Abstract
Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.
Highlights
It is well established that mesenchymal progenitor cells make an important contribution to angiogenesis during repair and regeneration
To identify paracrine factors that may be associated with an angiogenic response, secretory profiles of EDK and iPDK cells were assessed using an antibody array designed to detect soluble mediators of angiogenesis (Fig. 2A)
Analysis of the secretory profile of control, dermal-derived fibroblasts (BJ) using the same antibody array showed that the levels of key angiogenic regulators VEGF, PDGFAA, HGF, Ang-1, AR, Endostatin, and TIMP-4 were significantly lower when compared to secretory profiles of EDK and iPDK cells (Fig. 2A, 2B, Table 1)
Summary
It is well established that mesenchymal progenitor cells make an important contribution to angiogenesis during repair and regeneration. The biological plasticity of mesenchymal progenitor cells is linked to their ability to promote angiogenesis and vascular regeneration which is essential for defining their therapeutic utility. The broad-scale use of mesenchymal cells for regenerative therapies remains somewhat limited due to their heterogeneity in vivo that complicates the isolation of well-defined populations of mesenchymal progenitor cells. The development of functional mesenchymal progenitor cells for specific therapeutic applications has been further complicated by their inherent plasticity. While pericytes and other stromal cell types of mesenchymal origin play a central role in neovascularization, this uncertainty about their cellular origins and fate currently limit their applications for regenerative therapies
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