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

Pluripotent stem cell-derived myogenic progenitors change from an embryonic to a postnatal molecular signature upon engrafting as satellite cells, which coincides with upregulation of Notch3. Since a role for Notch3 in skeletal muscle maturation is unknown, here we investigate whether Notch3 is required for this in vivo molecular maturation switch.

Our results show that lack of Notch3 in transplanted progenitors (N3KO) does not impact degree of engraftment, but leads to increased numbers of embryonic myofibers. Conversely, transplantation of Notch3 overexpressing (N3OE) myogenic progenitors results in lower numbers of embryonic myofibers, but diminished muscle grafts when compared to empty vector (EV) controls. Secondary transplantation studies confirmed these effects, whereby Notch3 overexpression significantly reduced secondary engraftment. Further characterization of N3OE donor-derived satellite cells revealed reduced proliferation and downregulation of cell cycle genes. Importantly, secondary grafts from N3KO satellite cells had increased numbers of embryonic myofibers compared to N3OE and EV controls.

Taken together, these findings demonstrate that Notch3 signaling is required for myofiber maturation, and that constant activation of Notch3 impairs proliferation and muscle regeneration. Transcriptional profiles of N3OE donor-derived satellite cells suggest that dampened regeneration may be driven by inhibitory alterations in cell cycle regulation.