used as a mechanical biomarker for cell lineage diversification and cell fate specification during embryogenesis. - [101] Magnetothermal heating facilitates cryogenic recovery of stem cell-laden alginate-Fe3O4 nanocomposite hydrogel
Xiaozhang Zhang, Gang Zhao, Yuan Cao, Zeeshan Haider, Meng Wang, and Jianping Fu
Biomaterials Science, vol. 6, pp. 3139-3151, 2018.
DOI: 10.1039/C8BM01004H.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the front cover story by Biomaterials Science.
Constructs of magnetic nanocomposite hydrogels microencapsulated with stem cells are of great interest as smart materials for tissue engineering and regenerative medicine. Due to short shelf life of such biocomposites at ambient temperature, their long-term storage and banking at cryogenic temperatures are essential for “off-the-shelf” availability of such biocomposites for widespread clinical applications. However, high-quality cryogenic recovery of stem cell-nanocomposite hydrogel constructs has not yet been achieved due to damage to cells and/or microstructures of hydrogel constructs caused by ice formation, particularly during warming from cryogenic temperature. Herein, stem cell-magnetic nanocomposite hydrogel constructs, which have an inherent magnetothermal property provided by embedded magnetic nanoparticles, are explored to achieve ultra-rapid cryogenic warming. Binding of water molecules by hydrogel combined with magnetothermal heating greatly suppressed ice formation during both cryogenic cooling and warming. Thus, cryogenic recovery of nanocomposite hydrogel constructs with intact microstructures and fully functional stem cells from ultra-low temperatures was successfully achieved. We further demonstrated that magnetic nanocomposite hydrogels microencapsulated with stem cells could be conveniently manipulated for self-assembled 3D culture. Together, we have developed a highly efficient and easy-to-perform approach for cryogenic recovery of stem cell-encapsulated magnetic nanocomposite hydrogel constructs. Our results will facilitate applications of such stem cell-magnetic nanocomposite hydrogels in regenerative medicine and tissue engineering applications. - [100] Acoustic tweezing cytometry induces rapid initiation of human embryonic stem cell differentiation
Tugba Topal, Adeline X. Hong, Xufeng Xue, Zhenzhen Fan, Ninad Kanetkar, Joe T. Nguyen, Jianping Fu, Cheri X. Deng, and Paul H. Krebsbach
Scientific Reports, vol. 8, 12977, 2018.
DOI:10.1038/s41598-018-30939-z.
Abstract | PDF | Supplemental Materials | PubMed
Mechanical forces play critical roles in human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces can influence hESC behaviors in vitro. Here, we used an ultrasound technique, acoustic tweezing cytometry (ATC), to exert cyclic subcellular forces to hESCs via integrin-bound microbubbles. We found that ATC-mediated cyclic forces applied to a subpopulation of hESCs induced global responses in hESC colonies, including increased contractile force, enhanced calcium activity, decreased nuclear expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase activation and cytoskeleton tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces. - [99] A systems mechanobiology model to predict cardiac reprogramming outcomes on different biomaterials
Yen P. Kong, Ana Y. Rioja, Xufeng Xue, Yubing Sun, Jianping Fu, and Andrew J. Putnam
Biomaterials, vol. 181, pp. 280-292, 2018.
DOI: 10.1016/j.biomaterials.2018.07.036.
Abstract | PDF | Supplemental Materials | PubMed
During normal development, the extracellular matrix (ECM) regulates cell fate mechanically and biochemically. However, the ECM’s influence on lineage reprogramming, a process by which a cell’s developmental cycle is reversed to attain a progenitor-like cell state followed by subsequent differentiation into a desired cell phenotype, is unknown. Using a material mimetic of the ECM, here we show that ligand identity, ligand density, and substrate modulus modulate indirect cardiac reprogramming efficiency, but were not individually correlated with phenotypic outcomes in a predictive manner. Alternatively, we developed a data-driven model using partial least squares regression to relate short-term cell states, defined by quantitative mechanosensitive responses to different material environments, with long-term changes in phenotype. This model accurately predicted the reprogramming outcomes on a different material platform. Collectively, these findings demonstrate the utility of this approach to rapidly screen candidate biomaterials that support reprogramming with high efficiency, without subjecting cells to the entire three-week process. - [98] Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells
Xufeng Xue, Yubing Sun, Agnes M. Resto-Irizarry, Ye Yuan, Koh Meng Aw Yong, Yi Zheng, Shinuo Weng, Yue Shao, Yimin Chai, Lorenz Studer, and Jianping Fu
Nature Materials, vol. 17, pp. 633-641, 2018.
DOI: 10.1038/s41563-018-0082-9.
Abstract | PDF | Supplemental Materials | PubMed
Recommended in F1000Prime by David Schaffer and Rocio Sampayo. [PDF]
Commentary by Mukul Tewary and Peter W. Zandstra, Nature Materials, vol. 17, pp. 571-572, 2017. [PDF]
Highlighted by
Toward a stem cell model of human nervous system development - Michigan News
Toward a stem cell model of human nervous system development - Michigan Engineering News
UMass Amherst’s Yubing Sun part of research team exploring how mechanical signals help develop the human nervous system - UMass Amherst News
Embryonic stem cells form useful proto-nervous system - Futurity
Toward a stem cell model of human nervous system development - Phys.org
Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals - cell shape and cytoskeletal contractile force - instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease. - [97] Carbon nanotube strain sensor based hemoretractometer for blood coagulation testing
Zida Li, Yize Wang, Xufeng Xue, Brendan McCraken, Kevin Ward, and Jianping Fu
ACS Sensors, vol. 3, pp. 670-676, 2018.
DOI: 10.1021/acssensors.7b00971.
Abstract | PDF | Supplemental Materials | PubMed
Coagulation monitoring is essential for perioperative care and thrombosis treatment. However, existing assays for coagulation monitoring have limitations such as large footprint and complex setup. In this work, we developed a miniaturized device for point-of-care blood coagulation testing by measuring dynamic clot retraction force development during blood clotting. In this device, a blood drop was localized between a protrusion and a flexible force-sensing beam to measure clot retraction force. The beam was featured with micropillar arrays to assist the deposition of carbon nanotube films, which served as a strain sensor to achieve label-free, electrical readout of clot retraction force in real time. We characterized mechanical and electrical properties of the force sensing beam and optimized its design. We further demonstrated that this blood coagulation monitoring device could obtain results that were consistent with those using an imaging method, and that the device was capable of differentiating blood samples with different coagulation profiles. Owing to its low fabrication cost, small size, and low consumption of blood samples, the blood coagulation testing device using carbon nanotube strain sensors holds great potential as a point-of-care tool for future coagulation monitoring. - [96] News and Views: Highly parallel single-cell force cytometry
Agnes M. Resto Irizarry and Jianping Fu
Nature Biomedical Engineering, vol. 2, pp. 60-61, 2018.
DOI: 10.1038/s41551-018-0198-x.
Abstract | PDF
Single-cell traction-force measurements performed on fluorescently-labelled elastomeric surfaces can probe the physiological and pathological behaviour of thousands of cells in a single experiment. - [95] Synthetic human embryology: The rise of a new era with new collaborations
Yue Shao, Kenichiro Taniguchi, Deborah Gumucio, and Jianping Fu
Development, 2018.
Abstract | PDF
In this commentary, we describe the collaborative work that made the discovery of the post-implantation human amniotic sac model possible. As synthetic human embryology is bound to be a highly interdisciplinary course of study, we hope our case will not only serve as a stepping stone to advance this type of research, but also an encouragement for fellow engineers, developmental and cell biologists, mathematicians, physicists, physicians, etc., to join forces and initiate fruitful interactions and collaborations to advance discovery. - [94] Nanotopography promotes motor neuron differentiation of human pluripotent stem cells
Weiqiang Chen, Shuo Han, Weyi Qain, Shinuo Weng, Haiou Yang, Yubing Sun, Luis G. Villa-Diaz, Paul H. Krebsbach, and Jianping Fu
Nanoscale, vol. 10, pp. 3556–3565, 2018.
DOI: 10.1039/c7nr05430k.
Abstract | PDF | Supplemental Materials | PubMed
Regulation of human pluripotent stem cell (hPSC) behaviors has been mainly studied through exploration of biochemical factors. However, current directed differentiation protocols for hPSCs that completely rely on biochemical factors remain suboptimal. It has recently become evident that coexisting biophysical signals in the stem cell microenvironment, including nanotopographic cues, can provide potent regulatory signals to mediate adult stem cell behaviors, including self-renewal and differentiation. Herein, we utilized a recently developed, large-scale nanofabrication technique based on reactive-ion etching (RIE) to generate random nanoscale structures on glass surfaces with high precision and reproducibility. We report here that hPSCs are sensitive to nanotopographic cues and such nanotopographic sensitivity can be leveraged for improving directed neuronal differentiation of hPSCs. We demonstrate motor neuron (MN) differentiation of hPSCs can be promoted by using nanoengineered topographic substrates. We further explore how hPSCs sense substrate nanotopography and relay this biophysical signal through a regulatory signaling network involving cell adhesion, actomyosin cytoskeleton, and Hippo/YAP signaling to mediate neuroepithelial induction of hPSCs. Our study provides an efficient method for large-scale production of MNs from hPSCs, useful for thereby helping advancing regenerative medicine and cell-based therapies. - [93] Modulation of miRNA expression and osteoblast differentiation by nanotopography
Elisa Mattias Sartori, Osvaldo Magro-Filho, Daniela B. Silveira Mendonça, Xiang Li, Jianping Fu, and Gustavo Mendonça
International Journal of Oral & Maxillofacial Implants, vol. 33, pp. 269-280, 2018.
DOI: 10.11607/jomi.5372.
Abstract | PDF | Supplemental Materials | PubMed
Purpose: This study evaluated the expression pattern of miRNAs on a surface with nanotopography compared to a smooth surface (control). Material and Methods: Human mesenchymal stem cells (hMSCs) were plated on different surfaces and compared at 3,7 and 14 days for alkaline phosphatase (ALP) activity, expression of genes (Osterix (OSX), Runx2, BMP2 and ALP) and expression of miRNAs. Western blot was also used to detect osteogenic proteins (BMP2, OSX and Osteocalcin (OCN)). Scanning electron microscopy of cells plated onto the surfaces were obtained. Results: ALP activity on different surfaces was significantly greater in the nanotopography surface. At day 14 there was a 3.5-fold and a 9-fold increase for Runx2 and OSX gene, respectively. BMP2 and ALP also increased by 4- and 7-fold compared to control. Protein levels for OSX and BMP2 were also upregulated compared to control group. Using RNA sequencing technology (RNA-Seq) a total of 117 miRNAs were found differentially expressed comparing control (day 7) to nanosurface (day 14). 45 miRNAs were upregulated and 72 were downregulated. Several of these miRNAs that were differently expressed regulate osteogenic genes. For example, hsa-miR-135b-5p that targets OCN, BSP, RUNX2, CO15A1 and OSX, hsa-miR-122-5p targets OPN, hsa-miR-196a-5p targets BMP4, hsa-miR-26b-5p targets BMP2 and hsa-miR-148b-3p targets OPN. Conclusion: Surfaces with nanotopography have the potential to improve osseointegration response in order to reduce the time of osseointegration and also increase bone formation around the implants improving low bone quality areas. Within the limitation of this study nanotopography surfaces affected mesenchymal stem cell differentiation to osteoblasts. Several miRNAs were differentially ly regulated by surface topography. These miRNAs could be related to the differentiation response to may also help control the osseointegration process.
2017
- [92] Capillary-facilitated deposition of carbon nanotube film for strain sensing
Zida Li, Xufeng Xue, Feng Lin, Yize Wang, Kevin Ward, and Jianping Fu
Applied Physics Letter, vol. 111, 173105, 2017.
DOI: 10.1063/1.5001754.
Abstract | PDF
Advances in stretchable electronics offer the possibility of developing skin-like motion sensors. Carbon nanotube (CNT), owing to its superior electrical properties, has great potential for applications in such sensors. In this paper, we report a method for deposition and patterning of CNT on soft, elastic polydimethylsiloxane (PDMS) substrates using capillary action. Micropillar arrays were generated on PDMS surfaces before treatment with plasma to render them hydrophilic. Capillary force enabled by the micropillar array spreads CNT solution evenly on PDMS surfaces. Solvent evaporation leaves a uniform deposition and patterning of CNT on PDMS surfaces. We studied the effect of CNT concentration and micropillar gap size on CNT coating uniformity, film conductivity, and piezoresistivity. Leveraging the piezoresistivity of deposited CNT film, we further designed and characterized a device for contraction force measurement. Our capillary assisted deposition method of CNT film showed great application potential in fabrication of flexible CNT thin films for strain sensing. - [91] An apicosome initiates self-organizing morphogenesis in human pluripotent stem cells
Kenichiro Taniguchi, Yue Shao, Ryan F. Townshend, Clair E. Harris, Sasha Meshinchi, Sundeep Kalantry, Jianping Fu, K. Sue O’Shea, and Deborah L. Gumucio
Journal of Cell Biology, vol. 216, pp. 3981-3990, 2017.
DOI: 10.1083/jcb.201704085.
Abstract | PDF | PubMed
Commentary by Alejandra I. Romero-Morales, Natalya A. Ortolano, and Vivian Gama, Journal of Cell Biology, vol. 216, pp. 3891-3893, 2017. [PDF]
Human pluripotent stem cells (hPSC) self-organize into apico-basally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSC, through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+. Time-lapse imaging of isolated hPSC reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multi-cellular aggregate of hPSC, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique pre-assembled apical structure that can be rapidly employed in single or clustered hPSC to initiate self-organized apical polarization and lumenogenesis. - [90] Tracking the tumor invasion front using long-term fluidic tumoroid culture
Koh Meng Aw Yong, Zida Li, Sofia D. Merajver, and Jianping Fu
Scientific Reports, vol. 7, 10784, 2017.
DOI: 10.1038/s41598-017-10874-1.
Abstract | PDF | Supplemental Materials | PubMed
Highlighted by
Tiny device offers insight into how cancer spreads - Michigan Health Lab Report
Tiny device offers insight into how cancer spreads - Michigan Engineering News Center
Tiny device offers insight into how cancer spreads - EurekAlert! by AAAS
Cancer invasion front gets new tracking device - Genetic Engineering & Biotechnology News
A tiny device offers insights to how cancer spreads - ScienceDaily
A tiny device offers insights to how cancer spreads - Nanowerk
A tiny device offers insights to how cancer spreads - Health Medicine Network
A tiny device offers insights to how cancer spreads - Health News Digest
A tiny device offers insights to how cancer spreads - eCancer News
New microfluidic device offers insights on cancer metastasis - The Medical News
Researchers develop a fluidic device to track over time which cancer cells lead the invasive march - Medical Xpress
The analysis of invading leader cells at the tumor invasion front is of significant interest as these cells may possess a coordinated functional and molecular phenotype which can be targeted for therapy. However, such analyses are currently limited by available technologies. Here, we report a fluidic device for long-term three-dimensional tumoroid culture which recapitulates the tumor invasion front, allowing for both quantification of invasive potential and molecular characterization of invasive leader cells. Preliminary analysis of the invasion front indicates its association with cell proliferation and expression of growth differentiation factor 15 (GDF15). This device makes real-time tracking of invading leader cell phenotypes possible and has potential for use with patient material for clinical risk stratification and personalized medicine. - [89] A pluripotent stem cell-based model for post-implantation human amniotic sac development
Yue Shao, Kenichiro Taniguchi, Ryan F. Townshend, Toshio Miki, Deborah L. Gumucio, and Jianping Fu
Nature Communications, vol. 8, 208, 2017.
DOI: 10.1038/s41467-017-00236-w.
Abstract | PDF | Supplemental Materials | PubMed
Recommended in F1000Prime by Magdalena Zernicka-Goetz. [PDF]
Highlighted by MIT Technology Review, "Artificial human embryos are coming, and no one knows how to handle them". (link)
Highlighted by
Amniotic sac in a dish: Stem cells form structures that could aid understanding of infertility - Michigan News
How stem cells grow into structures that could aid understanding of infertility - Michigan Health Lab Report
Stem cells mimic key parts of human embryonic development - Michigan Engineering News Center
Image of the Day: Embryoid - The Scientist
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - Phys.org
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - EurekAlert! by AAAS
Breakthrough in infertility research - Innovators Magazine
Amniotic weal in a dish: Stem cells form structures that might assist of infertility research - Health Medicine Network
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - BioSpace
Amniotic sac in a dish: Stem cells form structures that could aid understanding of infertility and more - e Science News
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - HiTech Days
Stem cells grow post-implantation amniotic sac embryoid - RELIAWIRE
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - ScienceDaily
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - TransBio-Tex
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - Science Newsline
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - MedWorm
Amniotic sac in a dish: Stem cells form structures that could aid understanding of infertility - World University News
Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - Jersey Tribune
Amniotic sac in a dish: Stem cells form structures that could aid understanding of infertility and more - News Wise
Tiny lab-grown structures that resemble amniotic sac may help in infertility research - News-Medical
Amniotic sac in a dish: Stem cells form structures that may aid understanding of infertility - My Science
Boost to infertility research – Stem cell derived amniotic sacs - MedIndia
Development of the asymmetric amniotic sac—with the embryonic disc and amniotic ectoderm occupying opposite poles—is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology. - [88] Notch signaling in regulating angiogenesis in a 3D biomimetic environment
Yi Zheng, Shue Wang, Xuefeng Xue, Alan Xu, Wei Liao, Alice Deng, Guohao Dai, Allen P. Liu, and Jianping Fu
Lab on a Chip, vol. 17, pp. 1948-1959, 2017.
DOI: 10.1039/c7lc00186j.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the inside back cover story by Lab on a Chip.
Angiogenesis is a complex cellular process involving highly orchestrated invasion and organization of endothelial cells (ECs) in a three-dimensional (3D) environment. Recent evidence indicates that Notch signaling is critically involved in regulating specialized functions and distinct fates of ECs in newly formed vasculatures during angiogenesis. Here we demonstrated, for the first time, the application of a microengineered biomimetic system to quantitatively investigate the role of Notch signaling in regulating early angiogenic sprouting and vasculature formation of ECs in 3D extracellular matrix. Morphological features of angiogenesis including invasion distance, invasion area, and tip cell number were quantified and compared under pharmacological perturbations of Notch signaling. In addition, influences of Notch signaling on EC proliferation in angiogenic vasculatures and directional invasion of tip cells were also investigated. Moreover, leveraging a novel nanobiosensor system, mRNA expression of Dll4, a Notch ligand, was monitored in invading tip cells using live cell imaging during dynamic angiogenic process. Our data showed that inhibition of Notch signaling resulted in hyper-sprouting endothelial structures, while activation of Notch signaling led to opposite effects. Our results also supported the role of Notch signaling in regulating EC proliferation and dynamic invasion of tip cells during angiogenesis. - [87] Microfluidic-based high-throughput optical trapping of nanoparticles
Abhay Kotnala, Yi Zheng, Jianping Fu, and Wei Cheng
Lab on a Chip, vol. 17, pp. 2125-2134, 2017.
DOI: 10.1039/C7LC00286F.
Abstract | PDF | Supplemental Materials | PubMed
Optical tweezers have emerged as a powerful tool for multiparametric analysis of individual nanoparticles with single-molecule sensitivity. However, its inherent low-throughput characterstic remains a major obstacle for its applications within and beyond the laboratory. This limitation is further exacerbated when working with low concentration nanoparticle samples. Here, we present a microfluidic-based optical tweezer system that can ‘actively’ deliver nanoparticles to be analyzed to a designated microfluidic region for optical trapping. Using active microfluidic nanoparticle delivery results in significantly improved throughput and efficiency for optical trapping of nanoparticles. We observed a more than tenfold increase of optical trapping throughput for nanoparticles as compared to conventional systems under the same nanoparticle concentration. To demonstrate the utility of this microfluidic-based optical tweezer system, we further utilized the back-focal plane interferometry coupled with the trapping laser for precise quantitation of nanoparticle size without a prior knowledge of the refractive index of nanoparticles. Development of this microfluidic-based active optical tweezer system thus opens doors for high-throughput multiparametric analysis of nanoparticles using precision optical trap in the future. - [86] Effects of substrate stiffness and actomyosin contractility on coupling between force transmission and vinculin-paxillin recruitment at single focal adhesions
Dennis W. Zhou, Ted T. Lee, Shinuo Weng, Jianping Fu, and Andrés J. García
Molecular Biology of the Cell, vol. 28, pp. 1901, 2017.
DOI: 10.1091/mbc.E17-02-0116.
Abstract | PDF | Supplemental Materials | PubMed
Focal adhesions (FAs) regulate force transfer between the cytoskeleton and ECM-integrin complexes. We previously showed that vinculin regulates force transmission at FAs. Vinculin residence time in FAs correlated with applied force, supporting a mechanosensitive model in which forces stabilize vinculin’s active conformation to promote force transfer. In the present study, we examined the relationship between traction force and vinculin-paxillin localization to single FAs in the context of substrate stiffness and actomyosin contractility. Substrate stiffness and contractility regulated vinculin localization to FAs, and vinculin auto-inhibition is a crucial regulatory step in this process that overrides the effects of cytoskeletal tension and substrate stiffness. Vinculin and paxillin FA area did not correlate with traction force magnitudes at single FAs, and this was consistent across different ECM stiffness and cytoskeletal tension states. Vinculin residence time at FAs linearly varied with applied force for stiff substrates, but this coupling was disrupted on soft substrates and in the presence of contractility inhibitors. In contrast, paxillin residence time at FAs was independent of force, substrate stiffness, and cytoskeletal contractility. Lastly, substrate stiffness and cytoskeletal contractility regulated whether vinculin and paxillin turnover dynamics are correlated to each other at single FAs. This analysis sheds new insights on the coupling among traction force, substrate stiffness, and FA dynamics. - [85] Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation
Xufeng Xue, Xiaowei Hong, Zida Li, Cheri X. Deng, and Jianping Fu
Biomaterials, vol. 134, pp. 22-30, 2017.
DOI: 10.1016/j.biomaterials.2017.04.039.
Abstract | PDF | PubMed
Human mesenchymal stem cells (hMSCs) have great potential for cell-based therapies for treating degenerative bone diseases. It is known that mechanical cues in the cell microenvironment play an important role in regulating osteogenic (bone) differentiation of hMSCs. However, mechanoregulation of lineage commitment of hMSCs in conventional two-dimensional (2D) monocultures or bioengineered three-dimensional (3D) tissue constructs remains suboptimal due to complex biomaterial design criteria for hMSC culture. In this study, we demonstrate the feasibility of a novel cell mechanics and mechanobiology tool, acoustic tweezing cytometry (ATC), for mechanical stimulation of hMSCs. ATC utilizes ultrasound (US) pulses to actuate functionalized lipid microbubbles (MBs) which are covalently attached to hMSCs via integrin binding to exert forces to the cells. ATC stimulation increases cytoskeletal contractility of hMSCs regardless of the cell area. Furthermore, ATC application rescues osteogenic differentiation of hMSCs in culture conditions that are intrinsically repressive for hMSC osteogenesis (e.g., soft cell culture surfaces). ATC application activates transcriptional regulator YAP to enhance hMSC osteogenesis. Our data further show that F-actin, myosin II, and RhoA/ROCK signaling function upstream of YAP activity in mediating ATC-stimulated hMSC osteogenesis. With the capability of applying controlled dynamic mechanical stimuli to cells, ATC provides a powerful tool for mechanoregulation of stem cell behaviors in tissue engineering and regenerative medicine applications. - [84] AC electroosmosis-enhanced nanoplasmofluidic detection of ultralow-concentration cytokine
Yujing Song, Pengyu Chen, Meng Ting Chung, Robert Nidetz, Younggeun Park, Zhenhui Liu, Walker McHugh, Timothy T. Cornell, Jianping Fu, and Katsuo Kurabayashi
Nano Letters, vol. 17, pp. 2374-2380, 2017.
DOI: 10.1021/acs.nanolett.6b05313.
Abstract | PDF | Supplemental Materials | PubMed
Label-free, nanoparticle-based plasmonic optical biosensing, combined with device miniaturization and microarray integration, has emerged as a promising approach for rapid, multiplexed biomolecular analysis. However, limited sensitivity prevents the wide use of such integrated label-free nanoplasmonic biosensors in clinical and life science applications where low-abundance biomolecule detection is needed. Here, we present a nanoplasmofluidic device integrated with microelectrodes for rapid, label-free analysis of a low-abundance cell signaling protein, detected by AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biofunctional nanoparticle imaging. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The AC electroosmosis (ACEO) based enhancement of the biosensor performance enables rapid (5–15 min) quantification of IL-1β, a pro-inflammatory cytokine biomarker, with a sensitivity down to 158.5 fg/mL (9.1 fM) for spiked samples in PBS and 1 pg/mL (58 fM) for diluted human serum. Together with the optimized detection sensitivity and speed, our study presents the first critical step toward the application of nanoplasmonic biosensing technology to immune status monitoring guided by low-abundance cytokine measurement. - [83] Mechanotransduction-induced reversible phenotypic switching in prostate cancer cells
Koh Meng Aw Yong, Yubing Sun, Sofia D. Merajver, and Jianping Fu
Biophysical Journal, vol. 112, pp. 1236-1245, 2017.
DOI: 10.1016/j.bpj.2017.02.012. PMCID: PMC5376107.
Abstract | PDF | Supplemental Materials | PubMed
Phenotypic plasticity is posed to be a vital trait of cancer cells such as circulating tumor cells (CTCs), allowing them to undergo reversible or irreversible switching between phenotypic states important for tumorigenesis and metastasis. While irreversible phenotypic switching can be detected by studying the genome, reversible phenotypic switching is often difficult to examine due to its dynamic nature and the lack of knowledge about its contributing factors. In this study, we demonstrate that culturing cells in different physical environments, stiff, soft or suspension, induced a phenotypic switch in prostate cancer cells via mechanotransduction. The mechanosensitive phenotypic switching in prostate cancer cells was sustainable yet reversible even after long term culture, demonstrating the impact of mechanical signals on prostate cancer cell phenotypes. Importantly, such a mechanotransduction-mediated phenotypic switch in prostate cancer cells was accompanied by decreased sensitivity of the cells to paclitaxel, suggesting a role of mechanotransduction in the evolution of drug resistance. Multiple signaling pathways such as p38MAPK, ERK, and Wnt were found to be involved in the mechanotransduction-induced phenotypic switching of prostate cancer cells. Given that cancer cells experience different physical environments during disease progression, this study provides useful information about the important role of mechanotransduction in cancer and how CTCs may be capable of continuously changing their phenotypes throughout the disease process. - [82] Microfluidics for biopreservation
Gang Zhao and Jianping Fu
Biotechnology Advances, vol. 35, pp. 323-336, 2017.
DOI: 10.1016/j.biotechadv.2017.01.006. PMCID: PubMed - in process.
Abstract | PDF | PubMed
Cryopreservation has utility in clinical and scientific research but implementation is highly complex and includes labor-intensive cell-specific protocols for the addition/removal of cryoprotective agents and freeze-thaw cycles. Microfluidic platforms can revolutionize cryopreservation by providing new tools to manipulate and screen cells at micro/nano scales, which are presently difficult or impossible with conventional bulk approaches. This review describes applications of microfluidic tools in cell manipulation, cryoprotective agent exposure, programmed freezing/thawing, vitrification, and in situ assessment in cryopreservation, and discusses achievements and challenges, providing perspectives for future development. - [81] Centrifugal microfluidics for sorting immune cells from whole blood
Zeta Tak For Yu, Jophin George Joseph, Shirley Xiaosu Liu, Mei Ki Cheung, Katsuo Kurabayashi, and Jianping Fu
Sensors & Actuators: B. Chemical, vol. 245, pp. 1050-1061, 2017.
DOI: 10.1016/j.snb.2017.01.113. PMCID: PMC5619665.
Abstract | PDF | Supplemental Materials | PubMed
Sorting and enumeration of immune cells from blood are critical operations involved in many clinical applications. Conventional methods for sorting and counting immune cells from blood, such as flow cytometry and hemocytometers, are tedious, inaccurate, and difficult for implementation for point-of-care (POC) testing. Herein we developed a microscale centrifugal technology termed Centrifugal Microfluidic Chip (CMC) capable of sorting immune cells from blood and in situ cellular analysis in a laboratory setting. Operation of the CMC entailed a blood specimen layered on a density gradient medium and centrifuged in microfluidic channels where immune cell subpopulations could rapidly be sorted into distinct layers according to their density differentials. We systematically studied effects of different blocking molecules for surface passivation of the CMC. We further demonstrated the applicability of CMCs for rapid separation of minimally processed human whole blood without affecting immune cell viability. Multi-color imaging and analysis of immune cell distributions and enrichment such as recovery and purity rates of peripheral blood mononuclear cells (PBMCs) were demonstrated using CMCs. Given its design and operation simplicity, portability, blood cell sorting efficiency, and in situ cellular analysis capability, the CMC holds promise for blood-based diagnosis and disease monitoring in POC applications. - [80] Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche
Yue Shao, Kenichiro Taniguchi, Katherine Gurdziel, Ryan F. Townshend, Xufeng Xue, Koh Meng Aw Yong, Jianming Sang, Jason R. Spence, Deborah L. Gumucio, and Jianping Fu
Nature Materials, vol. 16, pp. 419-425, 2017.
DOI: 10.1038/nmat4829. PMCID: PMC5374007.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Martin Pera, Nature Materials, vol. 16, pp. 394-395, 2017. [PDF]
Highlighted by
The beginning of the amniotic sac - Michigan Engineering
How does the amniotic sac form? U-M team uses stem cells to study earliest stages - Michigan News
Team uses stem cells to study earliest stages of amniotic sac formation - Phys.org
How does the amniotic sac form? Team uses stem cells to study earliest stages - Technology.org
Amniogenesis - the development of amnion - is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP–SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine. - [79] Controlled tubular unit formation from collagen film for modular tissue engineering
Jianming Sang, Xiang Li, Yue Shao, Zida Li, and Jianping Fu
ACS Biomaterials Science & Engineering, vol. 3, pp. 2860-2868, 2017.
DOI: 10.1021/acsbiomaterials.6b00468. PMCID: PubMed - in process.
Abstract | PDF
Bottom-up or modular tissue engineering is one of the emerging approaches to prepare biomimetic constructs in vitro, involving fabrication of small tissue units as building blocks before assembling them into functional tissue constructs. Herein, we reported a microscale tissue engineering approach to generate tubular tissue units through cellular contractile force induced self-folding of cell-laden collagen films in a controllable manner. Self-folding of cell-laden collagen films was driven by film contraction resulted from intrinsic contractile property of adherent mammalian cells seeded in collagen films. We explored in detail independent effects of collagen gel concentration, cell density, and intrinsic cellular contractility on self-folding and tubular structure formation of cell-laden collagen films. Through both experiments and theoretical modeling, we further demonstrated the effectiveness of integrating ridge array structures onto the backside of collagen films in introducing structural anisotropy and thus controlling self-folding directions of collagen films. Our approach of using ridge array structures to introduce mechanical anisotropy and thus promote tubular tissue unit formation can be extended to other biomaterial systems and thus provide a simple yet effective way to prepare tubular tissue units for modular tissue engineering applications.
2016
- [78] Surface micromachining of polydimethylsiloxane for microfluidics applications
Staci Hill, Weiyi Qian, Weiqiang Chen, and Jianping Fu
Biomicrofluidics, vol. 10, 054114, 2016.
DOI: 10.1063/1.4964717. PMCID: PMC5065565.
Abstract | PDF | PubMed
Polydimethylsiloxane (PDMS) elastomer has emerged as one of the most frequently applied materials in microfluidics. However, precise and large-scale surface micromachining of PDMS remains challenging, limiting applications of PDMS for microfluidic structures with high-resolution features. Herein, surface patterning of PDMS was achieved using a simple yet effective method combining direct photolithography followed by reactive-ion etching (RIE). This method incorporated a unique step of using oxygen plasma to activate PDMS surfaces to a hydrophilic state, thereby enabling improved adhesion of photoresist on top of PDMS surfaces for subsequent photolithography. RIE was applied to transfer patterns from photoresist to underlying PDMS thin films. Systematic experiments were conducted in the present work to characterize PDMS etch rate and etch selectivity of PDMS to photoresist as a function of various RIE parameters, including pressure, RF power, and gas flow rate and composition. We further compared two common RIE systems with and without bias power and employing inductively-coupled plasma (ICP) and capacitively-coupled plasma (CCP) sources, respectively, in terms of their PDMS etching performances. The RIE-based PDMS surface micromachining technique is compatible with conventional Si-based surface and bulk micromachining techniques, thus opening promising opportunities for generating hybrid microfluidic devices with novel functionalities. - [77] Multiplexed nanoplasmonic temporal profiling of T-cell response under immunomodulatory agent exposure
Bo-Ram Oh, Pengyu Chen, Robert Nidetz, Walker McHugh, Jianping Fu, Thomas P. Shanley, Timothy T. Cornell, and Katsuo Kurabayashi
ACS Sensors, vol. 1, pp. 941-948, 2016.
DOI: 10.1021/acssensors.6b00240. PMCID: PMC4960639.
Abstract | PDF | Supplemental Materials | PubMed
Immunomodulatory drugs—agents regulating the immune response—are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 μL), short assay time (∼30 min), heightened sensitivity (∼20–30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment. - [76] Accelerated biofluid filling in complex microfluidic networks by vacuum-pressure accelerated movement (V-PAM)
Zeta Tak For Yu, Mei Ki Cheung, Shirley Xiaosu Liu, and Jianping Fu
Small, vol. 12, pp. 4521-4530, 2016.
DOI: 10.1002/smll.201601231. PMCID: PMC6215695.
Abstract | PDF | PubMed
Selected as the inside cover story by Small.
Rapid fluid transport and exchange are critical operations involved in many microfluidic applications. However, conventional mechanisms used for driving fluid transport in microfluidics, such as micro-pumping and high pressure, can be inaccurate and difficult for implementation for integrated microfluidics containing control components and closed compartments. Herein we developed a technology termed Vacuum-Pressure Accelerated Movement (V-PAM) capable of significantly enhancing biofluid transport in complex microfluidic environments containing dead-end channels and closed chambers. Operation of the V-PAM entailed a pressurized fluid loading into microfluidic channels where gas confined inside could rapidly be dissipated through permeation through a thin, gas-permeable membrane sandwiched between microfluidic channels and a network of vacuum channels. We systematically studied effects of different structural and operational parameters of the V-PAM for promoting fluid filling in microfluidic environments. We further demonstrated the applicability of V-PAM for rapid filling of temperature-sensitive hydrogels and unprocessed whole blood into complex irregular microfluidic networks such as microfluidic leaf venation patterns and blood circulatory systems. Together, the V-PAM technology provides a promising generic microfluidic tool for advanced fluid control and transport in integrated microfluidics for different microfluidic diagnosis, organs-on-chips, and biomimetic studies. - [75] A miniaturized hemoretractometer for blood clot retraction testing
Zida Li, Xiang Li, Brendan McCracken, Yue Shao, Kevin Ward, and Jianping Fu
Small, vol. 12, pp. 3926-3934, 2016.
DOI: 10.1002/smll.201600274. PMCID: PMC4980575.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the frontispiece story by Small.
Blood coagulation is a critical hemostatic process that must be properly regulated to maintain a delicate balance between bleeding and clotting. Disorders of blood coagulation can expose patients to the risk of either bleeding disorders or thrombotic diseases. Coagulation diagnostics using whole blood is very promising for assessing the complexity of the coagulation system and for global measurements of hemostasis. Despite the clinic values that existing whole blood coagulation tests have demonstrated, these systems have significant limitations that diminish their potential for point-of-care applications. In this work, we leverage recent advancements in device miniaturization using functional soft materials to develop a miniaturized clot retraction force assay device termed mHemoRetractoMeter (mHRM). The mHRM is capable of precise measurements of dynamic clot retraction forces in real time using minute amounts of whole blood. To further demonstrate the clinical utility of the mHRM, we conduct systematic studies using the mHRM to examine the effects of assay temperature, treatments of clotting agents, and pro- and anti-coagulant drugs on clot retraction force developments of whole blood samples. The mHRM's low fabrication cost, small size, and consumption of only minute amounts of blood samples make the technology promising as a point-of-care tool for future coagulation monitoring. - [74] Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous molecular expression and metastatic characteristics
Weiqiang Chen, Steven G. Allen, Ajaya Kumar Reka, Weiyi Qian, Shuo Han, Jianing Zhao, Liwei Bao, Venkat Keshamouni, Sofia D. Merajver, and Jianping Fu
BMC Cancer, vol. 16, 614, 2016.
DOI: 10.1186/s12885-016-2638-x. PMCID: PMC4977622.
Abstract | PDF | PubMed
Circulating tumor cells (CTCs) have prognostic relevance in numerous cancers. However, many current CTC capture methods rely on positive selection techniques that require a priori knowledge about CTC surface protein expression, which is known to be dynamic. Herein, we developed a microfluidic CTC capture chip that incorporated nanoroughened glass to capture CTCs. Our chip utilized the differential adhesion preference of cancer cells to nanoroughened glass as compared to normal blood cells and thus did not depend on CTC size or marker expression. The microfluidic chip achieved high capture yields of both EpCAM+ and EpCAM- cancer cells in blood as well as CTCs undergoing TGF-beta-induced EMT with dynamically down-regulated EpCAM expression. In a mouse model of breast cancer, the number of CTCs captured correlated positively with primary tumor size and was independent of the xenografted cell lines’ EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with primary tumors independent of the cell lines’ metastatic ability. Thus, our nanoroughened-glass CTC capture chip is capable of capturing clinically interesting heterogeneous CTC populations independent of their surface marker expression and metastatic propensity. Our chip also captured CTCs from a non-metastatic lung cancer model, demonstrating its potential to collect the entirety of CTC populations including phenotypically diverse subgroups. - [73] Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis
Shinuo Weng, Yue Shao, Weiqiang Chen, and Jianping Fu
Nature Materials, vol. 15, pp. 961-967, 2016.
DOI: 10.1038/nmat4654. PMCID: PMC4996707.
Abstract | PDF | Supplemental Materials | PubMed
Mechanical homeostasis - a fundamental process by which cells maintain stable states under environmental perturbations - is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths via either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modeling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviors in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight previously underappreciated physical nature of the mechanical homeostasis of cells. - [72] Atomic force microscopy indentation and inverse analysis for non-linear viscoelastic identification of breast cancer cells
Nhung Nguyen, Yue Shao, Alan Wineman, Jianping Fu, and Anthony Waas
Mathematical Biosciences, vol. 277, pp. 77-88, 2016.
DOI: 10.1016/j.mbs.2016.03.015.
Abstract | PDF | PubMed
Breast cancer cells (MCF-7 and MCF-10A) are studied through indentation with spherical borosilicate glass particles in atomic force microscopy (AFM) contact mode in fluid. Their mechanical properties are obtained by analyzing the recorded reaction force-time response. The analysis is based on comparing experimental data with predictions from fnite element (FE) simulation. Here, FE modeling is employed to simulate the AFM indentation experiment which is neither a displacement nor a force control test. This approach is expected to overcome many underlying problems of the widely used models such as Hertz contact model due to its capability to capture the contact behaviors between the spherical indentor and the cell, account for cell geometry, and incorporate with large strain theory. In this work, a non-linear viscoelastic (NLV) model in which the viscoelastic part is described by Prony series terms is used for the constitutive model of the cells. The time-dependent material parameters are extracted through an inverse analysis with the use of a surrogate model based on a Kriging estimator. The purpose is to automatically extract the NLV properties of the cells with a more efficient process compared to the iterative inverse technique that has been mostly applied in the literature. The method also allows the use of FE modeling in the analysis of a large amount of experimental data. The NLV parameters are compared between MCF-7 and MCF-10A and MCF-10A treated and untreated with cytochalasin D to examine the possibility of using relaxation properties as biomarkers for distinguishing these types of breast cancer cells. The comparisons indicate that malignant cells (MCF-7) are softer and exhibit more relaxation than benign cells (MCF-10A). Disrupting the cytoskeleton using the drug cytochalasin D also results in a larger amount of relaxation in the cell's response. In addition, relaxation properties indicated larger differences as compared to the elastic moduli like instantaneous shear modulus. These results may be useful for disease diagnosing purposes. - [71] Multiparametric biomechanical and biochemical phenotypic profiling of single cancer cells using elasticity microcytometer
Shuhuan Hu, Guangyu Liu, Weiqiang Chen, Xiang Li, Wei Lu, Raymond H. W. Lam, and Jianping Fu
Small, vol. 12, pp. 2300-2311, 2016.
DOI: 10.1002/smll.201503620. PMCID: PubMed - in process.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the front cover story for 2016 May 4 issue of Small.
Deep phenotyping of cancer cells at the single-cell level is of critical importance in the era of precision medicine to advance understanding of the precise relationship between gene mutation and cell phenotype and to elucidate biological nature of tumor heterogeneity and their potential biological and clinical implications. Existing microfluidic single-cell phenotyping tools, albeit their high-throughput, high-resolution operation, are limited to phenotypic measurements of 1 - 2 selected morphological and physiological features of single cells. To address the critical need for multiplexed, informative phenotyping of live single cancer cells, herein we reported a microfluidic elasticity microcytometer for multiparametric biomechanical and biochemical phenotypic profiling of free-floating, live single cancer cells to obtain quantitative information of cell size, cell deformability / stiffness, and expression levels of surface receptors simultaneously for the same single live cancer cells. The elasticity microcytometer was implemented for single-cell measurements and comparisons of four human cell lines with distinct metastatic potentials and derived from different human tissues. An analytical model was developed from first principles for the first time to effectively convert cell deformation and adhesion information of single cancer cells encapsulated inside the elasticity microcytometer to cell deformability / stiffness and surface protein expression. Together, the elasticity microcytometer holds a great promise for comprehensive molecular, cellular, and biomechanical phenotypic profiling of live cancer cells at the single cell level, critical for studying intra-tumor cellular and molecular heterogeneity using low-abundance, clinically relevant human cancer cells. - [70] Angiogenesis in liquid tumors: An in-vitro assay for leukemic cell induced bone marrow angiogenesis
Yi Zheng, Yubing Sun, Xinwei Yu, Yue Shao, Ping Zhang, Guohao Dai, and Jianping Fu
Advanced Healthcare Materials, vol. 5, pp. 1014-1024, 2016.
DOI: 10.1002/adhm.201501007. PMCID: PMC4890157.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the front cover story for 2016 May 11 issue of Advanced Healthcare Materials.
The critical role of angiogenesis for solid tumor growth and metastatic spread has been well established. In contrast, even though increased vascularity has been commonly observed in bone marrows of patients with hematological malignancies (liquid tumors), the pathophysiology of leukemia induced angiogenesis in the bone marrow remains elusive. In this paper, we demonstrated the usage of a microengineered 3D biomimetic model to study leukemic cell induced bone marrow angiogenesis. Rational design of the 3D angiogenesis chip incorporating endothelial cells (ECs), leukemic cells, and bone marrow stromal fibroblasts provided an efficient biomimetic means to promote and visualize early angiogenic processes. Morphological features of angiogenesis induced by three different leukemic cell lines (U937, HL60, and K562) were investigated and compared. Quantitative measurements of angiogenic factors secreted from monocultures and cocultures of leukemic cells with bone marrow stromal fibroblasts suggested a synergistic relationship between ECs, leukemic cells, and bone marrow stromal fibroblasts for angiogenic induction, and also confirmed the necessity of conducting functional angiogenic assays in proper 3D biomimetic cell culture systems like the one developed in this work.
2015
- [69] Lumen formation is an intrinsic property of isolated human pluripotent stem cells
Kenichiro Taniguchi, Yue Shao, Ryan F. Townshend, Yu-Hwai Tsai, Cynthia J. DeLong, Shawn A. Lopez, Srimonta Gayen, Andrew M. Freddo, Deming J. Chue, Dennis J. Thomas, Jason R. Spence, Benjamin Margolis, Sundeep Kalantry, Jianping Fu, K. Sue O’Shea, and Deborah L. Gumucio
Stem Cell Report, vol. 5, pp. 954-962, 2015.
DOI: 10.1016/j.stemcr.2015.10.015. PMCID: PMC4682207.
Abstract | PDF | Supplemental Materials | PubMed
We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs. - [68] Supersoft lithography: Candy-derived soft microstructures to measure microtissue-generated stresses
Christopher Moraes, Joseph M. Labuz, Yue Shao, Jianping Fu, and Shuichi Takayama
Lab on Chip, vol. 15, pp. 3760-3765, 2015.
DOI: 10.1039/C5LC00722D. PMCID: PMC4550510.
Abstract | PDF | Supplemental Materials | PubMed
We designed a fabrication technique able to replicate microstructures in soft silicone materials (E < 1 kPa). Sugar-based ‘hard candy’ recipes from the confectionery industry were modified to be compatible with silicone processing conditions, and used as templates for replica molding. Microstructures fabricated in soft silicones can then be easily released by dissolving the template in water. We anticipate that this technique will be of particular importance in replicating physiologically soft, microstructured environments for cell culture, and demonstrate a first application in which intrinsically soft microstructures are used to measure forces generated by fibroblast-laden contractile tissues. - [67] Desktop aligner for fabrication of multilayer microfluidic devices
Xiang Li, Zeta Tak-For Yu, Dalton Geraldo, Shinuo Weng, Nitesh Alve, Andy Dun, Akshay Kini, Karan Patel, Roberto Shu, and Jianping Fu
Review of Scientific Instruments, vol. 86, 075008, 2015.
DOI: 10.1063/1.4927197. PMCID: PMC4522017.
Abstract | PDF | PubMed
Multilayer assembly is a commonly used technique to construct multilayer polydimethylsiloxane (PDMS)-based microfluidic devices with complex 3D architecture and connectivity for large-scale microfluidic integration. Accurate alignment of structure features on different PDMS layers before their permanent bonding is critical in determining the yield and quality of assembled multilayer microfluidic devices. Herein, we report a custom-built desktop aligner capable of both local and global alignments of PDMS layers covering a broad size range. Two digital microscopes were incorporated into the aligner design to allow accurate global alignment of PDMS structures up to 4 in. in diameter. Both local and global alignment accuracies of the desktop aligner were determined to be about 20 μm cm−1. To demonstrate its utility for fabrication of integrated multilayer PDMS microfluidic devices, we applied the desktop aligner to achieve accurate alignment of different functional PDMS layers in multilayer microfluidics including an organs-on-chips device as well as a microfluidic device integrated with vertical passages connecting channels located in different PDMS layers. Owing to its convenient operation, high accuracy, low cost, light weight, and portability, the desktop aligner is useful for microfluidic researchers to achieve rapid and accurate alignment for generating multilayer PDMS microfluidic devices. - [66] Biocompatible PEG-chitosan@carbon dots hybrid nanogels for optical pH-sensing, two-photon fluorescence imaging and near-infrared light/pH dual-responsive drug carrier
Hui Wang, Jing Di, Yubing Sun, Jianping Fu, Zengyan Wei, Hiroshi Matsui, Alejandra del C. Alonso, and Shuiqin Zhou
Advanced Functional Materials, vol. 25, pp. 5537-5547, 2015.
DOI: 10.1002/adfm.201501524.
Abstract | PDF | Supplemental Materials
This work designs a class of biocompatible PEG-chitosan@CDs hybrid nanogels by integrating nonlinear poly(ethylene glycol) (PEG), chitosan, and graphitic carbon dots (CDs) into a single nanoparticle for two-photon fluorescence (TPF) bioimaging, pH and near-infrared (NIR) light dual-responsive drug release, and synergistic therapy. Such hybrid nanogels can be simply prepared from a one-pot surfactant-free precipitation polymerization of the PEG macromonomers complexed with chitosan and CDs in water, resulting in a semi-interpenetration of chitosan chains and an immobilization of CDs in the nonlinear PEG networks. The embedded CDs in hybrid nanogels not only serve as an excellent confocal and TPF imaging contrast agent and fluorescent pH-sensing probe, but also enhance the loading capacity of the hybrid nanogels for hydrophobic anticancer drug. The chitosan can induce a pH-sensitive swelling/deswelling of the hybrid nanogels for pH-regulated drug release over the physiologically important range of 5.0–7.4 and surface modulation of embedded CDs to realize fluorescent pH sensing. The thermosensitive nonlinear PEG network can promote the drug release through the local heat produced by the embedded CDs under NIR irradiation. The in vitro results indicate that the hybrid nanogels demonstrated high therapeutic efficacy through the synergistic effect of combined chemo–photothermal treatments. - [65] Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA
Zeta Tak-For Yu, Huijiao Guan, Mei Ki Cheung, Walker McHugh, Timothy T. Cornell, Thomas P. Shanley, Katsuo Kurabayashi, and Jianping Fu
Scientific Reports, vol. 5, 11339, 2015.
DOI: 10.1038/srep11339. PMCID: PMC4466892.
Abstract | PDF | PubMed
Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology ('AlphaLISA') in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes in a free solution environment before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg per mL. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications. - [64] Emerging roles of YAP/TAZ in mechanobiology
Yubing Sun, Yue Shao, Xufeng Xue, and Jianping Fu
Molecular and Cellular Mechanobiology (edited by Shu Chien, Adam J. Engler, and Yingxiao Wang), the American Physiological Society, 2015.
Abstract | PDF
Understanding mechanotransduction principles is a major challenge in the mechanobiology field. YAP, and its paralog TAZ, are transcriptional coactivators at the core of the Hippo signaling pathway. Recent studies have identified YAP/TAZ as both mechano-sensors and -transducers that respond to multiple mechanical stimuli and relay them to downstream transcriptional signals to regulate cell functions such as proliferation, spreading, and differentiation. In this chapter, we discuss how different types of mechanical cues, including actin cytoskeleton, substrate rigidity, and external forces, mediate YAP/TAZ activities. We also highlight the possible mechanosensitive molecular machineries that function upstream of YAP/TAZ. - [63] Age-associated increase of skin fibroblast-derived prostaglandin E2 contributes to reduced collagen levels in elderly human skin
Yong Li, Dan Lei, William R. Swindell, Wei Xia, Shinuo Weng, Jianping Fu, Christal A. Worthen, Toru Okubo, Andrew Johnston, Johann E. Gudjonsson, John J. Voorhees, and Gary J. Fisher
Journal for Investigative Dermatology, vol. 135, pp. 2181-2188, 2015.
DOI: 10.1038/jid.2015.157. PMCID: PMC4537382.
Abstract | PDF | Supplemental Materials | PubMed
Production of type I collagen declines during aging, leading to skin thinning and impaired function. Prostaglandin E2 (PGE2) is a pleiotropic lipid mediator that is synthesized from arachidonic acid by the sequential actions of cyclooxygenases (COX) and PGE synthases(PTGES). PGE2 inhibits collagen production by fibroblasts in vitro. We report that PTGES1 and COX2 progressively increase with aging in sun-protected human skin. PTGES1 and COX2 mRNA was increased 3.4-fold and 2.7-fold, respectively, in the dermis of elderly (> 80 years) versus young (21 - 30 years) individuals. Fibroblasts were the major cell source of both enzymes. PGE2 levels were increased 70% in elderly skin. Fibroblasts in aged skin display reduced spreading due to collagen fibril fragmentation. To investigate the relationship between spreading and PGE2 synthesis, fibroblasts were cultured on micropost arrays or hydrogels of varying mechanical compliance. Reduced spreading/mechanical force resulted in increased expression of both PTGES1 and COX2 and elevated levels of PGE2. Inhibition of PGE2 synthesis by diclofenac enhanced collagen production in skin organ cultures. These data suggest that reduced spreading/mechanical force of fibroblasts in aged skin elevates PGE2 production, contributing to reduced collagen production. Inhibition of PGE2 production may be therapeutically beneficial for combating age-associated collagen deficit in human skin. - [62] Multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays
Pengyu Chen, Meng Ting Chuang, Walker McHugh, Robert Nidetz, Yuwei Li, Jianping Fu, Timothy T. Cornell, Thomas P. Shanley, and Katsuo Kurabayashi
ACS Nano, vol. 9, pp. 4173-4181, 2015.
DOI: 10.1021/acsnano.5b00396. PMCID: PMC4447431.
Abstract | PDF | Supplemental Materials | PubMed
Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making impractical and virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders, as access to their blood in large quantities is prohibited. Here, we developed a high-throughput, label-free, multi-arrayed optical biosensor device with 480 nanoplasmonic biosensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokine species in complex human serum samples with a detection sensitivity nearly 10 times higher than that of conventional refractometric optical biosensing methods. We demonstrated that our rapid and sample-sparing assay using the nanoplasmonic biosensor microarray could monitor inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking time-course variations of serum cytokine biomarkers. - [61] Fluorescent porous carbon nanocapsules for two-photon imaging, NIR/pH dual-responsive drug carrier, and photothermal therapy
Hui Wang, Yubing Sun, Jinhui Yi, Jianping Fu, Jing Di, Alejandra del Carmen Alonso, and Shuiqin Zhou
Biomaterials, vol. 53, pp. 117-126, 2015.
DOI: 10.1016/j.biomaterials.2015.02.087.
Abstract | PDF | Supplemental Materials | PubMed
An efficient nanomedical platform that can combine two-photon cell imaging, near infrared (NIR) light and pH dual responsive drug delivery, and photothermal treatment was successfully developed based on fluorescent porous carbon-nanocapsules (FPC-NCs, size ~ 100 nm) with carbon dots (CDs) embedded in the shell. The stable, excitation wavelength-tunable and upconverted fluorescence from the CDs embedded in the porous carbon shell enable the FPC-NCs to serve as an excellent confocal and two-photon imaging contrast agent under the excitation of laser with a broad range of wavelength from ultraviolet (UV) light (405 nm) to NIR light (900 nm). The FPC-NCs demonstrate a very high loading capacity (1335 mg/g) toward doxorubicin drug benefited from the hollow cavity structure, porous carbon shell, as well as the supramolecular π stacking and electrostatic interactions between the doxorubicin molecules and carbon shell. In addition, a responsive release of doxorubicin from the FPC-NCs can be activated by lowering the pH to acidic (from 7.4 to 5.0) due to the presence of pH-sensitive carboxyl groups on the FPC-NCs and amino groups on doxorubicin molecules. Furthermore, the FPC-NCs can absorb and effectively convert the NIR light to heat, thus, manifest the ability of NIR-responsive drug release and combined photothermal/chemotherapy for high therapeutic efficacy. - [60] Improving survival of disassociated human embryonic stem cells by mechanical stimulation using acoustic tweezing cytometry
Di Chen, Yubing Sun, Cheri X. Deng, and Jianping Fu
Biophysical Journal, vol. 108, pp. 1315-1317, 2015.
DOI: 10.1016/j.bpj.2015.01.033. PMCID: PMC4375556.
Abstract | PDF | Supplemental Materials | PubMed
Dissociation-induced apoptosis of human embryonic stem cells (hESCs) hampers their large-scale culture. Herein we leveraged the mechanosensitivity of hESCs and used a novel technique, acoustic tweezing cytometry (ATC), for subcellular mechanical stimulation of disassociated single hESCs to improve their survival. By acoustically actuating integrin-bound microbubbles (MBs) to live cells, ATC increased the survival rate and cloning efficiency of hESCs by 3-fold. A positive correlation was observed between increased hESC survival rate and total accumulative displacement of integrin-anchored MBs during ATC stimulation. Our study suggests ATC as a promising biocompatible tool to improve hESC culture. - [59] On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology
Yue Shao, Jianming Sang, and Jianping Fu
Biomaterials, vol. 52, pp. 26-43, 2015.
DOI: 10.1016/j.biomaterials.2015.01.078. PMCID: PMC4379448.
Abstract | PDF | PubMed
Human pluripotent stem cells (hPSCs) provide promising resources for regenerating tissues and organs and modeling development and diseases in vitro. To fulfill their promise, the fate, function, and organization of hPSCs need to be precisely regulated in a three-dimensional (3D) environment to mimic cellular structures and functions of native tissues and organs. In the past decade, innovations in 3D culture systems with functional biomaterials have enabled efficient and versatile control of hPSC fate at the cellular level. However, we are just at the beginning of bringing hPSC-based regeneration and development and disease modeling to the tissue and organ levels. In this review, we summarize existing bioengineered culture platforms for controlling hPSC fate and function by regulating inductive mechanical and biochemical cues coexisting in the synthetic cell microenvironment. We highlight recent excitements in developing 3D hPSC-based in vitro tissue and organ models with in vivo-like cellular structures, interactions, and functions. We further discuss an emerging multifaceted mechanotransductive signaling network – with transcriptional coactivators YAP and TAZ at the center stage – that regulate fates and behaviors of mammalian cells, including hPSCs. Future development of 3D biomaterial systems should incorporate dynamically modulated mechanical and chemical properties targeting specific intracellular signaling events leading to desirable hPSC fate patterning and functional tissue formation in 3D. - [58] Two bubble acoustic tweezing cytometry for biomechanical probing and stimulation of cells
Di Chen, Yubing Sun, Madhu S. R. Gudur, Yising Hsiao, Ziqi Wu, Jianping Fu, and Cheri X. Deng
Biophysical Journal, vol. 108, pp. 32-42, 2015.
DOI: 10.1016/j.bpj.2014.11.050. PMCID: PMC4286600.
Abstract | PDF | PubMed
Study of mechanotransduction relies on tools capable of applying mechanical forces to elicit and assess cellular responses. Here we report a new technique, two bubble acoustic tweezing cytometry (TB-ATC), for generating spatiotemporally controlled subcellular mechanical forces to live cells by acoustic actuation of paired microbubbles targeted to cell adhesion receptor integrin. By measuring ultrasound induced activities of cell-bound microbubbles and the actin cytoskeleton contractile force responses, we determine that TB-ATC elicited mechanoresponsive cellular changes via cyclic, paired displacements of integrin-bound microbubbles driven by the attractive secondary acoustic radiation force (sARF) between the bubbles in an ultrasound field. We demonstrate the feasibility of dual-mode TB-ATC for both subcellular probing and mechanical stimulation. By exploiting the robust and unique interaction of ultrasound with microbubbles, TB-ATC provides distinct advantages for experimentation and quantification of the applied mechanical forces and cellular responses for biomechanical probing and stimulating of cells.
2014
- [57] Themed issue on nanotechnology for biology and biomedical applications
Jianping Fu and Donglei Fan
ASME Journal of Nanotechnology in Engineering and Medicine, vol. 5, 040201, 2014.
DOI: 10.1115/1.4030870.
PDF
- [56] Single-crystalline, nanoporous gallium nitride films with fine tuning of pore size for stem cell engineering
Lin Han, Jing Zhou, Yubing Sun, Yu Zhang, Jung Han, Jianping Fu, and Rong Fan
ASME Journal of Nanotechnology in Engineering and Medicine, vol. 5, 041004, 2014.
DOI: 10.1115/1.4030615. PMCID: PMC4492071.
Abstract | PDF | PubMed
Single-crystalline nanoporous gallium nitride (GaN) thin films were fabricated with the pore size readily tunable in 20 - 100 nm. Adhesion and spreading of human mesenchymal stem cells (hMSCs) seeded on these substrates peak on the surface with pore size of 30 nm. Substantial cell elongation was observed on the films with pore size of 80 nm. The osteogenic differentiation of hMSCs occurs preferentially on the films with 30 nm sized nanopores, which is correlated with the conditions for optimum cell spreading, which suggests that adhesion, spreading, and stem cell differentiation are interlinked and might be co-regulated by nanotopography. - [55] Nanotopograghical surfaces for stem cell fate control: Engineering mechanobiology from the bottom
Weiqiang Chen, Yue Shao, Xiang Li, and Jianping Fu
Nano Today, vol. 9, pp. 759-784, 2014.
DOI: 10.1016/j.nantod.2014.12.002. PMCID: PMC4394389.
Abstract | PDF | PubMed
During embryogenesis and tissue maintenance and repair in an adult organism, a myriad of stem cells are regulated by their surrounding extracellular matrix (ECM) enriched with tissue/organ-specific nanoscale topographical cues to adopt different fates and functions. Attributed to their capability of self-renewal and differentiation into most types of somatic cells, stem cells also hold tremendous promise for regenerative medicine and drug screening. However, a major challenge remains as to achieve fate control of stem cells in vitro with high specificity and yield. Recent exciting advances in nanotechnology and materials science have enabled versatile, robust, and large-scale stem cell engineering in vitro through developments of synthetic nanotopographical surfaces mimicking topological features of stem cell niches. In addition to generating new insights for stem cell biology and embryonic development, this effort opens up unlimited opportunities for innovations in stem cell-based applications. This review is therefore to provide a summary of recent progress along this research direction, with perspectives focusing on emerging methods for generating nanotopographical surfaces and their applications in stem cell research. Furthermore, we provide a review of classical as well as emerging cellular mechano-sensing and -transduction mechanisms underlying stem cell nanotopography sensitivity and also give some hypotheses in regard to how a multitude of signaling events in cellular mechanotransduction may converge and be integrated into core pathways controlling stem cell fate in response to extracellular nanotopography. - [54] Encoding through the host/guest structure: Construction of multiplexed fluorescent beads
Ying Zhu, Hong Xu, Kaimin Chen, Jianping Fu, and Hongchen Gu
Chemical Communications, vol. 50, pp. 14041-14044, 2014
DOI: 10.1039/c4cc05793g.
Abstract | PDF | Supplemental Materials | PubMed
A new encoding strategy based on the host–guest structure is developed, where encoding is achieved by combinational use of a set of guest particles with different fluorescence intensity levels at various ratios. This system is proven to be well-defined and highly controllable. - [53] Emerging microengineering tools for functional analysis and phenotyping of blood cells
Xiang Li, Weiqiang Chen, Zida Li, Ling Li, Hongchen Gu, and Jianping Fu
Trends in Biotechnology, vol. 32, pp. 586-594, 2014.
DOI: 10.1016/j.tibtech.2014.09.003. PMCID: PMC4252757.
Abstract | PDF | PubMed
The available techniques for assessing blood cell functions are limited considering the various types of blood cells and their diverse functions. In the past decade, rapid advancement in microengineering has enabled an array of blood cell functional measurements that are difficult or impossible to achieve using conventional bulk platforms. Such miniaturized blood cell assay platforms also provide attractive capabilities of reducing chemical consumption, cost, and assay time, as well as exciting opportunities of device integration, automation, and assay standardization. This review summarizes these contemporary microengineering tools and discusses their promising potential for constructing accurate in vitro models and rapid clinical diagnosis using minimal amount of whole blood samples. - [52] Stretchable micropost array cytometry: A powerful tool for cell mechanics and mechanobiology research
Yue Shao, Shinuo Weng, and Jianping Fu
Integrative Mechanobiology: Micro and Nano Techniques in Cell Mechanobiology (edited by Yu Sun, Craig Simmons, and Deok-Ho Kim). Cambridge University Press, 2014.
Abstract | PDF
It has become increasingly appreciated that living mammalian cells are not just complex biochemical reactors but also sophisticated biomechanical systems that can adapt their mechanical properties to various signals and perturbations from the extracellular space and integrate with intracellular signaling events through a process called mechanotransduction to regulate cell behaviors. To gain fundamental insights into such biomechanical nature of mammalian cells, many biomechanical tools have been developed with unprecedented spatiotemporal resolutions covering both molecular and cellular length scales. In this chapter, we describe a recently developed biomechanical tool, termed stretchable micropost array cytometry (SMAC), which is capable of quantitative control and real-time measurements of both mechanical stimuli and cellular biomechanical responses with a high spatiotemporal subcellular resolution. We further discuss implementations of the SMAC for characterizing cell cytoskeletal contractile force, cell stiffness, and cell adhesion signaling and dynamics at both whole-cell and subcellular scales in real time. We conclude with remarks regarding future improvements and applications of the SMAC for cell mechanics and mechanobiology studies. - [51] Ultrasensitive ELISA using enzyme-loaded nanospherical brushes as labels
Zhenyuan Qu, Hong Xu, Ping Xu, Kaimin Chen, Rong Mu, Jianping Fu, and Hongchen Gu
Analytical Chemistry, vol. 86, pp. 9367-9371, 2014.
DOI: 10.1021/ac502522b.
Abstract | PDF | Supplemental Materials | PubMed
Improving the detection sensitivity of ELISA is utmost important for meeting the demand of early disease diagnosis. Herein we report an ultrasensitive ELISA system using horseradish peroxidase (HRP)-loaded nanospherical poly(acrylic acid) brushes (SPAABs) as labels. HRP was covalently immobilized in SPAABs with high capacity and activity via an efficient “chemical conjugation after electrostatic entrapment” (CCEE) process, thus endowing SPAABs with high amplification capability as labels. The periphery of SPAAB-HRP was further utilized to bind a layer of antibody with high density for efficient capture of analytes owing to the three-dimensional architecture of SPAABs. Using human chorionic gonadotrophin (hCG) as a model analyte, the SPAAB-amplified system drastically boasted the detection limit of ELISA with a 267-fold improvement. - [50] Harnessing mechanobiology of human pluripotent stem cells for regenerative medicine
Yubing Sun and Jianping Fu
ACS Chemical Neuroscience, vol. 5, pp. 621-623, 2014.
DOI: 10.1021/cn5001155. PMCID: PMC4140584.
Abstract | PDF | PubMed
Selected as the front cover story for 2014 Aug. 20 issue of ACS Chemical Neuroscience.
Recent advances in human pluripotent stem cells (hPSCs) open new doors for therapeutics of motor neuron (MN)-associated neurodegenerative diseases. However, the MN differentiation process is not yet completely understood. In this Viewpoint, we stress the concept of designing synthetic cell culture surfaces with precisely controlled mechanical properties (such as rigidity) to improve the efficiency of MN differentiation from hPSCs. Emerging evidence strongly supports the potent role of mechanobiology in controlling stem cell fate. Leveraging the intrinsic mechanosensitive properties of hPSCs in conjunction with a synthetic elastomeric micropost array system, we have recently demonstrated significantly improved MN differentiation from hPSCs. Mechanotransduction mechanisms of hPSCs are an unexplored territory and likely involve coordination and crossregulations of multiple targets and pathways including cell surface receptors, signaling transduction molecules, and nuclear proteins. We envision that research in hPSCs for MN degenerative diseases will benefit from accumulating knowledge of mechanobiology of hPSCs. - [49] Continuous-flow microfluidic blood cell sorting for unprocessed whole blood using surface-micromachined microfiltration membranes
Xiang Li, Weiqiang Chen, Guangyu Liu, Wei Lu, and Jianping Fu
Lab on a Chip, vol. 14, pp. 2565-2575, 2014.
DOI: 10.1039/c4lc00350k. PMCID: PMC4106416.
Abstract | PDF | Supplemental Materials | PubMed
White blood cells (WBCs) constitute about 0.1% of the blood cells, yet they play a critical role in innate and adaptive immune responses against pathogenic infections, allergic conditions, and malignancies and thus contain rich information about the immune status of the body. Rapid isolation of WBCs directly from whole blood is a prerequisite for any integrated immunoassay platform designed for examining WBC phenotypes and functions; however, such functionality is still challenging for blood-on-a-chip systems, as existing microfluidic cell sorting techniques are inadequate for efficiently processing unprocessed whole blood on chip with concurrent high throughput and cell purity. Herein we report a microfluidic chip for continuous-flow isolation and sorting of WBCs from whole blood with high throughput and separation efficiency. The microfluidic cell sorting chip leveraged the crossflow filtration scheme in conjunction with a surface-micromachined poly(dimethylsiloxane) (PDMS) microfiltration membrane (PMM) with high porosity. With a sample throughput of 1 mL h−1, the microfluidic cell sorting chip could recover 27.4 ± 4.9% WBCs with a purity of 93.5 ± 0.5%. By virtue of its separation efficiency, ease of sample recovery, and high throughput enabled by its continuous-flow operation, the microfluidic cell sorting chip holds promise as an upstream component for blood sample preparation and analysis in integrated blood-on-a-chip systems. - [48] Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells
Yubing Sun, Koh Meng Aw Yong, Luis G. Villa-Diaz, Xiaoli Zhang, Weiqiang Chen, Renee Philson, Shinuo Weng, Haoxing Xu, Paul H. Krebsbach, and Jianping Fu
Nature Materials, vol. 13, pp. 599-604, 2014.
DOI: 10.1038/nmat3945. PMCID: PMC4051885.
Abstract | PDF | Supplemental Materials | PubMed
Commentary by Emily Rhodes Lowry & Christopher E. Henderson, “Stem cell differentiation: Yielding substrates for neurons”, Nature Materials, vol. 13, pp. 543-544, 2014. [web link | PDF]
Commentary by Ning Wang, “Stem cells go soft: Pliant substrate surfaces enhance motor neuron differentiation”, Cell Stem Cell, vol. 14, pp. 701-703, 2014. [web link | PDF]
Highlighted by
How a Silly Putty ingredient could advance stem cell therapies - Michigan News
How Silly Putty may advance stem cell research - Detroit Local 4 News
Not so Silly Putty: UM researchers use one of its components to turn stem cells into spinal cord cells - Crain's Detroit Business
Silly Putty ingredient could advance stem cell therapies - ABC 7 Sarasota - WWSB
No so silly: Ingredient in Silly Putty boosts stem cell growth - ABC News Radio
Silly Putty ingredient creates 'soft carpets' for better nerve cell growth - Headlines & Global News
Silly Putty ingredient could help stem cells become motor neurons - Red Orbit
Growing stem cells may be child's play - The Doctor Will See You Now
Silly Putty may help treat neurological disorders - Consultant
Stem cell culture growth improved by Silly Putty - Guardian Liberty Voice
Stem cell research uses Silly Putty - The Australian
Silly Putty stem cells '10 times larger' than those grown in traditional lab conditions - International Business Times UK
Stem cells grown on 'soft carpets' function better - The Times of India
How a Silly Putty ingredient could advance stem cell therapies - Israel Herald
Stem cells grown on 'soft carpets' function better - Business Standard
Stem cells get a boost from material in Silly Putty - Milwaukee Wisconsin Journal Sentinel
Could Silly Putty help treat neurological disorders? - Medical News Today
Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive1. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs. - [47] Integrated nanoplasmonic sensing for cellular functional immunoanalysis using human blood
Bo-Ram Oh, Nien-Tsu Huang, Weiqiang Chen, Jungwhan Seo, Pengyu Chen, Timothy T. Cornell, Thomas P. Shanley, Jianping Fu, and Katsuo Kurabayashi
ACS Nano, vol. 8, pp. 2667-2676, 2014.
DOI: 10.1021/nn406370u. PMCID: PMC4004291.
Abstract | PDF | Supplemental Materials | PubMed
Localized surface plasmon resonance (LSPR) nanoplasmonic effects allow for label-free, real-time detection of biomolecule binding events on a nanostructured metallic surface with simple optics and sensing tunability. Despite numerous reports on LSPR bionanosensing in the past, no study thus far has applied the technique for cytokine secretion assay using clinically relevant immune cells from human blood. Cytokine secretion assays - a technique to quantify intercellular-signaling proteins secreted by blood immune cells - allow determination of the functional response to the donor’s immune cells, thus providing valuable information about the immune status of the donor. However, implementation of LSPR bionanosensing in cellular functional immunoanalysis based on cytokine secretion assay poses major challenges primarily owing to its limited sensitivity and a lack of sufficient sample handling capability. In this paper, we have developed a novel label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-alpha in clinical blood samples. Our approach integrates LSPR bionanosensors in an optofluidic platform that permits trapping and stimulation of target immune cells in a microfluidic chamber with optical access for subsequent cytokine detection. The on-chip spatial confinement of the cells is the key for rapidly increasing a cytokine concentration high enough for detection by the LSPR setup, thereby allowing the assay time and sample volume to be significantly reduced. We have successfully applied this approach first to THP-1 cells and then later to CD45-positive cells isolated directly from human blood. Our LSPR optofluidics device allows for detection of TNF-alpha secreted from cells as few as 1,000, which translates into a nearly 100 times decrease in sample volume than conventional cytokine secretion assay techniques require. We achieved cellular functional immunoanalysis with a minimal blood sample volume (3 microlitre) and a total assay time three times shorter than that of conventional enzyme-linked immunosorbent assay (ELISA). - [46] Global architecture of F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction
Yue Shao, Jennifer M. Mann, Weiqiang Chen, and Jianping Fu
Integrative Biology, vol. 6, pp. 300-311, 2014.
DOI: 10.1039/c3ib40223a. PMCID: PMC3963173.
Abstract | PDF | Supplemental Materials | PubMed
Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch. - [45] Microfluidic blood cell sorting: Now and beyond
Zeta Tak-For Yu, Koh Meng Aw Yong, and Jianping Fu
Small, vol. 10, pp. 1687-1703, 2014.
DOI: 10.1002/smll.201302907. PMCID: PMC4013196.
Abstract | PDF | PubMed
Blood plays an utmost important role in homeostatic regulation with each of its components (either cellular or non-cellular) having great significance for therapeutic and diagnostic purposes. Therefore, separation and sorting of blood cells has been of great interest to clinicians and researchers. However, while conventional methods of processing blood have been successful in generating relatively pure fractions, they are time consuming, labor intensive, and are not optimal for processing small volume blood samples. In recent years, microfluidics has garnered great interest as a powerful technology for separating blood into different cell fractions. As microfluidics involves fluid manipulation at the microscale level, it has the potential for achieving high-resolution separation and sorting of blood cells down to the single-cell level, with an added benefit of integrating physical and biological methods of blood cell separation and analysis on the same single chip platform. This review will first review the conventional methods of processing and sorting blood cells, followed by a discussion on how microfluidics is emerging as an efficient tool to rapidly change the field of blood cell sorting for blood-based therapeutic and diagnostic applications. - [44] Integrated micro/nanoengineered functional biomaterials for cell mechanics and mechanobiology: A materials perspective
Yue Shao, and Jianping Fu
Advanced Materials, vol. 26, pp. 1494-1533, 2014.
DOI: 10.1002/adma.201304431. PMCID: PMC4076293.
Abstract | PDF | PubMed
The rapid development of micro/nanoengineered functional biomaterials in the last two decades has empowered materials scientists and bioengineers to precisely control different aspects of the in vitro cell microenvironment. Following a philosophy of reductionism, many studies using synthetic functional biomaterials have revealed instructive roles of individual extracellular biophysical and biochemical cues in regulating cellular behaviors. Development of integrated micro/nanoengineered functional biomaterials to study complex and emergent biological phenomena has also thrived rapidly in recent years, revealing adaptive and integrated cellular behaviors closely relevant to human physiological and pathological conditions. Working at the interface between materials science and engineering, biology, and medicine, we are now at the beginning of a great exploration using micro/nanoengineered functional biomaterials for both fundamental biology study and clinical and biomedical applications such as regenerative medicine and drug screening. In this review, we present an overview of state of the art micro/nanoengineered functional biomaterials that can control precisely individual aspects of cell-microenvironment interactions and highlight them as well-controlled platforms for mechanistic studies of mechano-sensitive and -responsive cellular behaviors and integrative biology research. We also discuss the recent exciting trend where micro/nanoengineered biomaterials are integrated into miniaturized biological and biomimetic systems for dynamic multiparametric microenvironmental control of emergent and integrated cellular behaviors. The impact of integrated micro/nanoengineered functional biomaterials for future in vitro studies of regenerative medicine, cell biology, as well as human development and disease models are discussed. - [43] Substrates with engineered step changes in rigidity suggest a role for subcellular biases in traction force in driving durotaxis
Mark T. Breckenridge, Ravi A. Desai, Michael T. Yang, Jianping Fu, and Christopher S. Chen
Cellular and Molecular Bioengineering, vol. 7, pp. 26-34, 2014.
DOI: 10.1007/s12195-013-0307-6. PMCID: PMC5055071.
Abstract | PDF | Supplemental Materials | PubMed
Rigidity sensing plays a fundamental role in multiple cell functions ranging from migration, to proliferation and differentiation. During migration, single cells have been reported to preferentially move toward more rigid regions of a substrate in a process termed durotaxis. Durotaxis could contribute to cell migration in wound healing and gastrulation, where local gradients in tissue rigidity have been described. Despite the potential importance of this phenomenon to physiology and disease, it remains unclear how rigidity guides these behaviors and the underlying cellular and molecular mechanisms. To investigate the functional role of subcellular distribution and dynamics of cellular traction forces during durotaxis, herein we have developed a unique microfabrication strategy to generate elastomeric micropost arrays patterned with regions exhibiting two different rigidities juxtaposed next to each other. After initial cell attachment on the rigidity boundary of the micropost array, NIH 3T3 fibroblasts are observed to preferentially migrate toward the rigid region of the micropost array, indicative of durotaxis. Additionally, cells bridging two rigidities across the rigidity boundary on the micropost array develop stronger traction forces on the more rigid side of the substrate indistinguishable from forces generated by cells exclusively seeded on rigid regions of the micropost array. Together, our results highlight the utility of step-rigidity micropost arrays to investigate the functional role of traction forces in rigidity sensing and durotaxis, suggesting that cells can sense substrate rigidity locally to induce an asymmetrical intracellular traction force distribution to contribute to durotaxis.
2013
- [42] Uniaxial cell stretching device for live-cell imaging of mechanosensitive cellular functions
Yue Shao, Xinyu Tan, Roman Novitski, Misha Muqaddam, Paul T. List, Laura Williamson, Jianping Fu, and Allen P. Liu
Review of Scientific Instruments, vol. 84, 114304, 2013.
DOI: 10.1063/1.4832977. PMCID: PMC3862604.
Abstract | PDF | PubMed
External mechanical stretch plays an important role in regulating cellular behaviors through intracellular mechanosensitive and mechanotransductive machineries such as the F-actin cytoskeleton (CSK) and focal adhesions (FAs) anchoring the F-actin CSK to the extracellular environment. Studying the mechanoresponsive behaviors of the F-actin CSK and FAs in response to cell stretch has great importance for further understanding mechanotransduction and mechanobiology. In this work, we have developed a novel cell stretching device combining dynamic directional cell stretch with in situ subcellular live-cell imaging. Using a cam and follower mechanism and applying a standard mathematical model for cam design, we can generate different dynamic stretch outputs. By examining stretch-mediated FA dynamics under step-function static stretch and the realignment of cell morphology and the F-actin CSK under cyclic stretch, we have demonstrated successful applications of our cell stretching device for mechanobiology studies where external stretch plays an important role in regulating subcellular molecular dynamics and cellular phenotypes. - [41] Cellular enrichment from clinical samples
Koh Meng Aw Yong, Zeta Tak-For Yu, Huijiao Guan, and Jianping Fu
Micro- and Nanosystems for Biotechnology (edited by J. Christopher Love). Wiley Biotechnology Series, 2013.
Abstract | PDF
The study of cells on either the tissue or single cell level yields important biological information useful to both clinicians and researchers. There are many approaches to study cells and technological improvements have made it now possible to image or study the molecular makeup of a single cell. While such high sensitivity allows the analysis of rare cells, the sample analyzed must be pure and free of any contamination from unwanted cells. Therefore, there is a need for technologies that enable enrichment for the cell of interest from clinical samples in a specific, reliable and consistent manner. To achieve this, an understanding of different tissue types as well as their complexity is first required. In this chapter, we will first introduce to readers the types of clinical samples typically handled, their complexity as well as some of the conventional methods of cellular enrichment. The latter part of this chapter will delve into micro- or nano-scale devices and how they are revolutionizing cellular enrichment from clinical samples. - [40] Biosample preparation by lab-on-a-chip devices
Yong-Ak Song, Jianping Fu, Ying-Chih Wang, and Jongyoon Han
Encyclopedia of Microfluidics and Nanofluidics (edited by Dongqing Li), Springer-Verlag, 2013.
DOI: 10.1007/SpringerReference_66329.
Abstract | PDF
In this book chapter, we review state of the art micro/nanofluidic techniques for different biosample preparation appplications, including separation and preconcentration. - [39] Acoustic tweezing cytometry for live-cell subcellular control of intracellular cytoskeleton contractility
Zhenzhen Fan, Yubing Sun, Di Chen, Donald Tay, Weiqiang Chen, Cheri X. Deng, and Jianping Fu
Scientific Reports, vol. 3, 2176, 2013.
DOI: 10.1038/srep02176. PMCID: PMC3709169.
Abstract | PDF | Supplemental Materials | PubMed
Mechanical forces are critical to modulate cell spreading, contractility, gene expression, and even stem cell differentiation. Yet, existing tools that can apply controllable subcellular forces to a large number of single cells simultaneously are still limited. Here we report a novel ultrasound tweezing cytometry utilizing ultrasound pulses to actuate functionalized lipid microbubbles covalently attached to single live cells to exert mechanical forces in the pN - nN range. Ultrasonic excitation of microbubbles could elicit a rapid and sustained reactive intracellular cytoskeleton contractile force increase in different adherent mechanosensitive cells. Further, ultrasound-mediated intracellular cytoskeleton contractility enhancement was dose-dependent and required an intact actin cytoskeleton as well as RhoA/ROCK signaling. Our results demonstrated the great potential of ultrasound tweezing cytometry technique using functionalized microbubbles as an actuatable, biocompatible, and multifunctional agent for biomechanical stimulations of cells. - [38] How vinculin regulates traction forces and adhesion strength
David W. Dumbauld, Ted T. Lee, Ankur Singh, Jan Scrimgeour, Charles A. Gersbach, Evan A. Zamir, Jianping Fu, Christopher S. Chen, Jennifer E. Curtis, Susan W. Craig, and Andrés J. García
Proceedings of the National Academy of Sciences of the United States of America, vol. 110, pp. 9788-9793, 2013.
DOI: 10.1073/pnas.1216209110. PMCID: PMC3683711.
Abstract | PDF | Supplemental Materials | PubMed
Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces, but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (VH) and tail domains. VH enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared to VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; VH has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or VH, correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.. - [37] Microfluidic modeling of cancer metastasis
Zeta Tak-For Yu, Koh Meng Aw Yong, and Jianping Fu
Cells, Forces and the Microenvironment (edited by Andrew E. Pelling and Charles M. Cuerrier). Pan Stanford Publishing, 2013.
Abstract | PDF
Metastatic disease remains the primary cause of death for patients with most types of cancer. Metastasis occurs through a series of interrelated steps, including cancer invasion and intravasation, survival of cancer cells in the circulation, adhesion to endothelial cells lining blood vessels, and growth of cancer cells to form metastases. Successfully interrupting any one of these steps will stop metastatic disease and potentially cure cancer. Recent advances in microfluidics have demonstrated promising applications of microfluidics for studying the metastatic cascade with well-controlled microfluidic environment. This review will highlight the recent developments of microfluidic platforms for studying cancer metastasis. We will briefly describe some of the crucial steps of metastasis: invasion, intravasation into the circulatory system and finally colonization of distal sites. We will further describe the conventional methods used to study these steps, their limitations and how the advent of microfluidics is facilitating a better understanding of the metastatic process. - [36] Emerging microfluidic tools for cellular functional immunophenotyping: A new potential paradigm for immune status characterization
Weiqiang Chen, Nien-Tsu Huang, Xiang Li, Zeta Tak-For Yu, Katsuo Kurabayashi, and Jianping Fu
Frontiers in Oncology, vol. 3, 98, 2013.
DOI: 10.3389/fonc.2013.00098. PMCID: PMC3631762.
Abstract | PDF | PubMed
Rapid, accurate, and quantitative characterization of immune status of patients is of utmost importance for disease diagnosis and prognosis, evaluating efficacy of immunotherapeutics and tailoring drug treatments. Immune response of patients is often dynamic and patient-specific, and such complex heterogeneity has made accurate, real-time measurements of patient immune status challenging in the clinical setting. Recent advances in microfluidics have demonstrated promising applications of microfluidics for immune monitoring with minimum sample requirement and rapid functional immunophenotyping capability. This review will highlight the recent developments of microfluidic platforms that can perform rapid and accurate cellular functional assays on patient immune cells. We will also discuss the future potential of integrated microfluidics to perform rapid, accurate, and sensitive cellular functional assays at a single-cell resolution on different types or subpopulations of immune cells, to provide an unprecedented level of information depth on the distribution of immune cell functionalities. We envision that such microfluidic immunophenotyping tools will allow comprehensive and systems-level immunomonitoring, unlocking the potential to transform experimental clinical immunology into an information-rich science. - [35] Adhesive signature-based, label-free isolation of human pluripotent stem cells
Ankur Singh, Shalu Suri, Ted T. Lee, Jamie M. Chilton, Marissa T. Cooke, Weiqiang Chen, Jianping Fu, Steven L. Stice, Hang Lu, Todd C. McDevitt, and Andrés J. García
Nature Methods, vol. 10, pp. 438-444, 2013.
DOI: 10.1038/nmeth.2437. PMCID: PMC3641175.
Abstract | PDF | Supplemental Materials | PubMed
Highlighted by NIH Director's Blog.
Commentary by Oscar J. Abilez & Joseph C. Wu, “Stem cell isolation: Differential stickiness”, Nature Materials, vol. 12, pp. 474-476, 2013; web link.
The ability to efficiently isolate undifferentiated human pluripotent stem cells (hPSCs) as colonies from contaminating non-pluripotent cells is a crucial step in the stem cell field to maintain survival efficiency and karyotype stability. Currently, this isolation relies primarily on time-intensive manual isolation or single cell sorting. Here we demonstrate significant differences in focal adhesion assembly and adhesion strength among undifferentiated hPSCs (hiPSC, hESC) and parental/feeder layer cells, partially reprogrammed cells, spontaneously differentiated and directly differentiated progenitor cells. This distinct ‘adhesive signature’ of hPSCs is exploited to rapidly (< 10 min) and efficiently isolate fully reprogrammed undifferentiated hiPSCs as intact colonies from partially reprogrammed cells, parental cells, feeder layer cells, spontaneously differentiated cells, and terminally differentiated cardiomyocytes using microfluidics technology termed µSHEAR (micro Stem cell High-Efficiency Adhesion-based Recovery). hPSCs, irrespective of source, passage number, and feeder-free matrix, are isolated in a label-free fashion and enriched to > 95-99% purity and survival without adversely affecting the transcriptional profile, differentiation potential or karyotype of the pluripotent cells. This low-cost, rapid, label-free, high-throughput strategy is applicable to isolate undifferentiated hiPSCs from heterogeneous reprogramming cultures and differentiating cell populations during routine hPSC culture and can be expanded to purify stem cells of specific lineages, such as neurons and cardiomyocytes. - [34] Mechanobiology: A new frontier for human pluripotent stem cells
Yubing Sun and Jianping Fu
Integrative Biology, vol. 5, pp. 450-457, 2013.
DOI: 10.1039/c2ib20256e. PMCID: PMC4116275.
Abstract | PDF | PubMed
Selected as the front cover story and HOT article for 2013, Issue 3 of Integrative Biology.
Top 10 Integrative Biology articles for the month of Jan. 2013.
Research on human pluripotent stem cells (hPSCs) has expanded rapidly over the last two decades, owing to the promises of hPSCs for applications in regenerative medicine, disease modeling, and developmental biology studies. A few promising studies have emerged in recent years to reveal some unique mechano-sensitive and -responsive properties of hPSCs and the effect of the physical aspects of the local cellular microenvironment on regulating hPSC behaviors. This Frontier Review is to highlight these recent studies of mechanobiology in hPSCs and to discuss the impact of advancing our understanding of mechanoregulation of hPSC behaviors on improving survival, self-renewal and differentiation of hPSCs using well-controlled synthetic micro/nanoscale cell culture tools. - [33] Micromachined elastomeric microposts and their applications for mechanotransduction research
Shinuo Weng, Yue Shao, Yubing Sun, and Jianping Fu
Dynamic Control of the Cellular Microenvironment (edited by Wendy Liu and Elliot Hui). Springer, 2013.
Abstract | PDF
We focus this book chapter on discussing a synthetic micromechanical system based on microfabricated elastomeric micropost arrays that have been proven ideal for studying functional roles of mechanical forces and substrate rigidity in regulating cellular behaviors and the mechanotransductive mechanisms involving cytoskeleton contractile forces. We first review the development of the elastomeric micropost array system, followed by a discussion of its applications in different mechanobiology studies. We will then discuss recent trends in integrating the elastomeric micropost array with other force actuation mechanisms as well as microfluidic tools to apply well-controlled external dynamical subcellular mechanical forces to cells, thus demonstrating the broad functionalities enabled by integrated elastomeric micropost array systems for studying cellular mechanotransduction in vitro. - [32] Surface-micromachined microfiltration membranes for efficient isolation and functional immunophenotyping of subpopulations of immune cells
Weiqiang Chen, Nien-Tsu Huang, Boram Oh, Raymond Hiu-Wai Lam, Rong Fan, Timothy T. Cornell, Thomas P. Shanley, Katsuo Kurabayashi, and Jianping Fu
Advanced Healthcare Materials, vol. 2, pp. 965-975, 2013.
DOI: 10.1002/adhm.201200378. PMCID: PMC4459734.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the front cover story for 2013 July issue of Advanced Healthcare Materials.
Highlighted by Materials View and UM ME Dept. News.
An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. In this work, we have developed a large surface micromachined poly-dimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we have demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Further, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay ("AlphaLISA"), has enabled us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples. - [31] Nanoroughened surfaces for efficient capture of circulating tumor cells without using capture antibodies
Weiqiang Chen, Shinuo Weng, Feng Zhang, Steven Allen, Liwei Bao, Raymond Hiu-Wai Lam, Jill A. Macoska, Sofia D. Merajver, and Jianping Fu
ACS Nano, vol. 7, pp. 566-575, 2013.
DOI: 10.1021/nn304719q. PMCID: PMC3962680.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the front cover story for 2013 Jan. issue of ACS Nano.
Highlighted by UM Homepage, UM News Service, UM Health System, Medicine @ Michigan, UM ME Dept. News, UM BME Dept. News, C&EN Online Story, ACS Nano Podcast, NSF News, Medical News Today, MediLexicon, ScienceDaily, PhysOrg, and Bio-Medicine.Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. In this work we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilizes the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus does not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques. - [30] Microfabricated nanotopological surfaces for study of adhesion-dependent cell mechanosensitivity
Weiqiang Chen, Yubing Sun, and Jianping Fu
Small, vol. 9, pp. 81-89, 2013.
DOI: 10.1002/smll.201201098. PMCID: PMC4113413.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the front cover story for 2013 Jan. issue of Small.
A simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces is reported here by using photolithography and reactive ion etching. Using these nanorough glass surfaces, we demonstrate that local nanoroughness as a biophysical cue can regulate a diverse array of NIH/3T3 fi broblast behaviors, including cell morphology, adhesion, proliferation, migration, and cytoskeleton contractility.
2012
- [29] Live-cell subcellular measurement of cell stiffness using a microengineered stretchable micropost array membrane
Raymond Hiu-Wai Lam, Shinuo Weng, Wei Lu, and Jianping Fu
Integrative Biology, vol. 4, pp. 1289-1298, 2012.
DOI: 10.1039/C2IB20134H. PMCID: PMC4088946.
Abstract | PDF | PubMedHere we report a completely new whole-cell cell stiffness measurement technique with a subcellular spatial resolution. This technique is based on a novel cell stretching device that allows for quantitative control and real-time measurements of mechanical stimuli and cellular biomechanical responses. A robust computation scheme is developed and implemented for subcellular quantifications of cell stiffness using the data of local cell stretching forces and cell area increments generated from the cell stretch assays. - [28] An integrated microfluidic platform for in-situ cellular cytokine secretion immunophenotyping
Nien-Tsu Huang, Weiqiang Chen, Boram Oh, Timothy T. Cornell, Thomas P. Shanley, Jianping Fu, and Katsuo Kurabayashi
Lab on a Chip, vol. 12, pp. 4093-4101, 2012.
DOI: 10.1039/C2LC40619E. PMCID: PMC3508001.
Abstract | PDF | Supplemental Materials | PubMedRapid, quantitative detection of cell-secreted biomarker proteins with a low sample volume holds great promise to advance cellular immunophenotyping techniques for personalized diagnosis and treatment of infectious diseases. Here we report such an assay using a highly integrated microfluidic platform incorporating a surface micromachined polydimethylsiloxane (PDMS) microfiltration membrane (PMM) and a no-wash bead-based chemiluminescence immunodetection scheme (AlphaLISA). - [27] UV-modulated substrate mechanics for multiscale study of mechanoresponsive cellular behaviors
Yubing Sun, Liang-Ting Jiang, Ryoji Okada, and Jianping Fu
Langmuir, vol. 28, pp. 10789-10796, 2012.
DOI: 10.1021/la300978x. PMCID: PMC4123631.
Abstract | PDF | Supplemental Materials | PubMedIn this work we have applied novel photosensitive polydimethylsiloxane (photoPDMS) chemistry to create photosensitive, biocompatible photoPDMS as a rigidity-tunable material for study of mechanoresponsive cellular behaviors. We have further fabricated photoPDMS micropost arrays for multiscale study of mechanoresponsive cellular behaviors. Our results have suggested that adherent cells could sense and respond to changes of substrate rigidity at a subfocal adhesion resolution. - [26] Microengineered synthetic cellular microenvironment for stem cells
Yubing Sun, Shinuo Weng, and Jianping Fu
Wiley Interdisciplinary Reviews (WIREs): Nanomedicine & Nanobiotechnology, vol. 4, pp. 414-427, 2012.
DOI: 10.1002/wnan.1175. PMCID: PMC4109891.
Abstract | PDF | PubMedPrecise control and regulation of the biochemical and biomechanical stimuli to stem cells have been successfully achieved using emerging micro/nanoengineering techniques. This review provides insights into how these micro/nanoengineering approaches, particularly microcontact printing and elastomeric micropost array, are applied to create dynamic and complex environment for stem cells culture. - [25] Matrix mechanics regulates fate decisions of human embryonic stem cells
Yubing Sun, Luis G Villa-Diaz, Raymond Hiu-Wai Lam, Weiqiang Chen, Paul H. Krebsbach, and Jianping Fu
PLoS ONE, vol. 7, e37178, 2012.
DOI: 10.1371/journal.pone.0037178. PMCID: PMC3353896.
Abstract | PDF | Supplemental Materials | PubMed
Highlighted by ESC & iPSC News, UM ME Dept News, and UM CoE News.The mechanoresponsive behaviors of human embryonic stem cells (hESCs) are elusive. We have demonstrated here that hESCs can increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates can support maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility may be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. - [24] Nanotopography influences adhesion, spreading, and self-renewal of human embryonic stem cells
Weiqiang Chen, Luis G Villa-Diaz, Yubing Sun, Shinuo Weng, Raymond Hiu-Wai Lam, Lin Han, Rong Fan, Paul H. Krebsbach, and Jianping Fu
ACS Nano, vol. 6, pp. 4094-4103, 2012.
DOI: 10.1021/nn3004923. PMCID: PMC3358529.
Abstract | PDF | Supplemental Materials | PubMed
Highlighted by ESC & iPSC News, C&EN Online Story, UM ME Dept News, and UM CoE News.Here we investigate the mechanosensitivity of human embryonic stem cells (hESCs) to nanotopography using nanorough glass surfaces. Our results demonstrate that nanotopography can provide a potent regulatory signal over different hESC behaviors including adhesion, spreading, and self-renewal. Our results indicate that topological sensing of hESCs may include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. - [23] Elastomeric microposts integrated into microfluidics for flow-mediated endothelial mechanotransduction analysis
Raymond Hiu-Wai Lam, Yubing Sun, Weiqiang Chen, and Jianping Fu
Lab on a Chip, vol. 12, pp. 1865-1873, 2012.
DOI: 10.1039/c2lc21146g. PMCID: PMC4120067.
Abstract | PDF | Supplemental Materials | PubMed
Selected for the May 1 2012 Issue of Virtual Journal of Biological Physical Research.Here we report an integrated soft lithography-compatible microfluidic methodology that can enable independent controls and modulations of fluid shear, substrate rigidity, and adhesive pattern in a microfluidic environment, by integrating micromolded elastomeric micropost arrays and microcontact printing with microfluidics. We have demonstrated its application to investigate quantitatively the involvement of cytoskeletal contractile forces in the flow-mediated mechanotransduction process of endothelial cells. - [22] Forcing stem cells to behave: A biophysical perspective of the cellular microenvironment
Yubing Sun, Christopher S. Chen, and Jianping Fu
Annual Review of Biophysics, vol. 41, pp. 519-542, 2012.
DOI: 10.1146/annurev-biophys-042910-155306. PMCID: PMC4123632.
Abstract | PDF | PubMedThis review highlights how stem cells sense and respond to the insoluble biophysical signals in the local cellular microenvironment. We also discuss the mechanotransduction processes and their functional crosstalk with other potent growth-factor-mediated signaling pathways to regulate stem cell fate. - [21] In silico experimentation of glioma microenvironment development and anti-tumor therapy
Yu Wu, Yao Lu, Weiqiang Chen, Jianping Fu, and Rong Fan
PLoS Computational Biology, vol. 8, e1002355, 2012.
DOI: 10.1371/journal.pcbi.1002355. PMCID: PMC3271023.
Abstract | PDF | Supplemental Materials | PubMedTumor cells co-evolve with stromal cells and tumor-associated immune cells in a tumor microenvironment. Here we report an unbiased, generic model to integrate prior biochemical data and the constructed brain tumor microenvironment in silico. Our model results show that glioma develops through three distinct phases: pre-tumor, rapid expansion, and saturation. We have designed a microglia depletion therapy and observed significant benefit for virtual patients treated at the early stages but strikingly no therapeutic efficacy at all when therapy is given at a slightly later stage. Cytokine combination therapy exhibits more focused and enhanced therapeutic response even when microglia depletion therapy already fails. Our model further reveals that the optimal combination depends on the molecular profile of individual patients. - [20] A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response
Jennifer M. Mann, Raymond Hiu-Wai Lam, Shinuo Weng, Yubing Sun, and Jianping Fu
Lab on a Chip, vol. 12, pp. 731-740, 2012.
DOI: 10.1039/c2lc20896b. PMCID: PMC4120061.
Abstract | PDF | Supplemental Materials | PubMed
Selected as one of the top 10% of all Lab on Chip articles published this year. See Lab on Chip Top 10%.Here we report a cell stretching device that allows for quantitative control and real-time measurement of mechanical stimuli and cellular biomechanical responses. Our strategy involves a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. This micropost array membrane (mPAM) can be activated to apply equibiaxial cell stretching forces to adherent cells attached to the microposts. Using the mPAM, we have studied the live-cell subcellular dynamic responses of contractile forces in vascular smooth muscle cells (VSMCs) to cell stretches. - [19] Photolithographic surface micromachining of polydimethylsiloxane (PDMS)
Weiqiang Chen, Raymond Hiu-Wai Lam, and Jianping Fu
Lab on a Chip, vol. 12, pp. 391-395, 2012.
DOI: 10.1039/c1lc20721k. PMCID: PMC4120064.
Abstract | PDF | Supplemental Materials | PubMed
Selected for the Jan. 9 2012 Issue of Virtual Journal of Nanoscale Science & Technology.
This paper was in Lab on Chip's top ten most accessed articles during the months of Nov. and Dec. of 2011.
Here we report a simple yet highly precise and repeatable surface micromachining method for polydimethylsiloxane (PDMS) using photolithography followed by reactive ion etching (RIE). Our method to achieve surface patterning of PDMS applies an oxygen plasma treatment to PDMS to activate its surface to overcome the challenge of poor photoresist adhesion on PDMS for photolithography.
2011
- [18] Nanofluidic devices for bioseparation
Pan Mao, and Jianping Fu
Nanoproteomics: Methods and Protocols (edited by Steven A. Toms and Robert J. Weil)
Methods in Molecular Biology, vol. 790, pp. 127-140, Humana Press, 2011.
Abstract | PDF | PubMedIn this book chapter, we detail the fabrication process of a two-dimensional nanofluidic filter array device and its implementation for rapid continuous-flow separation of biomolecules such as proteins. - [17] Synergistic regulation of cell function by matrix rigidity and adhesive pattern
Shinuo Weng, and Jianping Fu
Biomaterials, vol. 32, pp. 9584-9593, 2011.
DOI: 10.1016/j.biomaterials.2011.09.006. PMCID: PMC4120063.
Abstract | PDF | PubMedIn this work, we have generated a library of microfabricated polydimethylsiloxane (PDMS) micropost arrays to study the synergistic and independent effects of matrix rigidity and adhesive ECM pattern on mechanoresponsive behaviors of both NIH/3T3 fibroblasts and human umbilical vein endothelial cells (HUVECs). - [16] Cell shape and substrate rigidity both regulate cell stiffness
Shang-You Tee, Jianping Fu, Christopher S. Chen, and Paul A. Janmey
Biophysical Journal, vol. 100, pp. L25-27, 2011.
DOI: 10.1016/j.bpj.2010.12.3744. PMCID: PMC3043219.
Abstract | PDF | Supplemental Materials | PubMed
Selected by the Faculty of 1000 (F1000) for open peer evaluation; web link.Cells from many different tissues sense the stiffness and spatial patterning of their microenvironment to modulate their shape and cortical stiffness. It is currently unknown how substrate stiffness, cell shape, and cell stiffness modulate or interact with one another. Here, we use microcontact printing and microfabricated arrays of elastomeric posts to independently and simultaneously control cell shape and substrate stiffness. Our experiments show that cell cortical stiffness increases as a function of both substrate stiffness and spread area. - [15] Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity
Michael T. Yang, Jianping Fu, Yang-Kao Wang, Ravi A. Desai, and Christopher S. Chen
Nature Protocols, vol. 6, pp. 187-213, 2011.
DOI: 10.1038/nprot.2010.189.
Abstract | PDF | PubMedIn this protocol, we describe the design, fabrication, and use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function.
2010
- [14] Mechanical regulation of cell function with geometrically modulated elastomeric substrates
Jianping Fu, Yang-Kao Wang, Michael T. Yang, Ravi A. Desai, Xiang Yu, Zhijun Liu, and Christopher S. Chen
Nature Methods, vol. 7, pp. 733-736, 2010.
DOI: 10.1038/nmeth.1487. PMCID: PMC3069358.
Abstract | PDF | Supplemental Materials | PubMed
Selected as the front cover story.
Commentary by Amnon Buxboim & Dennis Discher, “Stem cell feels the difference”, Nature Methods, vol. 7, pp. 695-697, Aug. 2010. [web link | PDF]
Addendum, Nature Methods, vol. 8, pp.184, Feb. 2011. [web link | PDF]
Highlighted by UMich News Service, UMich ME Dept, Penn News, Popular Science, ScienceDaily, EurekAlert by AAAS, and PhysOrg.
We report here the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We have demonstrated that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility can predict later stem cell fate decisions in single cells.
2009
- [13] Continuous-flow bioseparation using microfabricated anisotropic nanofluidic sieving structures
Jianping Fu, Pan Mao, and Jongyoon Han
Nature Protocols, vol. 4, pp. 1681-1698, 2009.
DOI: 10.1038/nprot.2009.176. PMCID: PMC2896887.
Abstract | PDF | Supplemental Materials | PubMedIn this protocol we describe the fabrication of planar and vertical anisotropic nanofluidic-filter (nanofilter) array (ANA) chips and how to perform continuous-flow bioseparation using them. - [12] Simulation of the contractile response of cells on an array of micro-posts
Patrick J. McGarry, Jianping Fu, Michel T. Yang, Christopher S. Chen, Robert M. McMeeking, Vikram S. Deshpande, and Anthony G. Evans
Proceedings of the Royal Society A, vol. 367, pp. 3477-3497, 2009.
DOI: 10.1098/rsta.2009.0097.
Abstract | PDFIn this work, a bio-chemo-mechanical model has been developed and used to predict the contractile responses of smooth muscle cells on a bed of micro-posts. Predictions obtained for smooth muscle cells reveal that the model captures all of the following responses in a self-consistent manner: (i) the scaling of the force exerted by the cells with the number of posts; (ii) actin distributions within the cells, including the rings of actin around the micro-posts; (iii) the curvature of the cell boundaries between the posts; and (iv) the higher post forces towards the cell periphery. Consistent with measurements, the model predicts that the forces exerted by the cells will increase with both increasing post stiffness and cell area. - [11] Quantitative immunological detection of tumor-marker proteins in anisotropic nanofluidic sieving structures
Masumi Yamada, Pan Mao, Jianping Fu, and Jongyoon Han
Analytical Chemistry, vol. 81, pp. 7067-7074, 2009.
DOI: 10.1021/ac901226z. PMCID: PMC2846189.
Abstract | PDF | PubMedHere we demonstrate the quantitative analysis of disease-marker proteins by continuously separating the antibody-protein immunocomplexes from the unbound antibodies, utilizing the anisotropically patterned nanosieve array (ANA) structures. We have examined two proteins used as disease markers, human C-reactive protein (CRP) and human chorionic gonadotropin (hCG), by using fluorescent-labeled polyclonal antibodies. We have shown that the size of the immunocomplex and the field strength are the critical factors for the separation. Additionally, this device allows a convenient measurement of homogeneous binding kinetics, without the need for repeated binding experiments and immobilizing the molecules.
2008
- [10] Decreasing effective nanofluidic filter size by modulating electrical double layers: Separation enhancement in microfabricated nanofluidic filters
Hansen Bow, Jianping Fu, and Jongyoon Han
Electrophoresis, vol. 29, pp. 4646-4651, 2008.
DOI: 10.1002/elps.200800256.
Abstract | PDF | PubMedIn this work, we present an experimental investigation of a method that can be used in the nanofluidic sieves to increase separation selectivity and resolution. This method exploits the electrostatic repulsion between the charged molecules and the charged nanofluidic structure. - [9] Integrated electroplated heat spreaders for high power semiconductor lasers
Jianping Fu, Ronggui Yang, Gang Chen, Jean Pierre Fleurial, and G. Jeffrey Snyde
Journal of Applied Physics, vol. 104, 064907, 2008.
DOI: 10.1063/1.2986888.
Abstract | PDFIn this work, we demonstrate the use of a microfabricated laser test device to study the thermal management of edge emitting semiconductor lasers. The effects of various structural parameters of the heat spreader on the reduction of the thermal resistance of the laser test device are demonstrated both experimentally and theoretically. Without resolving to computational costive simulations, we have developed two independent analytical models to verify the experimental data and further utilized them to identify the dominant thermal resistance under different laser mounting configurations. - [8] Artificial molecular sieves and filters: a new paradigm for biomolecule separation
Jianping Fu, Pan Mao and Jongyoon Han
Trends in Biotechnology, vol. 26, pp. 311-320, 2008.
DOI: 10.1016/j.tibtech.2008.02.009.
Abstract | PDF | PubMedIn this review, we discuss recent developments of microfabricated nanofluidic sieves and filters that have demonstrated superior performance for both analytical and preparative separation of various physiologically relevant macromolecules, including proteins. The insights gained from designing these artificial molecular sieves and filters, along with the promising results gathered from their first applications, serve to illustrate the impact that they can have on improving future separation of complex biological samples. - [7] Engineering tissue form and function
Colette Shen, Jianping Fu, and Christopher S. Chen
Cellular and Molecular Bioengineering, vol. 1, pp. 15-23, 2008.
DOI: 10.1007/s12195-008-0005-y.
Abstract | PDFIn this review, we discuss available strategies that have been developed for spatially arranging cells within extracellular matrix (ECM) environments by patterning cell–ECM and cell–cell adhesion, soluble cues, and substrate mechanical propertie and how such strategies can subsequently affect cell and tissue function. - [6] Sample preparation by lab-on-a-chip devices
Jongyoon Han, Jianping Fu, Ying-Chih Wang, and Yong-Ak Song
Encyclopedia of Microfluidics and Nanofluidics (edited by Dongqing Li), Springer-Verlag, 2008.
Abstract | web linkIn this book chapter, we review state of the art micro/nanofluidic techniques for different biosample preparation appplications, including separation and preconcentration. - [5] Molecular sieving using nanofilters: past, present and future
Jongyoon Han, Jianping Fu, and Reto B. Schoch
Lab on a Chip, vol. 8, pp. 23-33, 2008.
DOI: 10.1039/B714128A. PMCID: PMC2365755.
Abstract | PDF | PubMedHere we review the important engineering question of microfiltration of molecules by nanometer-sized structures with well-defined sizes, shapes, and surface properties. Using these nanoscale molecular filters, we are well positioned to engineer better functionality in molecular sieving, separation and other membrane applications. We also review past theoretical developments and connect them to the most recent advances in the field.
2000 - 2007
- [4] A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and protein
Jianping Fu, Reto B. Schoch, Anna L. Stevens, Steven R. Tannenbaum, and Jongyoon Han
Nature Nanotechnology, vol. 2, pp. 121-128, 2007.
DOI: 10.1038/nnano.2006.206. PMCID: PMC2621439.
Abstract | PDF | Supplemental Materials | PubMed
Cover story; Commentary by Robert Austin, “Nanofluidics: a fork in the nano-road", Nature Nanotechnology, vol. 2, pp. 121-128, 2007. [web link | PDF]
Highlighted by MIT News Office / MIT Tech Talk, ScienceDaily, EurekAlert by AAAS, and PhysOrg.com.We report here a microfabricated anisotropic sieving structure consisting of a two-dimensional periodic nanofluidic filter array. The designed structural anisotropy causes different-sized or -charged biomolecules to follow distinct trajectories, leading to efficient separation. Continuous-flow size-based separation of DNA and proteins, as well as electrostatic separation of proteins, have been achieved, demonstrating the potential use of this device as a generic molecular sieving structure for an integrated biomolecule sample preparation and analysis system. - [3] Molecular sieving in periodic free-energy landscapes created by patterned nanofilter arrays
Jianping Fu, Juhwan Yoo, and Jongyoon Han
Physical Review Letters, vol. 97, 018103, 2006.
DOI: 10.1103/PhysRevLett.97.018103. PMCID: PMC1752241.
Abstract | PDF | PubMed
Selected for the July 15 2006 issue of Virtual Journal of Biological Physical Research and for the July 24 2006 issue of Virtual Journal of Nanoscale Science and Technology.
Highlighted by MIT News Office.We present an experimental study of Ogston-like sieving process of rod-like DNA in patterned periodic nanofluidic filter arrays. The electrophoretic motion of DNA through the array is described as a biased Brownian motion overcoming periodically modulated free-energy landscape. A kinetic model, constructed based on the equilibrium partitioning theory and the Kramers theory, explains the field-dependent mobility well. - [2] Nanofilter array chip for fast gel-free biomolecule separation
Jianping Fu, Pan Mao, and Jongyoon Han
Applied Physics Letters, vol. 87, 263902, 2005.
DOI: 10.1063/1.2149979. PMCID: PMC2564606.
Abstract | PDF | PubMed
Selected for the Jan. 1 2006 issue of Virtual Journal of Biological Physical ResearchWe report here a microfabricated anisotropic sieving structure consisting of a two-dimensional periodic nanofluidic filter array. The designed structural anisotropy causes different-sized or -charged biomolecules to follow distinct trajectories, leading to efficient separation. Continuous-flow size-based separation of DNA and proteins, as well as electrostatic separation of proteins, have been achieved, demonstrating the potential use of this device as a generic molecular sieving structure for an integrated biomolecule sample preparation and analysis system.We report here a microfabricated nanofilter array chip that can fractionate denatured protein complexes and small DNA molecules based on Ogston sieving. Nanofilter arrays with a gap size of 40 - 180 nm have been fabricated and characterized. Complete separation of protein complexes and small DNA molecules can be achieved in several minutes with a separation length of 5 mm. - [1] Numerical simulation and experiment research on flow characteristics of capillary in refrigerator
Chaoguang Lin, Peng Hu, Zeshao Chen, Wenlong Chen, and Jianping Fu
Heat Transfer Science and Technology (edited by Buxuan Wang), pp. 555-562, Higher Education Press of China, 2000.
Conference Proceedings
- [18] Xiang Li, Weiqiang Chen, Guangyu Liu, Wei Lu, and Jianping Fu, "Continuous-flow microfluidic blood cell sorting for unprocessed whole blood using surface-micromachined microfiltration membranes", Proc. 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2014), San Antonio, Texas, Oct. 2014, pp. 1151-1153. (PDF)
- [17] Weiqiang Chen, Nien-Tsu Huang, Boram Oh, Timothy T. Cornell, Thomas P. Shanley, Katsuo Kurabayashi, and Jianping Fu, "Microfluidic immunophenotyping assay platform for immunomonitoring of subpopulations of immune cells", Proc. 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2013), Freiburg, Germany, Oct. 2013, pp. 1761-1763. (PDF)
- [16] Bo-Ram Oh, Nien-Tsu Huang, Weiqiang Chen, Jungwhan Seo, Jianping Fu, and Katsuo Kurabayashi, "Localized surface plasmon resonance (LSPR) optofluidic biosensor for label-free cellular immunophenotyping", Proc. 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2013), Freiburg, Germany, Oct. 2013, pp. 92-94. (PDF)
- [15] Yubing, Sun, Luis G. Villa-Diaz, Raymond Hiu-Wai Lam, Weiqiang Chen, Paul H. Krebsbach, and Jianping Fu, "Micromechanical elastomeric devices for investigations of mechanobiology in human embryonic stem cells", Proc. 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2012), Okinawa, Japan, Oct. 2012, pp. 1714-1716. (PDF)
- [14] Weiqiang Chen, Nien-Tsu Huang, Katsuo Kurabayashi, and Jianping Fu, "Surface micromachining of polydimethylsiloxane (PDMS) for microfluidic biomedical applications", Proc. 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2012), Okinawa, Japan, Oct. 2012, pp. 1849-1851. (PDF)
- [13] Nien-Tsu Huang, Weiqiang Chen, Boram Oh, Jianping Fu, and Katsuo Kurabayashi, "An integrated microfluidic platform for in-situ cellular cytokine secretion immunophenotyping", Proc. 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2012), Okinawa, Japan, Oct. 2012, pp. 989-991. (PDF)
- [12] Jennifer M. Mann, Raymond Hiu-Wai Lam, Yubing Sun, Shinuo Weng, and Jianping Fu, “A microengineered stretching platform for live-cell mechanotransductive response analysis”, Proc. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2011), Seattle, USA, Oct. 2011, pp. 9-11. (PDF)
- [11] Shinuo Weng, and Jianping Fu, “Synergistic regulation of cell functions by matrix rigidity and adhesive pattern using an elastomeric micropost array system”, Proc. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2011), Seattle, USA, Oct. 2011, pp. 157-159. (PDF)
- [10] Weiqiang Chen, Yubing Sun, and Jianping Fu, “Nanotopographic control of human embryonic stem cell function”, Proc. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2011), Seattle, USA, Oct. 2011, pp. 36-38. (PDF)
- [9] Masumi Yamada, Pan Mao, Jianping Fu, and Jongyoon Han, “Continuous-flow immunoseparation and rapid quantification of protein binding kinetics in anisotropically-patterned nano-sieve structures”, Proc. 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2009), Jeju, Korea, Nov. 2009.
- [8] Jianping Fu, Yang-Kao Wang, Michael T. Yang, Ted T. Lee, and Christopher S. Chen, “Mechanical control of stem cell differentiation using micro-engineered matrix”, Proc. 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2008), San Diego, California USA, Oct. 2008, pp. 1229-1231. (PDF)
- [7] Jianping Fu, and Jongyoon Han, “Nanofluidic devices for rapid analysis of DNA and proteins”, 2007 Digest of the IEEE/LEOS Summer Topical Meeting, Portland, Oregon, July 2007, pp. 115-116. (PDF)
- [6] Jianping Fu, and Jongyoon Han, “Continuous-flow biomolecule separation through patterned anisotropic nanofluidic sieving structure”, Proc. 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2006), Tokyo, Japan, Nov. 2006, pp. 519-521. (PDF)
- [5] Jianping Fu, and Jongyoon Han, “A nanofilter array chip for fast gel-free biomolecule separation”, Proc. 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2005), Boston, Massachusetts USA, Oct. 2005, pp. 1531-1533. (PDF)
- [4] Ying-Chih Wang, Jianping Fu, Pan Mao, and Jongyoon Han, “Nanofluidic molecular filters for efficient protein separation and preconcentration”, Proc. 13th International Conference on Solid-State Sensors, Actuators and Microsystems (Transducers'05), Seoul, Korea, June 2005, pp. 352-355. (PDF)
- [3] Jongyoon Han, and Jianping Fu, “Biomolecule Separation by steric hindrance using nanofluidic filters”, Proc. 26th IEEE-EMBS conference, San Francisco, California USA, Sept. 2004, pp. 2611-2614. (PDF)
- [2] Jianping Fu, and Jongyoon Han, “Biomolecule separation in nanofluidic filters by steric hindrance mechanism”, Proc. 8th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2004), Malmö, Sweden, Sept. 2004, pp. 285-287. (PDF)
- [1] Jianping Fu, Ronggui Yang, Gang Chen, Jean Pierre Fleurial, and Jeffrey G. Snyder, “Integrated electroplated heat spreader for high power semiconductor laser”, Proc. 6th ASME-JSME Thermal Engineering Joint Conference, Hawaii Island, Hawaii USA, March 2003, pp. 332-337.