Mash4 induces Pax7 expression and myogenic differentiation in ESCs
Tomohide Takaya, Shuichi Watanabe, Yoko Asakura, Michael Kyba, Atsushi Asakura.
Stem Cell institute, Paul & Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School, Minneapolis, MN, USA.
The 17th Annual Minnesota Muscle Symposium (Minneapolis, USA), 2013/06/07 (Poster).
Abstract
Cell therapy using skeletal muscle satellite cell-derived myoblasts (MBs) into Duchenne muscular dystrophy patients has shown that the transplanted MBs can participate in muscle regeneration and improve muscle function. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are possible cell sources of satellite cells and MBs for therapy. However, the efficiency of satellite cell and MB differentiation from ESCs/iPSCs is extremely low unless myogenic regulatory factors such as Pax7 or MyoD are used to be over-expressed in these cells. Therefore, it is crucial to understand molecular cascades of myogenic development in ESC cultures. Recently, we noticed that mouse achaete-scute complex homolog-4 (Mash4), a basic helix-loop-helix transcription factor, is expressed in quiescent satellite cells and plays essential roles in satellite cell self-renewal by promoting Pax7 expression in adult skeletal muscle.
In this study, we investigated whether Mash4 is also able to induce Pax7 and myogenic differentiation in mouse ESCs. For this purpose, we recently established iMash4 ESCs in which exogenous Mash4 expression can be induced by treatment with doxycycline (Dox). To induce myogenic differentiation, iMash4 ESCs were 2-dimensionally cultured with or without Dox treatment for 21 days. Myogenic differentiation was examined by immunofluorescent staining for Pax7 and MyoD expression. In the presence of Dox, Pax7 but not MyoD was clearly detected from day 5 and these Pax7(+)/MyoD(-) myogenic progenitor-like cells formed clusters. Subsequently, Pax7(+)/MyoD(+) MB-like cells can be detected at day 17, and Pax7(-)/MyoD(+) differentiated muscle cells were observed at day 21. On the other hand, Pax7 expression was disappeared after Dox removal, indicating that continuous Mash4 expression is required for Pax7 expression in the myogenic progenitor-like cells. These results strongly indicate that Mash4 is an up-stream myogenic regulatory factor which induces Pax7 expression followed by myogenic differentiation in mouse ESCs.