Necessity of Notch3 signaling in myofiber maturation in a pluripotent stem cell transplant model

This study investigates the role of Notch3 signaling in muscle regeneration using iPax3-derived myogenic progenitors. We generated iPax3-N3KO (Notch3-deficient) and iPax3-N3OE (Notch3-overexpressing) progenitors to assess how Notch3 influences muscle regeneration and myofiber maturation. In vivo transplantation of iPax3-N3KO progenitors showed normal myofiber engraftment but increased expression of embryonic myosin heavy chain (eMHC), suggesting delayed myofiber maturation. Conversely, iPax3-N3OE progenitors exhibited reduced myofiber engraftment and decreased eMHC expression, indicating that Notch3 activation inhibits myogenic progenitors differentiation and myofiber maturation. Secondary transplantation experiments showed similar engraftment rates between iPax3 (empty vector control, “EV”) and iPax3-N3KO satellite cells, but significantly reduced engraftment from iPax3-N3OE satellite cells. Additionally, fiber type composition analysis revealed increased eMHC expression in Pax3-N3KO grafts and no differences in iPax3-N3OE grafts compared to control. These findings suggest that Notch3 signaling plays a critical role in myofiber maturation and differentiation. Absence of Notch3 results in less mature fibers, while its activation impairs differentiation. Transcriptional profiling of iPax3-N3OE donor-derived satellite cells indicates that these effects may be driven by alterations in cell cycle regulation. Overall design: To investigated the effect of Notch3 GOF in iPax3 myogenic progenitors, we overexpress NICD3 (iPax3-N3OE) in PSC-derived iPax3 myogenic progenitors. To determine the molecular changes underlying Notch3 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-N3OE and iPax3 cohorts, we performed RNA sequencing analysis in donor-derived satellite cells.