Introduction: Human amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages,\r\nincluding myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and\r\ntheir regenerative potential for muscle injury remain to be elucidated.\r\nMethods: In order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and\r\ntransduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression\r\nof molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western\r\nblotting. For in vivo muscle regeneration, MYOD transduced hAFS cells were injected into left tibialis anterior\r\n(TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxylin and eosin,\r\nimmunocytochemistry and formation of neuro-muscular junction.\r\nResults: MYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells.\r\nConsistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD and MYH, was induced by MYOD.\r\nAnalysis of pre-myogenic factors showed that expression of PAX3, MEOX1 and EYA2 was significantly increased\r\nby MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells,\r\nMYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in\r\nmyogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them\r\ninto injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA\r\nmuscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more numbers of\r\nneuro-muscular junctions compared to controls, indicating functional restoration of muscle injury by MYOD-hAFS cells.\r\nConclusions: Collectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal\r\nmyogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.
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