Human pluripotent stem cell culture density modulates YAP signaling.

Human pluripotent stem cell (hPSC) density is an important factor in self-renewal and differentiation fates; however, the mechanisms through which hPSCs sense cell density and process this information in making cell fate decisions remain to be fully understood. One particular pathway that may prove important in density-dependent signaling in hPSCs is the Hippo pathway, which is regulated by cell-cell contact and mechanosensing through the cytoskeleton and has been linked to the maintenance of stem cell pluripotency. To probe regulation of Hippo pathway activity in hPSCs, we assessed whether Hippo pathway transcriptional activator YAP was differentially modulated by cell density. At higher cell densities, YAP phosphorylation and localization to the cytoplasm increased, which led to decreased YAP-mediated transcriptional activity. Furthermore, total YAP protein levels diminished at high cell density due to the phosphorylation-targeted degradation of YAP. Inducible shRNA knockdown of YAP reduced expression of YAP target genes and pluripotency genes. Finally, the density-dependent increase of neuroepithelial cell differentiation was mitigated by shRNA knockdown of YAP. Our results suggest a pivotal role of YAP in cell density-mediated fate decisions in hPSCs.

α-5 Laminin Synthesized by Human Pluripotent Stem Cells Promotes Self-Renewal.

Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation, but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify α-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore, treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous α-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing.

Effects of 3D microwell culture on initial fate specification in human embryonic stem cells.

Several studies have demonstrated that 3D culture systems influence human embryonic stem cell (hESC) phenotypes and fate choices. However, the effect that these microenvironmental changes have on signaling pathways governing hESC behaviors is not well understood. Here, we have used a 3D microwell array to investigate differences in activation of developmental pathways between 2D and 3D cultures of both undifferentiated hESCs and hESCs undergoing initial differentiation in embryoid bodies (EBs). We observed increased induction into mesoderm and endoderm and differences in expression of genes from multiple signaling pathways that regulate development, including Wnt/ß-catenin, TGF-ß superfamily, Notch and FGF during EB-mediated differentiation, in 3D microwells as compared with the 2D substrates. In undifferentiated hESCs, we also observed differences in epithelial-mesenchymal transition phenotypes and the TGFß/BMP pathway between cultures in 3D and 2D. These results illustrate that 3D culture influences multiple pathways that may regulate the differentiation trajectories of hESCs.

Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification.

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (>75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound.

A small molecule inhibitor of SRC family kinases promotes simple epithelial differentiation of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs, the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of ß-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated ß-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8), which are expressed in simple epithelial cells, while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications.

Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/ß-catenin signaling under fully defined conditions.

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of ß-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.

Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.

Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of ß-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of ß-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.

Microwell regulation of pluripotent stem cell self-renewal and differentiation.

The fates of pluripotent stem cells (PSCs), including survival, self-renewal, and differentiation, are regulated by chemical and mechanical cues presented in the three-dimensional (3D) microenvironment. Most PSC studies have been performed on two-dimensional substrates. However, 3D culture systems have demonstrated the importance of intercellular interactions in regulating PSC self-renewal and differentiation. Microwell culture systems have been developed to generate homogenous PSC colonies of defined sizes and shapes and to study how colony morphology affects cell fate. Using microwells, researchers have demonstrated that PSCs remain in a self-renewing undifferentiated state as a result of autocrine and paracrine signaling. Other studies have shown that microwell regulation of embryoid body size affects lineage commitment during differentiation via cell-cell contact and expression of soluble signals. In this review, we discuss recent advances in the design and utilization of 3D microwell platforms for studying intercellular regulation of PSC cell fate decisions and the underlying molecular mechanisms.