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Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury + mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4β1 or αVβ1, but not α5β1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation., Competing Interests: JZ, ZG, RT, GK, PL, JC, YM, DM, SP No competing interests declared, TK is a consultant for Fujifilm Cellular Dynamics Incorporated, a stem cell company, (© 2022, Zhang et al.)

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gαo and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L-AP4, a type III mGluR-selective agonist, enhances Ca(2+) uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L-AP4-induced Ca(2+) influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gαo protein is absent in melanocytes. However, forced expression of Gαo restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of Gi /Go proteins, did not affect basal or mGluR6-induced Ca(2+) uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin., (© 2013 John Wiley & Sons A/S.)