Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific and as such, we could generate a spatially segregated co-culture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.

We report a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), which sustains long-term human embryonic stem (hES) cell growth in several different culture media, including commercially available defined media. The development of a standardized, controllable and sustainable culture matrix for hES cells is an essential step in elucidating mechanisms that control hES cell behavior and in optimizing conditions for biomedical applications of hES 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 (MNs) derived from hPSCs within 23 days of culture using soft PMAs were improved more than 4- and 10-fold, respectively, compared to 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.