Abstract
BackgroundMyogenic transdifferentiation can be accomplished through ectopic MYOD1 expression, which is facilitated by various signaling pathways associated with myogenesis. In this study, we attempted to transdifferentiate pig embryonic fibroblasts (PEFs) myogenically into skeletal muscle through overexpression of the pig MYOD1 gene and modulation of the FGF, TGF-β, WNT, and cAMP signaling pathways.ResultsThe MYOD1 overexpression vector was constructed based on comparative sequence analysis, demonstrating that pig MYOD1 has evolutionarily conserved domains across various species. Although forced MYOD1 expression through these vectors triggered the expression of endogenous muscle markers, transdifferentiated muscle cells from fibroblasts were not observed. Therefore, various signaling molecules, including FGF2, SB431542, CHIR99021, and forskolin, along with MYOD1 overexpression were applied to enhance the myogenic reprogramming. The modified conditions led to the derivation of myotubes and activation of muscle markers in PEFs, as determined by qPCR and immunostaining. Notably, a sarcomere-like structure was observed, indicating that terminally differentiated skeletal muscle could be obtained from transdifferentiated cells.ConclusionsIn summary, we established a protocol for reprogramming MYOD1-overexpressing PEFs into the mature skeletal muscle using signaling molecules. Our myogenic reprogramming can be used as a cell source for muscle disease models in regenerative medicine and the production of cultured meat in cellular agriculture.
Highlights
Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression, which is facilitated by various signaling pathways associated with myogenesis
It demonstrates that the WNT signaling induces the commitment of myogenic precursors and has been substantiated by previous research showing that the WNT activator CHIR99021 with SB431542 and fibroblast growth factor 2 (FGF2) induces myogenic specification from human induced pluripotent stem cells (iPSCs) [14]
Pig MYOD1 overexpression vector construction Myod1/MYOD1 is reportedly identified as a master transcription factor in myogenesis, thereby inducing myogenic transdifferentiation in non-muscle cells [9, 24,25,26,27]
Summary
Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression, which is facilitated by various signaling pathways associated with myogenesis. The WNT activator secreted from the notochord, neural tube, and surrounding tissue is involved in a series of specifications along with the presomitic mesoderm, somite, dermomyotome, and myotome, as determined by in vitro directed differentiation in human and mouse PSCs [3, 6, 7]. It demonstrates that the WNT signaling induces the commitment of myogenic precursors and has been substantiated by previous research showing that the WNT activator CHIR99021 with SB431542 and FGF2 induces myogenic specification from human iPSCs [14]. The combination of FGF2, a WNT activator, and forskolin stimulated skeletal muscle differentiation in human iPSCs and especially forskolin improved satellite cell expansion in mice [16]
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