Abstract 16362: Serum-Free Differentiation and Enrichment of Functional Human Vascular Smooth Muscle Cells From Embryonic Stem Cells
Background: Currently, surface markers enabling cell sorting of viable vascular smooth muscle cells (SMCs) from mixed cell populations or tissues for therapeutic or diagnostic purposes are not available. Here we define a method to enrich for functional human vascular SMCs derived from human embryonic stem cells (hESCs).
Methods & Results: Embryoid bodies (EBs) subjected to serum-free cardiovascular-directed differentiation were characterized over 30 days for SMC-specific gene expression by qRT-PCR, immunohistochemistry (IHC), and flow cytometry. EBs were composed of endothelial cells, fibroblasts, cardiomyocytes, and SMCs. Flow cytometry of day 28 EBs reveals that the SMCs form a distinct sub-population, which can integrate with endothelial cells in an in vitro angiogenesis assay. To enrich for SMCs, day 28 EBs were dissociated and cultured as monolayers. Passaging monolayers in serum-free conditions resulted in the loss of cardiomyocyte, endothelial and fibroblast populations. qRT-PCR and IHC analyses showed over three passages that mRNA and protein levels of cardiomyocyte-, endothelial- and fibroblast-specific markers (TNNI3, Desmin, PECAM1, DDR2) were abolished, while those of mature SMCs (ACTA2, TAGLN, CNN1, SMTN) were unchanged. Next, we examined the function of ESC-derived SMCs in vitro and in vivo. Calcium cycling and cell shortening responses to phenylephrine and norepinephrine in passaged SMC monolayers were similar to those of primary human coronary artery SMCs. Passaged SMCs derived from GFP-expressing hESCs functionally integrated into the vasculature and contributed to new vessel formation when subcutaneously injected into the dorsal skin fold of nude mice.
Conclusion: The ability to enrich for a mature functional human vascular SMC lineage in serum-free conditions has implications for drug screening and a wide-range of regenerative therapies.
- © 2011 by American Heart Association, Inc.