Vascularization of PEGylated Fibrin Hydrogels Increases Proliferation of Human iPSC-Cardiomyocytes

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. The goal of this study was to test whether vascular cells or a formed vascular network in a fibrin-based hydrogel would alter the proliferation of human iPSC-derived cardiomyocytes. First, vascular network formation in a slowly degrading PEGylated fibrin hydrogel was optimized by altering the cell ratio of human umbilical vein endothelial cells (HUVEC) to human dermal fibroblasts (hDF), the inclusion of growth factors, and the total cell concentration. An endothelial to fibroblast ratio of 5:1 and a total cell concentration of 1.1x106 cells/mL without additional growth factors generated robust vascular networks while minimizing the number of cells required. Using this optimized system, human iPSC-derived cardiomyocytes were cultured on hydrogels without vascular cells, hydrogels with unorganized encapsulated vascular cells, or hydrogels with encapsulated vascular cells organized into networks for 7 days. Cardiomyocyte proliferation and gene expression were assayed following 7 days of culture on the hydrogels. The presence of vascular cells in the hydrogel, whether unorganized or in vascular networks, significantly increased cardiomyocyte proliferation compared to an acellular hydrogel. Hydrogels with unorganized vascular cells resulted in lower cardiomyocyte maturity evidenced by decreased expression of cardiac troponin t (TNNT2), myosin light chain 7 (MYL7), and phospholamban (PLN) compared to hydrogels without vascular cells and hydrogels with vascular networks.

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Images were quantified by hand using ImageJ (National Institutes of Health) by counting nuclei and EdU-labeled nuclei inside cells that were stained positive for cTnT, reported in figures as %EdU+ CMs. Expression of the cardiac genes TNNT2 (HS00943911_m1), MYH6 (HS01101425_m1), MYH7 (HS01110632_m1), MYL2 (HS00166405_m1), MYL7 (HS01085598_g1), CACNA1c (HS00167681_m1), and PLN (HS01848144_s1) were measured by quantitative polymerase chain reaction and normalized to GAPDH (HS02786624_g1). Fold change was normalized to the acellular (CM only) hydrogel group. Statistical analysis was performed using GraphPad Prism software. One way ANOVA followed by Fisher’s LSD test was used for statistical comparisons with P<0.05 considered significant.

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