Induced pluripotent stem cell reporter systems for smooth muscle cell sheet engineering
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Smooth muscle cells exist in many different locations within the body, including blood vessels and airways, where their principal function is contraction and relaxation. The heterogeneity of smooth muscle cells has been related to their embryological origins and could have implications in many diseases, including atherosclerosis, pulmonary hypertension, and asthma. Many of these diseases require an expandable cell source of smooth muscle cells for regenerative medicine or disease modeling. Here, we have developed Acta2hrGFP and ACTA2eGFP (GFP reporters for smooth muscle α-actin) reporter mouse and human induced pluripotent stem cells lines to track and isolate populations of smooth muscle-like cells. iPSCs were patterned to a KDR-expressing (kinase insert domain receptor) mesodermal progenitor, which was further specified towards a smooth muscle-like lineage through exposure to platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-β). The Acta2hrGFP+ or ACTA2eGFP+ cells were enriched for characteristic markers of smooth muscle cells, and these cells expressed low levels of contractile markers, reminiscent of an immature or synthetic smooth muscle cell. Aligned smooth muscle-like cell sheets were generated using these iPSC-derived populations in an enzymatically degradable hydrogel system. The cell sheets displayed mechanical behavior similar to native blood vessels, with the Acta2hrGFP+ cell sheets displaying a higher ultimate tensile strength than Acta2hrGFP- cell sheets. Furthermore, we performed global transcriptomic profiling of primary adult mouse lung vascular (Acta2hrGFP+ Cspg4DsRed+) and airway (Acta2hrGFP+ Cspg4DsRed-) smooth muscle cells from a double transgenic reporter mouse, where we identified distinct gene signatures of lung vascular SMCs and airway SMCs, with Hhip and Acta2 co-expression distinguishing airway SMCs from lung vascular SMCs. When comparing our miPSC-derived Acta2hrGFP+ cells to these primary SMC signatures, the in vitro derived cells cluster closer to aortic SMCs and lung vascular SMCs, but their transcriptomic signatures still remain significantly distinct. In addition, we have generated an Acta2hrGFP Cspg4DsRed reporter mouse iPSC line, which can be used to understand the signaling pathways involved in specification of these different smooth muscle cell subtypes. Thus, we have developed systems for isolating smooth muscle-like populations which have potential in tissue engineering applications, and we have identified gene signatures of adult lung vascular and airway smooth muscle cells to begin to address the heterogeneity of smooth muscle cell lineages.