Assistant Professor University of California, Irvine, California, United States
Abstract: Skeletal muscle regeneration is made possible by the muscle’s endogenous stem cells (SCs); which are supported by a dynamic microenvironment and immediate SC niche. Without the SC niche, SCs rapidly activate and lose their regenerative potential both in vitro and in vivo. Cell therapies which commonly rely on amplifying progenitors detached from their niche would greatly improve if we could replicate SC niches in vitro or understand how niches form following transplantation. Directed differentiation of human pluripotent stem cells (hPSCs) to skeletal muscle is among the few robust in vitro systems able to increase PAX7 expression by 1,000-fold. We thus performed bioinformatic lineage tracing using single nucleus RNA seq of hPSC undergoing directed differentiation to identity how muscle progenitor cells arise commitment and are supported by their unique in vitro niches. Analysis using CellRank identified the myogenic lineage commitment signature and supportive cell types that arise shortly after mesoderm induction, which we traced through several stages of in vitro myogenesis. We then transplanted hPSC-derived muscle progenitors into NSG mice and performed spatial transcriptomics at 5 time points post engraftment to determine how progenitors form and interact with their niches. We found that within a subset of muscle progenitors the expression of the stem cell marker PAX7 increases between days 1-5 in vivo and concurrently the stem cell niche receptor gene MEGF10 was highly expressed by these cells. We thus engineered a tetracycline inducible MEGF10 system in hPSCs, differentiated hPSCs to muscle progenitors, and induced MEGF10 at several time intervals following transplantation in NSG mice with doxycycline. We found that the first 10 days of MEGF10 overexpression in vivo significantly increased niche formation and PAX7 numbers, and demonstrate that MEG10 activates Notch signaling as a potential mechanism of action. Just as adult stem cells are dynamically regulated by their niche, hPSC-derived niches formed in development control cell fate decisions to mature progenitor cells. Identifying the unique ligands and targets can help transplanted progenitors colonize available niches to enable effective long term cell therapies skeletal muscle regeneration.
