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

Abstract

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.

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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.

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Acknowledgements

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

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.

Correspondence to Jianping Fu.

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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). https://doi.org/10.1038/nmat3945

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