Synthetic Embryology: Post-Implantation Development

Synthetic Embryology: Neural System Development

Mechanobiology of Human Pluripotent Stem Cells

Cell Mechanics and Mechanotransduction

Microfluidic Systems Immunology for Transformative Diagnostics

Nanofluidics for Single Molecule Analysis

 

Synthetic Embryology: Post-Implantation Development

Human post-implantation development remains mysterious. Recent advances in the development of human embryo models (or embryoids) from human pluripotent stem cells (hPSCs) have sparked great interest in using such models to advance human embryology. In this research, we have leveraged the developmental potential and self-organizing property of hPSCs in conjunction with biomimetic culture systems to develop different embryoid systems to study the early post-implantation human development. Specifically, we have developed the first hPSC-based embryoid system that recapitulates successive embryogenic events during the early post-implantation human development, including amniotic cavity formation, amnion-epiblast patterning, specification of primordial germ cells, and onset of the gastrulation. Together, our findings provide insight into previously inaccessible but critical embryogenic events in the post-implantation human development.


During the first three weeks of human embryo development, a few key embryogenic events occur, including blastocyst formation, implantation and gastrulation. Gastrulation is the fundamental organizational event that generates the basic body plan and provides the building blocks for all the tissues in the human embryo.

References:

  1. 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. Human primordial germ cells are specified from lineage-primed progenitors. Cell Reports, vol. 29, pp. 4568-4582, 2019. [PDF]
  2. 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. Controlled modeling of human epiblast and amnion development using stem cells. Nature, vol. 573, pp. 421-425, 2019. [PDF]
  3. 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. Commentary: Debate ethics of embryo models from stem cells. Nature, vol. 564, pp. 183-185, 2018. [PDF]
  4. 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. Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells. Nature Materials, vol. 17, pp. 633-641, 2018. [PDF | Supplemental Materials]
  5. Yue Shao, Kenichiro Taniguchi, Ryan F. Townshend, Toshio Miki, Deborah L. Gumucio, and Jianping Fu. A pluripotent stem cell-based model for post-implantation human amniotic sac development. Nature Communications, vol. 8, 208, 2017. [PDF | Supplemental Materials]
  6. 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. Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche. Nature Materials, vol. 16, pp. 419-425, 2017. [PDF | Supplemental Materials]
  7. 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. Lumen formation is an intrinsic property of isolated human pluripotent stem cells. Stem Cell Reports, vol. 5, pp. 954-962, 2015. [PDF | Supplemental Materials]

Synthetic Embryology: Neural System Development

Neurulation is a key developmental process that gives rise to the neural tube (NT), the embryonic precursor to the central nervous system (CNS). Neurulation is initiated after the gastrulation by a neural induction process, during which the dorsal ectoderm is specified into a patterned tissue containing the neural plate (NP) and the non-neural ectoderm (NNE) separated by the neural plate border (NPB). After neural induction, the NP folds and fuses to form the tubular NT. The development of the NT continues with differentiation of distinct classes of neuronal progenitor cells located at defined positions within the NT along both the anterior-posterior (AP) and dorsal-ventral (DV) axes. Recently, we have leveraged the developmental potential and self-organization property of human pluripotent stem cells (hPSCs) in conjunction with 2D and 3D bioengineering tools to achieve the development of patterned multicellular tissues that mimic certain aspects of early human neurulation process, including neural induction and DV patterning of the NT. These stem cell-derived models offer promising trackable systems to study neural development and disease.


Stem cell-based models of human neural system development. (left) Neurulation in vertebrate embryos. (top right) hPSC-based model of neural induction. (bottom right) hPSC-based, dorsal-ventral patterned neural tube model.

References:

  1. Yuanyuan Zheng, Xufeng Xue, Agnes M. Resto Irizarry, Zida Li, Yue Shao, Yi Zheng, Gang Zhao, and Jianping Fu. Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche. Science Advances, vol. 5, eaax5933, 2019. [PDF | Supplemental Materials]
  2. 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. Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells. Nature Materials, vol. 17, pp. 633-641, 2018. [PDF | Supplemental Materials]
  3. 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. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nature Materials, vol. 13, pp. 599-604, 2014. [PDF | Supplemental Materials

Mechanobiology of Human Pluripotent Stem Cells

Research on human pluripotent stem cells (hPSCs) has expanded rapidly over the last two decades, owing to the promise of hPSCs for regenerative medicine, disease modeling, and developmental biology studies. In our research, we have uniquely focused on a high-risk, high-payoff concept to investigate an emerging functional connection between mechanobiology and some critical questions in the field of hPSCs, including pluripotency, directed differentiation, cell reprogramming and transdifferentiation, and functional maturation. We are also exploring intracellular molecular mechanisms underlying mechanosensitive properties of hPSCs. Our research on mechanobiology of hPSCs will potentially enable drastic advances in large-scale production of hPSCs and their derivatives and contribute significantly to future cell-based regenerative therapies and disease modeling. So far, our research has unambiguously unraveled the mechanosensitive properties of hPSCs and their roles in directed neural differentiation and subtype specification. Our mechanistic work has further led to the discovery of an intervened regulatory network emerged from converging and reinforcing signal integration of TGF-β, WNT, Hippo, Rho-GTPase, and the actomyosin cytoskeleton that forms a molecular framework required for contextual, integrated responses of hPSCs.


Micro/nanoengineered ex vivo stem cell niche

References:

  1. 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. Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells. Nature Materials, vol. 17, pp. 633-641, 2018. [PDF | Supplemental Materials]
  2. 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. Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche. Nature Materials, vol. 16, pp. 419-425, 2017. [PDF | Supplemental Materials]
  3. Yubing Sun and Jianping Fu. Harnessing mechanobiology of human pluripotent stem cells for regenerative medicine. ACS Chemical Neuroscience, vol. 5, pp. 621-623, 2014. [PDF]
  4. 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. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nature Materials, vol. 13, pp. 599-604, 2014. [PDF | Supplemental Materials
  5. Yubing Sun and Jianping Fu. Mechanobiology: A new frontier for human pluripotent stem cells. Integrative Biology, vol. 5, pp. 450-457, 2013. [PDF]
  6. Yubing Sun, Luis G Villa-Diaz, Raymond Hiu-Wai Lam, Weiqiang Chen, Pasul H. Krebsbach, and Jianping Fu. Matrix mechanics regulates fate decisions of human embryonic stem cells. PLoS ONE, vol. 7, e37178, 2012. [PDF]
  7. Weiqiang Chen, Luis G Villa-Diaz, Yubing Sun, Shinuo Weng, Raymond Hiu-Wai Lam, Lin Han, Rong Fan, Paul H. Krebsbach, and Jianping Fu. Nanotopography influences adhesion, spreading, and self-renewal of human embryonic stem cells. ACS Nano, vol. 6, pp. 4094-4103, 2012. [PDF | supplemental materials]

Cell Mechanics and Mechanotransduction

External forces and matrix mechanics play a key role in the regulation of cell function. Cells sense and response to external forces and changes in matrix mechanics by modulating their endogenous cytoskeleton (CSK) contractility, balanced by external forces or resistant forces generated by the deformation of the extracellular matrix (ECM). Dysregulation of the tensional homeostasis in cells contributes to atherosclerosis, osteoarthritis and osteoporosis, and cancer. To aid in the mechanistic investigation of mechanotransduction, we have developed different micromechanical tools that allow for quantitative controls and real-time measurements of mechanical stimuli and cellular biomechanical responses. Our micromechanical tools are particularly useful for investigations of mechanotransduction centering on the ECM-integrin-CSK signaling axis to generate quantitative descriptions of the functional relations between matrix mechanics, external forces, CSK contractility, cell stiffness, and adhesion signaling and morphogenesis.


Micromechanical tools for precise control and measurement of mechanical stimuli and responses

References:

  1. Shinuo Weng, Yue Shao, Weiqiang Chen, and Jianping Fu. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis. Nature Materials, vol. 15, pp. 961-967, 2016. [PDF | Supplemental Materials]
  2. Di Chen, Yubing Sun, Madhu S. R. Gudur, Yising Hsiao, Ziqi Wu, Jianping Fu, and Cheri X. Deng. Two bubble acoustic tweezing cytometry for biomechanical probing and stimulation of cells. Biophysical Journal, vol. 108, pp. 32-42, 2015. [PDF]
  3. Yue Shao, Jennifer M. Mann, Weiqiang Chen, and Jianping Fu. Global architecture of F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction. Integrative Biology, vol. 6, pp. 300-311, 2014. [PDF | Supplemental Materials]
  4. Zhenzhen Fan, Yubing Sun, Di Chen, Donald Tay, Weiqiang Chen, Cheri X. Deng, and Jianping Fu. Acoustic tweezing cytometry for live-cell subcellular control of intracellular cytoskeleton contractility. Scientific Reports, vol. 3, 2176, 2013. [PDF | Supplemental Materials]
  5. Raymond Hiu-Wai Lam, Shinuo Weng, Wei Lu, and Jianping Fu. Live-cell subcellular measurement of cell stiffness using a microengineered stretchable micropost array membrane. Integrative Biology, vol. 4, pp. 1289-1298, 2012. [PDF]
  6. Raymond Hiu-Wai Lam, Yubing Sun, Weiqiang Chen, and Jianping Fu. Elastomeric microposts integrated into microfluidics for flow-mediated endothelial mechanotransduction analysis. Lab on a Chip, vol. 12, pp. 1865-1873, 2012. [PDF | supplemental materials]
  7. Jennifer M. Mann, Raymond Hiu-Wai Lam, Shinuo Weng, Yubing Sun, and Jianping Fu. A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response. Lab on a Chip, vol. 12, pp. 731-740, 2012. [PDF | supplemental materials]
  8. Shang-You Tee, Jianping Fu, Christopher S. Chen, and Paul A. Janmey. Cell shape and substrate rigidity both regulate cell stiffness. Biophysical Journal, vol. 100, pp. L25-27, Mar. 2011. [PDF | supplemental materials]
  9. Michael T. Yang, Jianping Fu, Yang-Kao Wang, Ravi A. Desai, and Christopher S. Chen. Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity. Nature Protocols, vol. 6, pp. 187-213, 2011. [PDF]
  10. Jianping Fu, Yang-Kao Wang, Michael T. Yang, Ravi A. Desai, Xiang Yu, Zhijun Liu, and Christopher S. Chen. Mechanical regulation of stem cell function using geometrically modulated elastomeric substrates. Nature Methods, vol. 7, pp.733-736, 2010. [PDF | supplemental materials]

Microfluidic Systems Immunology for Transformative Diagnostics

We are interested in developing highly integrated, automated, multiplexed microfluidic molecular and functional phenotyping tools for high-dimensional, high-resolution, rapid analysis of immune cells isolated directly from human biological specimens such as blood. Such systems immunology research will allow studying human immunity as a whole functional system and elucidating molecular and cellular metrics that define human immune system in health and disease, valuable for advancing fundamental understanding of human immunity and equally important for standardized immune monitoring in healthy as well as disease conditions such as allergy, asthma, autoimmunity, acquired and primary immunodeficiency, transplantation, and infection. As a first step towards this direction, we have successfully developed an integrated microfluidic immunomonitoring platform that can perform rapid, accurate, and sensitive cellular functional assays down to the single-cell level on different types or subpopulations of immune cells. Given shortened assay time, enhanced sample efficiency, and the ability to determine functional status of different subpopulations of immune cells simultaneously, our microfluidic immunomonitoring platform can serve as a standardized immunoassay to provide transformative clinical diagnostics for immune diseases. 


Functional immunophenotyping of subpopulations of immune cells using microfluidics

References:

  1. Andrew Stephens, Robert Nidetz, Nicolas Mesyngier, Meng Ting Chung, Yujing Song, Jianping Fu, and Katsuo Kurabayashi. Mass-producible microporous silicon membranes for specific leukocyte subset isolation, immunophenotyping, and personalized immunomodulatory drug screening in vitro. Lab on a Chip, vol. 19, pp. 3065-3076, 2019. [PDF | Supplemental Materials]
  2. Yujing Song, Pengyu Chen, Meng Ting Chung, Robert Nidetz, Younggeun Park, Zhenhui Liu, Walker McHugh, Timothy T. Cornell, Jianping Fu, and Katsuo Kurabayashi. AC electroosmosis-enhanced nanoplasmofluidic detection of ultralow-concentration cytokine. Nano Letters, vol. 17, pp. 2374-2380, 2017. [PDF | Supplemental Materials]
  3. Zeta Tak For Yu, Jophin George Joseph, Shirley Xiaosu Liu, Mei Ki Cheung, Katsuo Kurabayashi, and Jianping Fu. Centrifugal microfluidics for sorting immune cells from whole blood. Sensors & Actuators: B. Chemical, vol. 245, pp. 1050-1061, 2017. [PDF | Supplemental Materials]
  4. Bo-Ram Oh, Pengyu Chen, Robert Nidetz, Walker McHugh, Jianping Fu, Thomas P. Shanley, Timothy T. Cornell, and Katsuo Kurabayashi. Multiplexed nanoplasmonic temporal profiling of T-cell response under immunomodulatory agent exposure. ACS Sensors, vol. 1, pp. 941-948, 2016. [PDF | Supplemental Materials]
  5. Zeta Tak For Yu, Mei Ki Cheung, Shirley Xiaosu Liu, and Jianping Fu. Accelerated biofluid filling in complex microfluidic networks by vacuum-pressure accelerated movement (V-PAM). Small, vol. 12, pp. 4521-4530, 2016. [PDF]
  6. Zeta Tak-For Yu, Huijiao Guan, Mei Ki Cheung, Walker McHugh, Timothy T. Cornell, Thomas P. Shanley, Katsuo Kurabayashi, and Jianping Fu. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA. Scientific Reports, vol. 5, 11339, 2015. [PDF]
  7. Pengyu Chen, Meng Ting Chuang, Walker McHugh, Robert Nidetz, Yuwei Li, Jianping Fu, Timothy T. Cornell, Thomas P. Shanley, and Katsuo Kurabayashi. Multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays. ACS Nano, vol. 9, pp. 4173-4181, 2015. [PDF | Supplemental Materials]
  8. Xiang Li, Weiqiang Chen, Zida Li, Ling Li, Hongchen Gu, and Jianping Fu. Emerging microengineering tools for functional analysis and phenotyping of blood cells. Trends in Biotechnology, vol. 32, pp. 586-594, 2014. [PDF]
  9. 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. Lab on a Chip, vol. 14, pp. 2565-2575, 2014. [PDF | Supplemental Materials]
  10. Bo-Ram Oh, Nien-Tsu Huang, Weiqiang Chen, Jungwhan Seo, Pengyu Chen, Timothy T. Cornell, Thomas P. Shanley, Jianping Fu, and Katsuo Kurabayashi. Integrated nanoplasmonic sensing for cellular functional immunoanalysis using human blood. ACS Nano, vol. 8, pp. 2667-2676, 2014. [PDF | Supplemental Materials]
  11. Weiqiang Chen, Nien-Tsu Huang, Xiang Li, Zeta Tak-For Yu, Katsuo Kurabayashi, and Jianping Fu. Emerging microfluidic tools for cellular functional immunophenotyping: A new potential paradigm for immune status characterization. Frontiers in Oncology, vol. 3, 98, 2013. [PDF]
  12. Weiqiang Chen, Nien-Tsu Huang, Boram Oh, Raymond Hiu-Wai Lam, Rong Fan, Timothy T. Cornell, Thomas P. Shanley, Katsuo Kurabayashi, and Jianping Fu. Surface-micromachined microfiltration membranes for efficient isolation and functional immunophenotyping of subpopulations of immune cells. Advanced Healthcare Materials, vol. 2, pp. 965-975, 2013. [PDF | Supplemental Materials]
  13. Nien-Tsu Huang, Weiqiang Chen, Timothy Cornell, Thomas Shanley, Jianping Fu, and Katsuo Kurabayashi. An integrated microfluidic platform for in-situ cellular cytokine secretion immunophenotyping. Lab on a Chip, vol. 12, pp. 4093-4101, 2012. [PDF | supplemental materials]
  14. Weiqiang Chen, Raymond Hiu-Wai Lam, and Jianping Fu. Photolithographic surface micromachining of polydimethylsiloxane (PDMS). Lab on a Chip, vol. 12, pp. 391-395, 2012. [PDF | supplemental materials]

Nanofluidics for Single Molecule Analysis

Direct analysis of biologically-relevant entities such as nucleic acids and proteins offers the potential to outperform conventional analysis techniques and diagnostic methods through enhancements in speed, accuracy, and sensitivity. Moreover, direct biomolecule observations and manipulations help investigators probe fundamental molecular processes in biochemistry and biophysics that are often obscured in ensemble assays. Nanofluidic systems with critical dimensions comparable to the molecular scale open up new possibilities for direct observation, manipulation, and analysis of biomolecules, thus providing a novel basis for ultra-sensitive and high-resolution sensors and medical diagnostic systems. Inspired by this concept, we have developed integrated techniques to investigate biomolecules confined in micro/nanofluidic environments. We have studied molecular interactions with nanofluidic structures and explored the opportunity of using nanofluidic devices for ultra-sensitive biosensing and detection.


Nanofluidics for biosensing and detection

References:

  1. Jianping Fu, Pan Mao, and Jongyoon Han. Artificial molecular sieves and filters: a new paradigm for biomolecule separation. Trends in Biotechnology, vol. 26, pp.311-320, 2008. [PDF]
  2. Jongyoon Han, Jianping Fu, and Reto B. Schoch. Molecular sieving using nanofilters: Past, present and future. Lab on a Chip, vol. 8, pp.23-33, 2008. [PDF]
  3. Masumi Yamada, Pan Mao, Jianping Fu, and Jongyoon Han. Quantitative immunological detection of tumor-marker proteins in anisotropic nanofluidic sieving structures. Analytical Chemistry, vol. 81, pp.7067-7074, 2009. [PDF]
  4. Jianping Fu, Reto B. Schoch, Anna L. Stevens, Steven R. Tannenbaum, and Jongyoon Han. A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins. Nature Nanotechnology, vol. 2, pp.121-128, 2007. [PDF | supplemental materials]
  5. Jianping Fu, Juhwan Yoo, and Jongyoon Han. Molecular sieving in periodic free-energy landscapes created by patterned nanofilter arrays. Physical Review Letters, vol. 97, 018103, 2006. [PDF]
  6. Jianping Fu, Pan Mao, and Jongyoon Han. Nanofilter array chip for fast gel-free biomolecule separation. Applied Physics Letters, vol. 87, 263902, 2005. [PDF]