Epigenetic and phenotypic characterization of iPSCs-derived smooth muscle cells: towards a cellular model for complex arterial diseases
Authors: Georges A , Liu L , Berrandou T , Jouve C , Hulot JS , Bouatia-Naji N 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): European Research Council

Introduction
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.

Purpose
We aimed at obtaining a deep characterization of cellular phenotypes during the differentiation into synthetic or contractile SMCs, and evaluate these cellular models in the context of complex cardiovascular diseases.

Methods
We differentiated 4 human iPSC lines (2 males, 2 females) towards either contractile (Repsox induced) or synthetic (PDGF-BB/TGF-β induced) SMC phenotypes using a 24-days protocol (Figure). We performed RNA-Seq and assay for transposase accessible chromatin (ATAC)-Seq at 6 time points of differentiation. We compared gene expression and open chromatin profiles between them and to existing datasets of primary human SMCs and artery tissues. We characterized the extracellular matrix (matrisome) generated by SMCs using mass spectrometry.

Results
iPSCs derived SMCs showed expected morphology and positive expression of SMC markers. Synthetic SMCs exhibited greater capacity of proliferation, migration, lower contractility and calcium release capacity, compared to contractile SMCs. RNA-Seq results showed that multiple disease-associated genes involved in the contractile function of arteries, including smooth-muscle myosin heavy chain (MYH11), myosin light chain kinase (MYLK) and angiotensin type 1 receptor (AGTR1) genes, were highly expressed in contractile compared to synthetic SMCs. Interestingly, multiple genes coding for extracellular matrix components were also enriched in contractile SMCs. Matrisome characterization confirmed that contractile SMCs generated a rich extracellular matrix, compared to synthetic cells. Analysis of transcriptomic and open chromatin profiles suggests contractile SMCs retained a higher level of activity for transcription factors involved in vascular smooth muscle development. Synthetic SMCs however presented open chromatin profiles similar to cultured primary SMCs. Open chromatin regions of contractile SMCs were highly enriched for variants associated with vascular diseases such as hypertension and intracranial aneurysm, whereas synthetic SMCs were more enriched for variants associated to peripheral artery disease and aortic aneurysm.

Conclusions
Differentiation of SMCs from iPSCs using two complementary protocols provides valid cellular models suitable for the study of a variety of vascular diseases. Utilization of these cells in combination with genome-editing tools is a promising approach to the study of complex regulatory mechanisms at genetic risk loci while considering phenotypic variability of arterial cellular components.