MuSC expansion through EGFR activity inhibition
Sadegh Davoudi 1, 2 ; Jing Yang 3 ; Chih-Ying Chin 1, 2 ; Penney M. Gilbert 1, 2
1. Institute for Biomaterials and Biomedical Engineering, University of Toronto; 2. Donnelly Centre for Cellular and Biomolecular Research; 3. Division of Engineering Science, University of Toronto
Murine muscle stem cell (MuSC) isolation is a relatively recent phenomenon, and protocols for human MuSC isolation are even more novel. Ex-vivo expansion of the stem cell population has provided to be a common challenge, with the cells losing their stem cell characteristics after a few passages. This is evident by reduced regenerative capacity of the expanded population upon transplantation compared to freshly isolated MuSCs. The inability to culture MuSCs places restrictions on skeletal muscle regeneration research. Additionally, considering the limited number of MuSCs in skeletal muscle, the inability to expand this population ex-vivo must be overcome before MuSC transplantation can be considered as an effective treatment for muscle diseases. Despite recent progress, there is much needed research on the ex-vivo expansion and culture of MuSCs.
We tested a library of 462 anti-cancer drugs targeting a variety of signaling pathways. A 12-point concentration curve of each drug was tested on murine MuSCs for 7 days. 214 compounds resulted in higher cell numbers and less death, i.e. were considered as hits.
EGFR was a common target in many of the successful compounds and was chosen as a follow up candidate. Clonal analysis of freshly isolated MuSCs exposed to Erlotinib Hydrochloride (E-HCl), an EGFR inhibitor, over a period of 4 days revealed an increase in the number of MuSCs that enter cell cycle and a reduction in time between divisions after the first division, ultimately leading to a higher number of cells over the same time period. We next assessed the regenerative capacity of freshly isolated MuSCs exposed to EGFR inhibitor by transplanting actin-GFP MuSCs treated with E-HCl for 7 days into the irradiated and injured tibialis anterior of NSG mice. Our results revealed an increase in the contribution towards regeneration of cells exposed to the EGFR inhibitors. In summary, in vitro inhibition of EGFR activity results in increased proliferation and regenerative capacity of freshly isolated MuSCs.