Induced pluripotent stem cell-derived smooth muscle cells to study cardiovascular calcification

Abstract

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date, only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs), which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture. There is an increasing, practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically, we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First, we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis, immunofluorescent staining, flow cytometry, and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust, well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs, and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation, we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions, with high potential for future applications in cardiovascular calcification research.

Keywords: calcification; cardiovascular diseases; induced pluripotent stem cells; proteomics and bioinformatics; smooth muscle cells (SMCs); stem cell differentiation and reprogramming.