(336g) Functional Smooth Muscle Cells Derived From Induced Pluripotent Stem Cells for Cardiovascular Tissue Engineering Applications

Tissue
engineered vessels (TEV) represent attractive alternative to the shortage of
autologous vascular grafts for coronary artery disease.  Less proliferative and
senescent autologous vascular cells from the aged patients have motivated
researchers to seek alternate autologous sources. In this light, induced
pluripotent stem cell (iPSC) technology bears great promise for regenerative
medicine as it may provide a potential inexhaustible source for autologous
cells. However, iPSC have not been studied in detail in the context of vascular
tissue engineering particularly iPSC derived smooth muscle cells (iPS-SMC)
haven't been rigorously characterized for their functionality in-vitro and
in-vivo which is imperative for their successful application in cardiovascular
tissue engineering. In the present work, we developed novel monolayer culture
protocol of human (h)iPSC differentiation into smooth muscle using extra
cellular matrix molecules and diffusible signals and characterized them with a
battery of biochemical and functional assays. To assess differentiation in
real-time and monitor its progression, two hiPSC lines (generated from Yamanaka
factors (OCT4, SOX2, cMYC, KLF4) and Thomson factors (OCT4, SOX2, NANOG, LIN28))
were transduced on matrigel (to limit contamination by MEF) with dual promoter
lentiviral construct (LVDP). LVDP encodes for DsRed2 under the constitutive
human phosphoglycerokinase (hPGK) promoter and ZsGreen under the smooth muscle
alpha actin (αSMA) promoter. Thus DsRed2 expression provides a measure of
transduction efficiency while ZsGreen expression works as differentiation
selection marker. Transduced hiPSC were sequentially cultured on matrigel (in MEF
conditioned hESC medium (MEF-CM) plus bFGF) followed by culture on collagen IV
or gelatin (in presence of SMC medium (M231 supplemented with 5%FBS, bFGF, EGF,
Insulin and heparin) and finally in maturation media ( M231 plus TGF- β1 and
heparin)). hiPS-SMC were characterized for known biochemical markers at the
transcriptional and translational level. TEV were fabricated from hiPS-SMC and
fibrin and were tested for mechanical strength and vasoreactivity to determine
functional status of hiPS-SMC. Histological analysis of TEV was performed to
examine collagen and elastin synthesis by hiPS-SMC. We found that by day 7 on
matrigel, ~25% of transduced cells (DsRed+) were also green (ZsGreen+)
suggesting that αSMA promoter was active and this fraction further
enriched to >95% by treatment with SMC medium for 8 days as determined
through fluorescence microscopy and flow cytometry. RT-PCR confirmed
upregulation of SMC specific genes and concomitant downregulation of pluripotent,
mesendodermal and ectodermal genes during differentiation.  Immunostaining for
early (αSMA), intermediate (calponin) and late (myosin heavy chain, MHC)
SMC markers showed that only small fraction of cells exhibited filamentous
protein organization after SMC medium treatment, suggesting that the
contractile apparatus of enriched hiPS-SMC was still immature. Indeed vascular
contractility of TEV made with hiPS-SMC at this stage of differentiation was
low as compared to mature SMC. However, after treatment with maturation media for
5 days, immunostaining for the same proteins showed markedly increased
filamentous organization. TEV fabricated from mature iPS-SMC showed increased
contractility in response to vasoagonists like KCl, U46619 (Thromboxane A2
agonist) and endothelin-1 suggestive of well developed receptor mediated and
non-receptor mediated pathways of contractility. Additionally, hiPS-SMC
embedded in the vascular wall aligned circumferentially and synthesized collagen
and tropoelastin as evidenced by histological analysis. Figure 1 depicts phase
contrast, fluorescent microscopic and immunocytochemistry images of hiPSC and
mature hiPS-SMC.

Figure1. hiPSC differentiation into SMC. iPSC colony
on feeder layer (A); Mature hiPS-SMC fluorescence microscopy (B and C); Mature
hiPS-SMC Immunocytochemistry for αSMA (D) and MHC (E). Bars, A 50μm; B and C 100μm; D and E 10μm.

Our results
suggest that hiPSC can be efficiently differentiated into functional SMC using
monolayer protocol and can serve as an untapped autologous cell source for
potential application in cardiovascular tissue engineering and regenerative
medicine.