Clonal Dedifferentiation of Human Myogenic Progenitors into Skeletal Muscle Stem Cells Induced by MAPK Inhibition.
Authors: Wang Lei , Walker Brandon L. , Bhatt Devang , Kennedy Patrick J. , Tse William T. 
Published: April 2020

Abstract
Understanding the biology of skeletal muscle stem cells can facilitate development of effective cellular therapy for muscle diseases such as muscular dystrophy. While studying human bone marrow stromal cells, we identified a stromal cell subclone, WB15-M, which has developed spontaneously into a skeletal muscle cell line. This subclone no longer expressed CD105, CD90 and CD44, cell surface markers typically found on bone marrow stromal cells. Instead they expressed alpha7-integrin, an antigen found on myoblasts and regenerating muscles. WB15-M cells were positive for the myogenic regulatory factors MyoD and Myf5 and, when cultured under a low-serum condition, matured into multinucleated myofibers that expressed sarcomeric alpha-actinin, myosin heavy chain, dystroglycan and dystrophin, indicating that WB15-M cells were committed myogenic progenitors. We asked if the WB15-M cells might contain skeletal muscle stem cells. Immunofluorescent microscopic studies revealed that rare WB15-M cells expressed Pax7, Pax3 and Msx1, nuclear factors found in skeletal muscle stem cells. When WB15-M cells were cultured in SB203580 or PD98059, inhibitors of the p38 and Erk mitogen-activated protein kinases (MAPK), respectively, they markedly enhanced expression of Pax7, Pax3 and Msx1. The increase in the expression of these nuclear factors could be blocked by simultaneous treatment of WB15-M cells with orthovanadate, a protein tyrosine phosphatase. Alternatively, the increase could be induced by chemically inhibiting Mnk1, a common downstream target of the p38 and Erk MAPK signaling cascades. When further stimulated with bone morphogenic protein-2, MAPK inhibitor-treated WB15-M cells acquired the ability to express alkaline phosphatase, an early osteoblast marker, a property also seen in skeletal muscle stem cells. In contrast, untreated WB15-M cells did not exhibit this property. Clonal analysis showed that the biological changes exhibited by WB15-M cells upon MAPK inhibition was not an artifact of cellular heterogeneity but the result of reversion of individual committed myogenic progenitors to stem cell-like precursors that were more primitive in their development. Purified myogenin-expressing cells that have already initiated their myogenic differentiation program could still revert clonally to these stem cell-like precursors upon MAPK inhibition, indicating a bona fide dedifferentiation process and a true reversal of developmental fate. When WB15-M cells treated with MAPK inhibitors were cultured clonally under conditions that promoted both myogenic and osteogenic development, they formed colonies that expressed either myogenin or alkaline phosphatase but not both; untreated WB15-M cells cultured under the same conditions formed only myogenin-expressing colonies. In conclusion, we found that human bone marrow stromal cell-derived myogenic progenitors could be induced by MAPK inhibition to dedifferentiate into precursors that exhibited properties of the skeletal muscle stem cells. This finding should facilitate the development of novel cellular therapy that utilizes skeletal muscle stem cells.