Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States; Stem Cell and Regenerative Medicine Center, University of Wisconsin - Madison, Madison, United States
Zachery R Gregorich
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States
Ran Tao
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States
Gina C Kim
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States
Pratik A Lalit
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States; Department of Genomic Sciences and Biotechnology, University of Brasília, Brasília, Brazil
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States
Deane F Mosher
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States; Morgridge Institute for Research, Madison, United States; Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, United States
Sean P Palecek
Stem Cell and Regenerative Medicine Center, University of Wisconsin - Madison, Madison, United States; Department of Chemical and Biological Engineering, College of Engineering, University of Wisconsin, Madison, United States
Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States; Stem Cell and Regenerative Medicine Center, University of Wisconsin - Madison, Madison, United States; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, United States
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.
