Effect of Notch1 signaling on muscle engraftment and maturation from pluripotent stem cells

Pluripotent stem cell-derived iPax3 myogenic progenitors display an embryonic molecular signature but become postnatal upon transplantation. Because this correlates with upregulation of Notch signaling, here we probed whether Notch1 is required for in vivo maturation by performing gain- and loss-of-function studies in iPax3 myogenic progenitors. Transplantation studies revealed comparable myofiber engraftment. However, assessment of fiber type composition revealed more mature myofiber grafts in the overexpression cohort, as shown by the lower expression of embryonic MHC and increased levels of type IIX MHC. Surprisingly, more mature myofiber were also observed in the absence of Notch1, suggesting its dispensability for in vivo maturation. Donor-derived Pax7+ cells were detected in all transplants, however, in the absence of Notch1, secondary grafts exhibited a high fraction of these cells in the interstitial space. RNA-sequencing showed changes in extracellular matrix organization, cell cycle, and metabolism, suggesting that Notch1 is required for satellite cell homing. To investigated the effect of Notch1 GOF and LOF in iPax3 myogenic progenitors, we overexpress NICD1 (iPax3-N1OE) or knocking-out (KO) Notch1 (iPax3-N1KO) in PSC-derived iPax3 myogenic progenitors. To determine the molecular changes underlying Notch1 GOF in iPax3 myogenic progenitors in an unbiased manner, we next performed RNA sequencing. To elucidate the molecular determinants underlining the differences in satellite cell engraftment between iPax3-N1OE, iPax3-N1KO and iPax3 cohorts, we performed RNA sequencing analysis in donor-derived satellite cells.