SummaryHuman stem cell-derived models of development and neurodegenerative diseases are challenged by cellular immaturity in vitro. Microengineered organ-on-chip (or Organ-Chip) systems are designed to emulate microvolume cytoarchitecture and enable co-culture of distinct cell types. Brain microvascular endothelial cells (BMECs) share common signaling pathways with neurons early in development, but their contribution to human neuronal maturation is largely unknown. To study this interaction and influence of microculture, we derived both spinal motor neurons and BMECs from human induced pluripotent stem cells and observed increased calcium transient function and Chip-specific gene expression in Organ-Chips compared with 96-well plates. Seeding BMECs in the Organ-Chip led to vascular-neural interaction and specific gene activation that further enhanced neuronal function and in vivo-like signatures. The results show that the vascular system has specific maturation effects on spinal cord neural tissue, and the use of Organ-Chips can move stem cell models closer to an in vivo condition.

No abstract

Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin a6b1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, a6b1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin a6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin a6 is upregulated and FAK is inactivated. Knockdown of integrin a6 and activation of b1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin a6 functions in inactivation of integrin b1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin a5, the primary ligand of integrin a6b1. Knockdown of laminin a5 resulted in reduction of integrin a6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin a6b1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. STEM CELLS 2016;34:1753-1764 SIGNIFICANCE STATEMENTVilla-Diaz et al. describe a new signaling pathway in which the expression of pluripotent transcription factors is linked to the expression of laminin a5 and integrin a6b1 and the inactivation of FAK signaling which allows self-renewal of human pluripotent stem cells.

SummarySubstrate 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.