RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/citologia , Comunicação Celular , Contagem de Células , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fosforilação , Transdução de Sinais , Fatores de Transcrição , Transcrição Gênica , Proteínas de Sinalização YAPRESUMO
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.