Novel Directly Reprogrammed Smooth Muscle Cells Promote Vascular Regeneration as Microvascular Mural Cells

BACKGROUND: Although cell therapy has emerged as a promising approach to promote neovascularization, its effects are mostly limited to capillaries. To generate larger or more stable vessels, layering of mural cells such as smooth muscle cells (SMCs) or pericytes is required. Recently, direct reprogramming approaches have been developed for generating SMCs. However, such reprogrammed SMCs lack genuine features of contractile SMCs, a native SMC phenotype; thus, their therapeutic and vessel-forming potential in vivo was not explored. Therefore, we aimed to directly reprogram human dermal fibroblasts toward contractile SMCs (rSMCs) and investigated their role for generating vascular mural cells in vivo and their therapeutic effects on ischemic disease. METHODS: retinoic acid with specific culture conditions to directly reprogram human dermal fibroblasts into rSMCs. We characterized their phenotype as contractile SMCs through quantitative reverse-transcriptase polymerase chain reaction, flow cytometry, and immunostaining. We then explored their contractility using a vasoconstrictor, carbachol, and through transmission electron microscope and bulk RNA sequencing. Next, we evaluated whether transplantation of rSMCs improves blood flow and induces vessel formation as mural cells in a mouse model of hindlimb ischemia with laser Doppler perfusion imaging and histological analysis. We also determined their paracrine effects. RESULTS: retinoic acid efficiently reprogrammed human dermal fibroblasts into SMCs. These rSMCs displayed characteristics of contractile SMCs at the mRNA, protein, and cellular levels. Transplantation of rSMCs into ischemic mouse hind limbs enhanced blood flow recovery and vascular repair and improved limb salvage. Histological examination showed that vascular density was increased and the engrafted rSMCs were incorporated into the vascular wall as pericytes and vascular SMCs, thereby contributing to formation of more stable and larger microvessels. Quantitative reverse-transcriptase polymerase chain reaction analysis revealed that these transplanted rSMCs exerted pleiotropic effects, including angiogenic, arteriogenic, vessel-stabilizing, and tissue regenerative effects, on ischemic limbs. CONCLUSIONS: retinoic acid in defined culture conditions efficiently reprogrammed human fibroblasts into contractile and functional SMCs. The rSMCs were shown to be effective for vascular repair and contributed to neovascularization through mural cells and various paracrine effects. These human rSMCs could represent a novel source for cell-based therapy and research.