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Human induced pluripotent stem cells (hiPSCs) can give rise to multiple lineages derived from three germ layers, endoderm, mesoderm and ectoderm. Definitive endoderm (DE) cell types and tissues have great potential for regenerative medicine applications. Current hiPSC differentiation protocols focus on the addition of soluble factors; however, extracellular matrix properties are known to also play a role in dictating cell fate. Matrigel™ is the gold standard for DE differentiation, but this xenogeneic, poorly defined basement membrane extract limits the clinical translatability of DE-derived tissues. Here we present a fully defined PEG-based hydrogel substrate to support hiPSC-derived DE differentiation. We screened hydrogel formulations presenting different adhesive peptides and matrix stiffness. Our results demonstrate that presenting a short peptide, cyclic RGD, on the engineered PEG hydrogel supports the transition from undifferentiated hiPSCs to DE using a serum-free, commercially available kit. We show that increasing substrate stiffness (G' = 1.0–4.0 kPa) results in an increased linear response in DE differentiation efficiency. We also include a temporal analysis of the expression of integrin and syndecan receptors as the hiPSCs undergo specification towards DE lineage. Finally, we show that focal adhesion kinase activity regulates hiPSC growth and DE differentiation efficiency. Overall, we present a fully defined matrix as a synthetic alternative for Matrigel™ supporting DE differentiation. [ABSTRACT FROM AUTHOR]

• Glycerylphytate-crosslinked chitosan membranes were prepared for guided bone regeneration • Electrostatic interactions between glycerylphytate and chitosan provided highly stable systems. • Swelling, crosslinking degree, and crosslinker release demonstrated a glycerylphytate-dependent content behavior. • All the crosslinked-systems showed high biocompatibility. • The incorporation of glycerylphytate to chitosan increased ALP activity and calcium deposition on hMSCs culture. Chitosan-based membranes are promising systems for guided bone regeneration. In this work, we used glycerylphytate as ionic crosslinker and osteinductor compound for the fabrication of chitosan membranes as supports for human mesenchymal stem cells. Three different glycerylphytate-crosslinked membranes were developed by changing the crosslinker concentration, from 2.5–10 wt-%, respect to chitosan. Physico-chemical characterization in terms of composition, morphology, and thermal behavior was further analyzed. Swelling degree, crosslinking density, and crosslinker release showed a glycerylphytate content-dependent behavior. Glycerylphytate suggested to improve osteointegration ability of chitosan surfaces by the formation of apatite-like aggregates after incubation in body simulated fluid. Stem cells cultured on the membranes increased their viability over time, and the incorporation of glycerylphytate improved osteogenic and osteoinductivity potential of chitosan by increasing calcium deposition and alkaline phosphatase (ALP) activity on cultured stem cells. These results demonstrated a potential application of glycerylphytate-crosslinked chitosan systems for promising bone tissue regeneration. [ABSTRACT FROM AUTHOR]

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity. [ABSTRACT FROM AUTHOR]