(489f) 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.