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

. 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.本研究旨在探究纤维蛋白基水凝胶中的血管细胞或已成型血管网络，是否会对人类诱导多能干细胞（iPSC）来源心肌细胞的增殖产生影响。首先，通过调控人脐静脉内皮细胞（HUVEC）与人真皮成纤维细胞（hDF）的接种比例、生长因子添加方案及总细胞浓度，优化了缓慢降解聚乙二醇化纤维蛋白水凝胶中的血管网络构建流程。当内皮细胞与成纤维细胞比例为5:1、总细胞浓度为1.1×10⁶ cells/mL且无需额外添加生长因子时，可形成稳定的血管网络，同时最大限度降低所需细胞用量。依托该优化体系，将人类iPSC来源的心肌细胞分别接种至三类水凝胶上培养7天：不含血管细胞的空白水凝胶、封装有无序分布血管细胞的水凝胶，以及封装有已组装为有序网络的血管细胞的水凝胶。培养7天后，对各组心肌细胞的增殖水平与基因表达特征进行检测分析。实验结果表明，与空白无细胞水凝胶组相比，水凝胶中存在血管细胞（无论是否形成有序血管网络）均可显著促进心肌细胞增殖。与不含血管细胞的水凝胶组以及形成血管网络的水凝胶组相比，封装有无序血管细胞的水凝胶组的心肌细胞成熟度更低，具体表现为心肌肌钙蛋白T（TNNT2）、肌球蛋白轻链7（MYL7）及受磷蛋白（PLN）的基因表达水平显著下调。