Thesis

Using Synthetic RNA to Overexpress PAX7 in Human Induced-Pluripotent Stem Cells to Produce Skeletal Muscle Progenitors

Skeletal muscle myofibers contain muscle stem cells called satellite cells that express the transcription factor PAX7, and regenerate muscle after acute injury. These skeletal muscle progenitor cells (SMPCs), or satellite cells in the adult muscle, lie outside the muscle fiber and refurbish the endogenous muscle stem cells upon damage. Human pluripotent stem cells (hPSCs) have enormous potential for use in regenerative medicine, especially for muscle wasting diseases like DMD. In DMD, the satellite cells become exhausted leading to failed regenerative ability. Once these muscle stem cells are depleted, damaged muscles are replaced by excessive fat and extracellular matrix deposition leading to muscle deterioration and fibrosis. One potential target to replenish exhausted muscle stem cells in patients with DMD is to generate an equivalent source from hPSCs. The myogenic activity from SMPCs derived from hPSCs is currently not well understood. We evaluated human fetal muscle in order to better understand the timing and specification of human SMPCs during human development and compare to hPSC-derived SMPCs for in vitro and in vivo myogenic activity. We have also shown that PAX7 is present in week 9 and 17 fetal skeletal muscle tissue, which may be indicative of when SMPC differentiation occurs. hPSC-derived SMPCs will be developed by the use of directed differentiation and synthetic mRNA. We have cloned PAX7 coding region into a VEE vector to enable synthetic RNA mediated overexpression (OE) of PAX7 into pre-differentiated hiPSCs in various plating conditions. PAX7 expression exceeded 700 fold in some conditions. Understanding the developmental and molecular underpinnings of human skeletal myogenesis will provide roads into generating SMPCs from hPSCs for use in regenerative medicine.

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