Myogenic differentiation of mesenchymal stem cells co-cultured with primary myoblasts
Myogenic differentiation of mesenchymal stem cells co-cultured with primary myoblasts
复制标题
DOI：
10.1042/cbi20100417
复制
发表时间：
2011-04-01
影响因子：
3.9
通讯作者：
Kneser, Ulrich
中科院分区：
文献类型：
--
作者：
Beier, Justus P.;Bitto, Franz F.;Kneser, Ulrich
骨骼肌组织工程是一种很有前途的肌肉组织重建方法。本研究评估了MSC（间充质干细胞）作为该应用的新细胞来源。与原代成肌细胞共培养作为一种新的诱导骨髓间充质干细胞向成肌细胞分化的方法，并通过免疫细胞化学（ICC）方法对GFP（绿色荧光蛋白）转导的大鼠骨髓间充质干细胞与原代大鼠成肌细胞共培养的成肌潜能进行了评价。通过ICC、FACS和qPCR（定量FOR）分析MSC的成肌潜能。MSC成肌细胞融合现象导致杂交肌管进行了评估，使用一种新的方法来评估肌管融合率的基础上相差和荧光显微镜。此外，MSC组成型表达肌源性标志物MEF 2（肌源性增强因子2）和α-肌节肌动蛋白，MEF 2表达上调后，与原代成肌细胞共培养，并添加肌源性培养基补充剂。当bFGF（碱性成纤维细胞生长因子）和地塞米松加入到共培养物中时，参与肌管形成的MSC细胞核数量显著增加。总之，我们已经确定了MSC成肌分化直至肌管形成的最佳共培养条件，这是MSC作为骨骼肌TE以及其他基于肌细胞的疗法的细胞来源的适用性的有希望的一步。
TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co-cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)-transduced rat MSC co-cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative FOR). MSC-myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and alpha-sarcomeric actin, and MEF2 expression was up-regulated upon co-cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co-cultures. In summary, we have determined optimal co-culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell-based therapies.