Although human pluripotent stem cells (hPSCs) can be used to regenerate neural tissues, inefficient protocols and poorly defined culture conditions have hindered their use. It is now shown that soft, micropatterned culture substrates can induce hPSCs to differentiate into motor neurons with significantly improved yields and purity in comparison to rigid substrates, and that such mechanotransductive process involves the Hippo/YAP pathway and phosphorylation of the intracellular protein Smad. 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.

Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self-renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self-renewal and differentiation. When combined with three-dimensional cultures in bioreactors, these systems will also enable large-scale expansion for future clinical applications.

Human-induced pluripotent stem cells (hiPSCs) may represent an ideal cell source for research and applications in regenerative medicine. However, standard culture conditions that depend on the use of undefined substrates and xenogeneic medium components represent a significant obstacle to clinical translation. Recently, we reported a defined culture system for human embryonic stem cells using a synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), in conjunction with xenogeneic-free culture medium. Here, we tested the hypothesis that iPSCs could be maintained in an undifferentiated state in this xeno-free culture system and subsequently be differentiated into mesenchymal stem cells (iPS-MSCs). hiPSCs were cultured on PMEDSAH and differentiated into functional MSCs, as confirmed by expression of characteristic MSC markers (CD166+, CD105+, CD90+,CD73+, CD31−, CD34−, and CD45−) and their ability to differentiate in vitro into adipogenic, chondrogenic, and osteoblastic lineages. To demonstrate the potential of iPS-MSCs to regenerate bone in vivo, the newly derived cells were induced to osteoblast differentiation for 4 days and transplanted into calvaria defects in immunocompromised mice for 8 weeks. MicroCT and histologic analyses demonstrated de novo bone formation in the calvaria defects for animals treated with iPS-MSCs but not for the control group. Moreover, positive staining for human nuclear antigen and human mitochondria monoclonal antibodies confirmed the participation of the transplanted hiPS-MSCs in the regenerated bone. These results demonstrate that hiPSCs cultured in a xeno-free system have the capability to differentiate into functional MSCs with the ability to form bone in vivo. Disclosure of potential conflicts of interest is found at the end of this article.

Stem cells have the capacity for self-renewal and differentiation into specialized cells that form and repopulated all tissues and organs, from conception to adult life. Depending on their capacity for differentiation, stem cells are classified as totipotent (ie, zygote), pluripotent (ie, embryonic stem cells), multipotent (ie, neuronal stem cells, hematopoietic stem cells, epithelial stem cells, etc.), and unipotent (ie, spermatogonial stem cells). Adult or tissue-specific stem cells reside in specific niches located in, or nearby, their organ or tissue of origin. There, they have microenvironmental support to remain quiescent, to proliferate as undifferentiated cells (self-renewal), and to differentiate into progenitors or terminally differentiated cells that migrate from the niche to perform specialized functions. The presence of proteins at the cell surface is often used to identify, classify, and isolate stem cells. Among the diverse groups of cell surface proteins used for these purposes, integrin α6, also known as CD49f, may be the only biomarker commonly found in more than 30 different populations of stem cells, including some cancer stem cells. This broad expression among stem cell populations indicates that integrin α6 may play an important and conserved role in stem cell biology, which is reaffirmed by recent demonstrations of its role maintaining self-renewal of pluripotent stem cells and breast and glioblastoma cancer stem cells. Therefore, this review intends to highlight and synthesize new findings on the importance of integrin α6 in stem cell biology.

Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.