Epigenetic and phenotypic characterization of iPSCs-derived smooth muscle cells: Towards a cellular model for complex arterial diseases

Abstract

Smooth muscle cells (SMCs) capacity to switch between proliferative (synthetic) and quiescent (contractile) phenotypes is a widely studied mechanism in cardiovascular disease. Primary SMCs tend to lose many physiological features in culture, which makes the study of their contractile function challenging. Recently, an optimized protocol of induced pluripotent stem cells (iPSCs) differentiation into contractile SMCs was described. We aimed at obtaining a deep phenotyping of SMCs derived from iPSCs and evaluating these cellular models in the context of complex cardiovascular diseases. We differentiated 3 human iPSCs lines towards SMC phenotypes using two alternative 24-day protocols, involving either RepSox (R-SMCs) or TGF-β/PDGF-BB (TP-SMCs) during late differentiation. We analyzed gene expression and open chromatin profiles at 6 time points of differentiation and compared them to primary human SMCs and artery tissue. We used CRISPR-Cas9 system to induce mutations at LRP1 cardiovascular disease locus. Both differentiation protocols provided SMCs with abundant expression of typical SMC markers. TP-SMCs exhibited greater capacity of proliferation, migration and lower calcium release in response to contractile signals compared to R-SMCs. RNA-Seq results showed that most genes involved in the contractile function of arteries were highly expressed in R-SMCs, while TP-SMCs were more similar to cultured primary human SMCs. Open chromatin regions of R-SMCs were highly enriched for variants associated with vascular diseases such as hypertension and intracranial aneurysm, whereas TP-SMCs were more enriched for variants associated to peripheral artery disease and aortic aneurysm. Deletion of a cardiovascular disease associated enhancer region led to a dysregulation of LRP1 expression in TP-SMCs, but not in R-SMCs. LRP1 inactivation in TP-SMCs led to a major decrease in SMC proliferation, migration, and deregulation of multiple genes involved in SMC function and extracellular matrix maintenance. Differentiation of SMCs from iPSCs using two complementary protocols provides cellular models suitable for the study of a variety of vascular diseases. We confirmed LRP1 as a regulatory target gene of a common variant associated to multiple cardiovascular diseases and identified multiple genes involved in SMC function downstream of LRP1.