Abstract 16976: Elastin Mediated Smooth Muscle Cell Differentiation in Human Embryonic Stem Cells and Induced-Pluripotent Stem Cells
Elastin, an extracellular matrix protein, is essential for arterial morphogenesis. Elastin haploinsufficiency in humans, as seen in Williams syndrome, leads to increased vascular smooth muscle cell (VSMC) proliferation and vascular stenoses. We investigated the role of elastin in VSMC proliferation and differentiation using VSMCs derived from human embryonic stem cells (hESC-VSMCs) and human-induced pluripotent stem cells (hiPSC-VSMCs).
Methods: VSMCs were derived from hESCs and from hiPSCs (derived from BJ fibroblasts using retrovirus containing the pluripotency factors Oct4, Sox2, Klf4, and c-Myc) on day 27 of cardiac differentiation and treated for 5 days with elastin-binding protein ligand, EBPL-2. Immunostaining was performed for elastin, smooth muscle α-actin (SMA), Ki67 (cell proliferation marker) and analyzed by high-content imaging (Cellomics Arrayscan). mRNA expression was measured using RT-PCR for elastin, SMA, and mediators of VSMC differentiation i.e. serum response factor (SRF), Collagen IV and microRNA-145 (miR-145).
Results: Treatment with EBPL-2 decreased the number of Ki67-positive hESC-VSMCs from 28±3% to 20±3% (p<0.05) and Ki67-positive hiPSC-VSMCs from 36±4% to 26±2% (p<0.05) (Figure). EBPL-2 treatment increased VSMC differentiation as evident by a 1.5-fold increase in SMA-positive cells (p<0.05), and by the upregulation of genes that mediate SMC differentiation i.e SRF (1.4-fold), collagen IV (2-fold), and miR-145 (7-fold) (p<0.05).
Conclusion: Our study identifies elastin as a key regulator of arterial morphogenesis in humans. This effect is mediated by an increase in VSMC differentiation and decrease in VSMC proliferation via upregulation of SRF, collagen IV, and miR-145. Our study is the first to utilize hESC and hiPSC-derived VSMCs to study elastin function and provide an in vitro cellular model that can be used to screen anti-proliferative compounds as potential drug targets for vascular stenoses.
- © 2010 by American Heart Association, Inc.