ABSTRACT Human pluripotent stem cells (hPSCs) offer enormous potential for understanding human myogenesis and modeling diseases in a dish. In the previous funding period, we have identified the developmental trajectory of skeletal muscle progenitor cells (SMPCs) derived from hPSCs to be around embryonic to fetal week 7-12 stage in human development. We showed that SMPCs do not engraft into the mouse niche efficiently whereas adult muscle stem cells (SCs) have this robust potential. Further as disease severity increases in mdx mouse models of Duchenne Muscular Dystrophy we found an increase in aberrant cells in the microenvironment altering robust engraftment. The next phase of this work in this proposal is now focused on improving our ability to modulate SMPC and SC states and improve engraftment into the mouse niche as true repopulating stem cells. By using human development as a model, our work has already yielded significant insights into our understanding of human myogenesis and the generation of myogenic cells. We will utilize the tools, sequencing datasets and knowledge gained in the previous funding period to modulate SMPC and SC states and generate a better therapeutic candidate in this renewal. We will define and functionally validate the key regulatory networks important for controlling embryonic, fetal SMPCs, or adult SCs and improve our basic knowledge of how progenitors and stem cells change over time during human myogenesis and from hPSCs (Aim 1). Further we will evaluate the parameters important for modulating niche occupancy from hPSC SMPCs (Aim 2). This work will improve our ability to generate a robust stem cell for improved disease modeling and use in regenerative medicine.