IBBL Publications

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 | PubMed
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.

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