Abstract 15560: Induced Pluripotent Stem Cell (iPSC) Derived Smooth Muscle Cells as a Model to Study Vascular Calcification

Abstract

Introduction: Vascular calcification is the top cause of mortality worldwide. Vascular smooth muscle cells (SMCs) are major contributors to vascular calcification. There is an urgent need for a large scale and inexhaustible source of functional SMCs to study mechanisms of cardiovascular calcification. Here, we describe a new platform utilizing iPSCs differentiated towards SMCs that can calcify, to model ectopic calcification and identify mechanisms of SMCs’ contribution in this pathological process. Methods: We differentiated human iPSCs towards an induced-SMC (iSMC) phenotype in a 10-day protocol. We verified successful iSMC differentiation through immunofluorescent staining for SMC marker proteins αSMA and MYH11, FACS using CD140b, and proteomics analysis on whole cell lysate at days 0, 3, 5, 7, and 10. A set of 19 canonical SMC proteins were used to generate a Pearson’s correlation over the differentiation time course. After differentiation, we cultured the iSMCs in osteogenic media (OM) for 14 days. Alizarin Red S staining performed on day 14 confirmed the presence of calcification. Three independent experiments were performed for all data analyses. Results: Typical SMC morphology with positive staining for αSMA and MYH11 was detected at day 10 by immunofluorescence (Fig 1A). FACS analysis on markers for iPSCs (TRA-1-60), ECs (CD31), and SMCs (CD140b) demonstrated that 93.9% of cells were positive for CD140b after 10 days of differentiation (Fig 1B, C). Time course proteomics showed a strong Pearson’s correlation with of iSMCs with SMC marker proteins (R = 0.89) by day 10 (Figure 1D). Calcification in osteogenic media was observed by day 14 in culture using Alizarin Red S staining (Fig 1E, F), with 4.8-fold higher calcification in OM as compared to normal media (NM). Conclusions: Investigated iSMCs have promising future applications in cardiovascular research and may serve as an inexhaustible resource for studying vascular SMC calcification.