Phenotypes and Cellular Mechanics of Primary Human Aorta- and Pluripotent Stem Cell-derived Vascular Smooth Muscle Cells

Abstract

AimsMature, contractile vascular smooth muscle cells (vSMC) in the medial layer of muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Our aim was to define the mechanical properties of well-characterized in vitro-differentiated vSMC compared to vSMC derived from adult human aorta (AoSMC). Methods and ResultsWe generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines, and we obtained proliferating AoSMC. Immature, synthetic hPSC-vSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMCs are non-proliferative, have elevated levels of contractile proteins, and can undergo phenotype switching in vitro into a proliferative form. Immunostaining of contractile proteins present in vSMCs (CNN1, MYH11, TAGLN, ACTA2) display similar cell-to-cell heterogeneities between AoSMC and hPSC-vSMCs. The fluorescent signals are relatively uniform at sub-confluency, but highly variable at confluency. Human PSC-vSMC maintain doubling rates more readily and are more easily enriched using lactate medium than AoSMC. The single cell mechanics of AoSMC and hPSC-vSMC, assessed using micropost array detectors (mPADs), are highly comparable and have an average force production per micropost in the nN range. When cultivated to form humanized smooth muscle cell microtissues (SMTs), developed forces normalized to cell numbers are similar to that seen using microarray post detectors. Maximum developed forces in these tissues could be measured in response to endothelin-1. ConclusionsThese results demonstrate that synthetic hPSC-vSMCs and proliferating AoSMCs are highly comparable, and mechanically similar, but results with hPSC-vSMC are more reproducible. This study establishes the hPSC-vSMCs as a reproducible system to study how changes in cellular mechanics may contribute to development and disease. Translational PerspectiveThe mechanical properties and responses of lineage-specific human vSMC undergoing phenotype switching during perinatal development and vascular pathologies remain poorly understood. We have employed paraxial-mesoderm derived hPSC-vSMC, which recapitulate phenotypic properties of AoSMC, to define the mechanical properties of synthetic and contractile vSMCs. We quantitatively measured the forces of individual vSMC and of multicellular vSMC constructs. By defining the mechanical changes of normal vSMCs to phenotype switching, this validated, adaptable system advance studies of vSMC mechanics that may contribute to vascular pathologies in patients.