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.