| We also performed quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry analysis to elucidate the gene and protein expression profile of the iMuSCs and compared these to embryonic stem cells (ESCs) and myogenic stem cells (C2C12 and MuSCs). The iMuSCs expressed Oct4, Ssea1 (Stage-specific embryonic antigen 1), Sox2, Cxcr4, Msx1, Pax7, and Sca1 (Fig. 3a and Supplementary Fig. S3a,b), similar to the ESCs, but at a lower expression level. QPCR analysis revealed that the iMuSCs expressed the majority of pluripotency marker genes, with the exception of Esg1 and Dax1 (Fig. 3b); however, unlike the ESCs, the iMuSCs expressed myogenic marker genes and interestingly some of the primordial germ-cell-related markers, e.g. Blimp1 and Fragilis, and did not express other lineage related genes, such as CD45 or CD90 (Fig. 3c). Moreover, the iMuSCs were positive for alkaline phosphatase (Fig. 3a). These results indicate that the iMuSCs are similar to, but not identical to the ESCs, since they maintain their myogenic memory (e.g., high expression of myogenic genes when compared to the ESCs, and are easily induced to differentiate into a myogenic lineage in vitro and in vivo).
To clarify the pluripotent potential of the iMuSCs, we performed differentiation assays6,7 in vitro that showed that the iMuSCs were able to form embryoid bodies (EBs) in a petri dish (Fig. 3d,e). After seven days in suspension culture, EBs were expanded and initiated spontaneous differentiation into a variety of ectodermal and mesodermal germ layer derivatives, and after an additional two weeks in culture, attached EBs formed contracting multinucleated myotubes encompassed with neural-like structures (Fig. 3f,g). |
