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

Abstract

Summary: 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 (Acta2hrGFP and ACTA2eGFP, respectively) that identify Acta2+/ACTA2+ cells as they emerge in vitro in real time during iPSC-directed differentiation. We find that Acta2hrGFP+ and ACTA2eGFP+ 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. : In this article, Wong, Kotton, and colleagues developed murine and human iPSC lines with fluorochrome reporters for ACTA2, allowing for isolation of putative iPSC-derived smooth muscle-like populations (iPSC-SMC) with transcriptomic profiles reminiscent of immature or synthetic SMCs. The iPSC-SMCs were used to generate cell sheets with mechanical properties within physiological ranges to serve as building blocks for multi-layered tissue constructs. Keywords: induced pluripotent stem cells, directed differentiation, smooth muscle cells, cell sheets, fluorochrome reporters