Human Induced Pluripotent Stem Cells Differentiate Into Contractile Vascular Smooth Muscle Fate Via Mesenchymal Stem Cell Intermediates: Implication for Cardiovascular Regeneration

Smooth muscle cells (SMC) maintain vascular homeostasis and play prominent role in vascular diseases. Induced pluripotent stem cells (iPSC) can provide an untapped resource of functional SMC for autologous cell based therapies and for creating models for studying vascular diseases. This would require efficient differentiation of iPSC towards SMC fate. However existing protocols of pluripotent stem cell differentiation are inefficient and primarily give rise to terminally differentiated SMC that critically limits their in-vitro expansion that is unequivocally desirable for regenerative medicine applications. To address this challenge, we developed a robust, stage-wise monolayer protocol of human (h)iPSC differentiation towards functional SMC fate. Employing this protocol, we demonstrate that hiPSC differentiate into functional smooth muscle cell fate via intermediate mesenchymal stem cell (MSC) stage where they are highly proliferative and amenable to differentiate along all mesenchymal cell types including osteogenic, chondrogenic and adipogenic lineages. Thus using this protocol, MSC can be kept in a proliferative state for 45 population doublings and tuned into contractile SMC with short-term induction treatment when required. In the first stage of differentiation hiPSC lines (generated from two different set of transcription factors) were induced to undergo epithelial to mesenchymal transition (EMT) by culturing them on matrigel-coated dishes in presence of EMT medium (MEF conditioned medium human embryonic stem cell medium supplemented plus FGF2). EMT was followed in real time using smooth muscle alpha actin (ACTA2) promoter driven ZsGreen lentiviral reporter. To ensure that ACTA2-ZsGreen reporter reliably captures hiPSC undergoing EMT, we derived stable ACTA2-ZsGreen lentiviral reporter hiPSC clones and by immunocytochemistry clearly demonstrated that undifferentiated OCT4+ colonies also lacked ACTA2-ZsGreen expression. However, as early as day 3 after induction of EMT cells in the periphery of hiPSC colonies assumed fibroblastic morphology and expressed ZsGreen, suggesting upregulation of ACTA2 promoter and EMT. Indeed, EMT was confirmed by RT-PCR for EMT markers like SNAIL, TWIST, SLUG, FOXC2, FSP1, CDH1, CDH2, fibronectin and vimentin. EMT cells (ZsGreen+) were enriched (stage 2) into MSC fate by culture on gelatin-coated surfaces in the presence of enrichment medium (M231 supplemented with 5%FBS, bFGF, EGF, insulin and heparin). During the enrichment stage, the fraction of ZsGreen+ cells was significantly increased for the both hiPSC lines tested as quantified by flow cytometry. Enriched cells displayed remarkable similarity with bone marrow derived MSC as they displayed MSC surface immunophenotype (CD105+/CD73+/CD44+/CD49b+/CD90+/CD45-/CD34-) and could be coaxed to differentiate into adipogenic, osteogenic and chondrogenic lineages as shown by oil red O, Von Kossa and alcian blue stains, respectively. Enriched cells were highly proliferative cells as they were kept in culture for at least 45 population doublings without reaching cellular senescence. While enriched cells were highly proliferative, they were weakly contractile, perhaps due to immature contractile machinery as evidenced by immunostaining for ACTA2, CNN1 and MYH11. To induce contractile phenotype (stage 3), enriched MSC were further treated with maturation medium (M231 plus TGF-β1 and heparin). Immunostaining of mature cells showed markedly increased filamentous organization of ACTA2, CNN1 and MYH11 proteins. Contractile phenotype was further confirmed by excellent contractility in response to receptor and non-receptor mediated vasoagonists exhibited by cylindrical tissue engineered vessels (TEV) made from mature cells.

Figure 1. RT-PCR analysis of SMC specific and pluripotency markers during differentiation (A)  Immunocytochemistry for SMC specific markers show filamentous organization of these contractile proteins after maturation treatment (B) Vasoconstriction of TEV made of enriched and mature cells for two hiPSC lines (C).  

In summary, this study demonstrate that hiPSC undergo functional SMC fate through an intermediate stage of multipotent MSC. The iPSC derived SMC may be useful for studying the pathology of vascular diseases especially in context of SMC phenotypic conversion between synthetic and contractile fates and engineering SMC-containing tissues such as vascular grafts.