MYOD 介导人羊水干细胞的骨骼肌肉生成分化和肌肉损伤的再生。

MYOD mediates skeletal myogenic differentiation of human amniotic fluid stem cells and regeneration of muscle injury.

出版信息

Stem Cell Res Ther. 2013;4(6):147. doi: 10.1186/scrt358.

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4054934/

Abstract

INTRODUCTION

Human 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.

METHODS

In 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.

RESULTS

MYOD 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.

CONCLUSIONS

Collectively, 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.

摘要

简介

已证实人羊水干细胞（hAFS）可分化为多种谱系，包括成肌细胞。然而，hAFS 细胞成肌分化的分子机制及其对肌肉损伤的再生潜力仍有待阐明。

方法

为了诱导 hAFS 细胞的成肌分化，构建了 MYOD 的慢病毒并转导至 hAFS 细胞。通过免疫细胞化学分析肌管样细胞的形成，通过逆转录聚合酶链反应和 Western blot 分析成肌细胞的分子标志物表达。为了进行体内肌肉再生，将转导 MYOD 的 hAFS 细胞注射到用心脏毒素损伤的左胫骨前肌（TA）中，并通过苏木精和伊红、免疫细胞化学和神经肌肉接头形成分析肌肉再生。

结果

hAFS 细胞中 MYOD 的表达成功诱导其分化为多核肌管样细胞。一致地，MYOD 显著诱导了成肌标志物基因，如 MYOG、DES、DMD 和 MYH 的表达。对前成肌因子的分析表明，MYOD 显著增加了 PAX3、MEOX1 和 EYA2 的表达。MYOD 被磷酸化并定位于细胞核。这些结果表明，在 hAFS 细胞中，MYOD 被磷酸化并定位于细胞核，从而诱导成肌因子的表达，导致 hAFS 细胞的成肌分化。为了测试转导 MYOD 的 hAFS 细胞的再生潜力，我们将其移植到免疫缺陷 BALB/cSlc-nu 小鼠的损伤肌肉中。结果表明，注射 MYOD-hAFS 细胞的 TA 肌肉体积显著增加。此外，与对照组相比，注射 MYOD-hAFS 细胞的 TA 肌肉组织具有更多的神经肌肉接头，表明 MYOD-hAFS 细胞对肌肉损伤的功能恢复。