Abstract 498: Combinatorial Extracellular Matrix Microarrays for Probing Endothelial Differentiation of Human Induced Pluripotent Stem Cells
Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) are a promising cell source for vascular regeneration in patients with peripheral arterial disease. However, a critical bottleneck to their clinical translation is the ability to differentiate the cells reproducibly at high yields. Since endothelial cells interact with the basement membrane extracellular matrix (ECM), we sought to examine the role of ECMs on endothelial differentiation using combinatorial ECM microenvironments. ECM microarrays were developed by covalent conjugation of ECMs (gelatin, fibronectin, laminin, heparin sulfate proteoglycans, collagen IV, matrigel) and the multi-component combinations thereof. The pluripotent stem cells attached to the ECMs and subsequently differentiated over the course of 5 days. Endothelial differentiation was semi-quantitatively scored based on the degree of CD31 staining. Our results demonstrated greater levels of CD31staining when cultured on gelatin + matrigel + laminin (G+M+L) or fibronectin + laminin + heparan sulfate (F+L+H), compared to other combinations across three human pluripotent stem cell lines (iPSC-Huf5, iPSC-CON1, and ESC-H1). This enhancement in endothelial differentiation on the microscale was confirmed at larger cell culture platforms in which a marked increase in CD31+ cells was observed in G+M+L modified-dishes (> 5 fold), and F+L+H combination (> 10 fold), compared to gelatin-modified dishes. RT-PCR further confirmed the transcriptional upregulation in endothelial markers for CD31 (> 2 fold) and VE-cadherin (> 4 fold) on G+M+L, compared to gelatin-modified dishes. To elucidate the role of cell-ECM interactions on endothelial differentiation, gene expression of integrin subunits were examined. Gene expression was markedly upregulated in integrins α1 (>10 fold); α4, α5, and αV (>5 fold); and β1, β3 (>50 fold), and β4, when comparing differentiated cells on day14 to undifferentiated cells. The upregulation of integrin subunits was concomitant with upregulation in endothelial genes. Together, this data suggested that combinatorial ECMs differentially promote endothelial differentiation, in part through integrin-mediated pathways.
Author Disclosures: L. Hou: None. J. Coller: None. V. Natu: None. N. Huang: None.
- © 2015 by American Heart Association, Inc.