Generation of a Purified iPSC-Derived Smooth Muscle-like Population for Cell Sheet Engineering

Induced pluripotent stem cells (iPSCs) provide a potential source for the derivation of smooth muscle cells (SMCs); however, current approaches are limited by the production of heterogeneous cell types and a paucity of tools or markers for tracking and purifying candidate SMCs. Here, we develop murine and human iPSC lines carrying fluorochrome reporters (Acta2^hrGFP and ACTA2^eGFP, respectively) that identify Acta2⁺/ACTA2⁺ cells as they emerge in vitro in real time during iPSC-directed differentiation. We find that Acta2^hrGFP+ and ACTA2^eGFP+ cells can be sorted to purity and are enriched in markers characteristic of an immature or synthetic SMC. We characterize the resulting GFP⁺ populations through global transcriptomic profiling and functional studies, including the capacity to form engineered cell sheets. We conclude that these reporter lines allow for generation of sortable, live iPSC-derived Acta2⁺/ACTA2⁺ cells highly enriched in smooth muscle lineages for basic developmental studies, tissue engineering, or future clinical regenerative applications.