Induced pluripotent stem cell iPSC technology holds great

Induced pluripotent stem cell (iPSC) technology holds great promise for future autologous cellular therapies for vascular diseases (Tavernier et al., 2013). However, a major concern is the availability of approaches to differentiate iPSCs into large quantities of functional VSMCs for research and therapeutic applications (Dash et al., 2015). VSMCs have been previously derived from human iPSCs (hiPSCs) with different approaches (Bajpai et al., 2012; Cheung et al., 2012; Lee et al., 2009; Patsch et al., 2015; Wanjare et al., 2013). However, an efficient, large-scale production of highly enriched, functional hiPSC-VSMCs suitable for vascular tissue engineering still awaits to be established. In this study, hiPSCs from fibroblast cell adhesion molecules were generated using Sendai virus (SeV) vectors. We also derived a large quantity of highly enriched, functional hiPSC-VSMCs based on a robust embryoid body (EB) approach. In addition, we used a scaffold-free, self-assembly approach to engineer robust 3D model vascular tissue constructs from both normal and disease-specific human VSMCs.
Results
Discussion In this study, we generated an abundant and renewable source of functional VSMCs from multiple hiPSC lines. The success of VSMC production appears to be independent of the somatic cell sources (Y6: fibroblasts; control 1, VSMCs; control 2, peripheral blood mononuclear cells) and derivation approaches (Y6 and control 2, non-integrating SeV; control 1, lentiviral) used to generate the hiPSC lines. The feeder-free culture approach resulted in healthier and robust EBs, enabling us to derive ∼40 million VSMCs within 21 days of differentiation time from a 6-well plate of hiPSCs. The VSMCs were highly enriched for SM-22α, SMA, and calponin and can further be induced into mature VSMCs expressing SM-MHC and elastin. The VSMC derivation method was found to be efficient with different hiPSC lines and also with different batches of FBS. The hiPSC-VSMCs were functional and responded to chemical stimuli such as carbachol and KCl. We have also investigated different VSMC subtypes using an assay described previously (Cheung et al., 2012). hiPSC-VSMCs showed significant proliferation in response to serum, but only exhibited a modest trend of response upon treatment with TGF-β1 or angiotensin II without reaching statistical significance. These results suggest that hiPSC-VSMCs derived in this study may contain a significant population of VSMCs derived from the lateral plate mesoderm. We then used a cellular self-assembly approach to create hiPSC-VSMC tissue rings. Histological analysis of 14-day-old rings showed high cellularity and VSMC marker expression, including SMA, SM-22α, calponin, and SM-MHC. Type I collagen was also abundantly found in these tissue rings. hiPSC-VSMC tissue rings are mechanically strong enough to endure physical handling and mechanical testing within 7–14 days of cell seeding. In addition, we measured normal and SVAS hiPSC-VSMC contractility in 3D tissue constructs, suggesting that the ring self-assembly system, coupled with patient-specific VSMCs, can be used to create functional model human vascular tissues. SVAS, an autosomal disease, is caused by loss-of-function mutations in the elastin gene and is characterized by hyperproliferation of VSMCs leading to blockage of the ascending aorta and other arterial vessels (Ge et al., 2012). Patients with Williams-Beuren syndrome (WBS) also display SVAS (Ge et al., 2012). Earlier 2D studies in cell culture (Ge et al., 2012; Kinnear et al., 2013) reveal that SVAS and WBS iPSC-VSMCs are less contractile and more proliferative than control iPSC-VSMCs. We were able to model these two important characteristics of SVAS in the physiologically more relevant 3D tissue rings. The SVAS tissue rings showed significantly less contractility and more proliferative cells than control rings. The lower contractile forces generated by SVAS rings might be due to decreased formation of actin filament bundles and fewer cells expressing SM-MHC in SVAS VSMCs compared with those in control VSMCs.