Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells


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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Soft substrates promote neuroepithelial conversion while inhibiting neural crest differentiation of hESCs in a BMP4-dependent manner.
Figure 2: Purity and yield of functional motor neurons (MNs) derived from hESCs are improved on soft substrates.
Figure 3: Soft substrates promote hESC neuroepithelial conversion through a multi-targeted mechanotransductive process involving mechanosensitive Smad phosphorylation and nucleocytoplasmic shuttling regulated by rigidity-dependent Hippo/YAP activities and the actomyosin cytoskeleton.

Similar content being viewed by others


  1. Discher, D. E., Mooney, D. J. & Zandstra, P. W. Growth factors, matrices, and forces combine and control stem cells. Science 324, 1673–1677 (2009).

    CAS  Google Scholar 

  2. Li, X. J. et al. Specification of motoneurons from human embryonic stem cells. Nature Biotechnol. 23, 215–221 (2005).

    Article  Google Scholar 

  3. Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of Smad signaling. Nature Biotechnol. 27, 275–280 (2009).

    Article  CAS  Google Scholar 

  4. Fu, J. P. et al. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nature Methods 7, 733–736 (2010).

    Article  CAS  Google Scholar 

  5. Keung, A. J., Asuri, P., Kumar, S. & Schaffer, D. V. Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells. Integr. Biol. 4, 1049–1058 (2012).

    Article  CAS  Google Scholar 

  6. Hitoshi, S. et al. Primitive neural stem cells from the mammalian epiblast differentiate to definitive neural stem cells under the control of Notch signaling. Genes Dev. 18, 1806–1811 (2004).

    Article  CAS  Google Scholar 

  7. Patani, R. et al. Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state. Nature Commun. 2, 214 (2011).

    Article  CAS  Google Scholar 

  8. Bean, B. P. The action potential in mammalian central neurons. Nature Rev. Neurosci. 8, 451–465 (2007).

    Article  CAS  Google Scholar 

  9. Miles, G. B., Dai, Y. & Brownstone, R. M. Mechanisms underlying the early phase of spike frequency adaptation in mouse spinal motoneurones. J. Physiol. 566, 519–532 (2005).

    Article  CAS  Google Scholar 

  10. Karumbayaram, S. et al. Human embryonic stem cell-derived motor neurons expressing SOD1 mutants exhibit typical signs of motor neuron degeneration linked to ALS. Dis. Model. Mech. 2, 189–195 (2009).

    Article  CAS  Google Scholar 

  11. Hester, M. E. et al. Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes. Mol. Ther. 19, 1905–1912 (2011).

    Article  CAS  Google Scholar 

  12. Derynck, R. & Zhang, Y. E. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 425, 577–584 (2003).

    Article  CAS  Google Scholar 

  13. Varelas, X. et al. TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal. Nature Cell Biol. 10, 837–848 (2008).

    Article  CAS  Google Scholar 

  14. Zhao, B. et al. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 21, 2747–2761 (2007).

    Article  CAS  Google Scholar 

  15. Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).

    Article  CAS  Google Scholar 

  16. Wada, K. et al. Hippo pathway regulation by cell morphology and stress fibers. Development 138, 3907–3914 (2011).

    Article  CAS  Google Scholar 

  17. Yu, F. X. et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 150, 780–791 (2012).

    Article  CAS  Google Scholar 

  18. Calvo, F. et al. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nature Cell Biol. 15, 637–646 (2013).

    Article  CAS  Google Scholar 

  19. Zhao, B. et al. Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev. 26, 54–68 (2012).

    Article  Google Scholar 

  20. Chang, T-C. et al. Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density–dependent manner. PLoS ONE 5, e9187 (2010).

    Article  Google Scholar 

  21. Krawetz, R. J. et al. Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells. PLoS ONE 6, e26484 (2011).

    Article  CAS  Google Scholar 

  22. Garcia-Gonzalo, F. R. & Izpisua Belmonte, J. C. Albumin-associated lipids regulate human embryonic stem cell self-renewal. PLoS ONE 3, e1384 (2008).

    Article  Google Scholar 

  23. Blauwkamp, T. A. et al. Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors. Nature Commun. 3, 1070 (2012).

    Article  Google Scholar 

  24. Visser-Grieve, S. et al. Lats1 tumor suppressor is a novel actin-binding protein and negative regulator of actin polymerization. Cell Res. 21, 1513–1516 (2011).

    Article  CAS  Google Scholar 

  25. Du, J. et al. Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity. Proc. Natl Acad. Sci. USA 108, 9466–9471 (2011).

    Article  CAS  Google Scholar 

  26. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

  27. Connelly, J. T. et al. Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions. Nature Cell Biol. 12, 711–718 (2010).

    Article  CAS  Google Scholar 

  28. Gilbert, P. M. et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).

    Article  CAS  Google Scholar 

  29. Holst, J. et al. Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. Nature Biotechnol. 28, 1123–1128 (2010).

    Article  CAS  Google Scholar 

  30. Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nature Mater. 9, 518–526 (2010).

    Article  CAS  Google Scholar 

Download references


This work is supported by the National Science Foundation (CMMI 1129611 and CBET 1149401 to J.F.), the National Institutes of Health (1R21HL114011 to J.F.; 2R01DE016530-06 to P.H.K.; R01NS062792 and R01AR060837 to H.X.), the American Heart Association (12SDG12180025 to J.F.), and the Department of Mechanical Engineering at the University of Michigan, Ann Arbor. The Lurie Nanofabrication Facility at the University of Michigan, a member of the National Nanotechnology Infrastructure Network (NNIN) funded by the National Science Foundation, is acknowledged for support in microfabrication.

Author information

Authors and Affiliations



Y.S. and J.F. designed experiments; Y.S. performed in vitro differentiation experiments and Western blotting; K.M.A.Y., L.G.V-D., and Y.S. generated and analysed gene expression data; K.M.A.Y. and Y.S. performed siRNA transfection; X.Z. performed electrophysiology measurements; L.G.V-D. derived hiPSCs; W.C. fabricated silicon masters; S.W. developed image processing program; Y.S., L.G.V-D., R.P., H.X., P.H.K. and J.F. analysed data and wrote the manuscript; J.F. supervised the project. All authors edited and approved the final manuscript.

Corresponding author

Correspondence to Jianping Fu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 3508 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, Y., Yong, K., Villa-Diaz, L. et al. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nature Mater 13, 599–604 (2014).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing