Abstract Human pluripotent stem cells (hPSCs) transplantation therapies offer an appealing avenue to combat skeletal muscle diseases. Directed differentiation of hPSCs to skeletal muscle is among the few robust in vitro systems able to increase PAX7 expression by 1,000-fold. However, a drawback of these hPSC-derived muscle cells is that they are functionally immature compared to true adult muscle stem cells. Just as adult stem cells are dynamically regulated by their niche, embryonic niches formed in development equivalently control cell fate decisions to mature from an early precursor or progenitor and eventually to an adult quiescent stem cell. Importantly, embryonic niches also change over developmental stages, but for hPSCs which form muscle the niche is understudied or has never been studied. My work is significant because niches which support muscle stem cells during the repair process, also develop in parallel with the maturation of progenitor and stem cells from development through to adulthood. This proposal consists of complementary in vitro and in vivo aims with the overall goal of supporting PAX7 cell functions and numbers from hPSCs. In Aim 1, we will interrogate how muscle cells arise in the first place and develop through key functional hallmarks of myogenesis. To accomplish this aim we will use a lineage tracer built of a key myogenic commitment factor, SIX1, and test whether SIX1 co-factors lead to functional maturation of hPSC myogenic cells. When hPSCs differentiate to muscle they also produce impure cultures containing non-myogenic lineages that we hypothesize support the muscle cells in culture. We will use genetic inhibition of master transcription factors of these non-myogenic lineages to test their need in the support and generation of hPSC muscle. Aim 2, seeks to understand how to support hPSC muscle cells once they are made. We will build a simplified in vitro niche made of myotubes, extracellular matrix taken from human tissue muscle, and PAX7 muscle progenitors, and over express key ligands and receptors that we have already identified by spatial RNA seq to test their potential to support PAX7 cells. We will also test the key role of hypoxia in hPSC PAX7 cell support in vitro. Lastly, in Aim 3, we will pivot to in vivo systems using a mouse model available in the Hicks lab that inducible ablates the mouse Pax7 cells and enables improved engraftment by hPSC PAX7 cells. We will first perform a series of in-depth time course experiments to identify the timing of skeletal muscle stem cell niche formation following hPSC PAX7 cell transplantation. We will then use an inducible overexpression system to test the function of a key hPSC muscle niche factor, MEGF10, for enabling gain-of-function in at multiple stages of PAX7 niche formation. Finally, we will test whether MEGF10 induction can improve the ability of hPSC PAX7 cells to repopulate new myofibers and re-establish in vivo niches after injuries once transplanted. Success...