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.

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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.

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.

Highlights d BRCA1 mut patient iPSC lines can be differentiated into fallopian tube epithelium d BRCA1 mut fallopian tubes recapitulate ovarian carcinogenesis in vitro and in vivo d BRCA1 mut fallopian tubes may provide a model to predict disease severity d BRCA1 mut fallopian tube organoids provide a platform to study efficacy of cancer drugs

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process.

Poly(ethylene glycol) (PEG) hydrogels are being developed as cell delivery vehicles that have great potential to improve neuronal replacement therapies. Current research priorities include (1) characterizing neural cell growth within PEG hydrogels relative to standard culture systems and (2) generating neuronal-enriched populations within the PEG hydrogel environment. This study compares the percentage of neural precursor cells (NPCs), neurons, and glia present when dissociated neural cells are seeded within PEG hydrogels relative to standard monolayer culture. Results demonstrate that PEG hydrogels enriched the initial cell population for NPCs, which subsequently gave rise to neurons, then to glia. Relative to monolayer culture, PEG hydrogels maintained an increased percentage of NPCs and a decreased percentage of glia. This neurogenic advantage of PEG hydrogels is accentuated in the presence of basic fibroblast growth factor and epidermal growth factor, which more potently increase NPC and neuronal expression markers when applied to cells cultured within PEG hydrogels. Finally, this work demonstrates that glial differentiation can be selectively eliminated upon supplementation with a g-secretase inhibitor. Together, this study furthers our understanding of how the PEG hydrogel environment influences neural cell composition and also describes select soluble factors that are useful in generating neuronal-enriched populations within the PEG hydrogel environment.