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Abstract
IntroductionHuman amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and their regenerative potential for muscle injury remain to be elucidated.MethodsIn order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and transduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression of molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western blotting. For in vivo muscle regeneration, MYOD transduced hAFS cells were injected into left tibialis anterior (TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxylin and eosin, immunocytochemistry and formation of neuro-muscular junction.ResultsMYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells. Consistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD and MYH, was induced by MYOD. Analysis of pre-myogenic factors showed that expression of PAX3, MEOX1 and EYA2 was significantly increased by MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells, MYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in myogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them into injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA muscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more numbers of neuro-muscular junctions compared to controls, indicating functional restoration of muscle injury by MYOD-hAFS cells.ConclusionsCollectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal myogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.
Highlights
Human amniotic fluid stem cells have been shown to differentiate into multiple lineages, including myoblasts
Characterization of Human amniotic fluid stem (hAFS) cells Human amniocentesis specimens were obtained from Kyungpook National University Hospital. hAFS cells were isolated as described in the Methods section
Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that hAFS cells express stem cell marker genes for SCF, GATA-4, VIM, CK18, OCT4 and FGF5 throughout the culture period (Figure 1a)
Summary
Human amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. Accumulating evidence has demonstrated the ability of amniotic fluid stem (AFS) cells to differentiate into multiple lineages [1,2,3,4]. AFS cells harbor some of the characteristics of embryonic stem cells. AFS cells express the transcription factor OCT4, which is known to be expressed in embryonic stem cells. AFS cells express the pluripotent stem cell marker, telomerase reverse transcriptase [9]. These results suggest that, in terms of their versatility, AFS cells have an intermediate potential between embryonic and adult stem cells [7,9]
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