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目的:比较骨髓来源的胚胎样干细胞(ELSCs)与间充质干细胞(MSCs)的体外成肌分化能力。方法:采用胚胎干细胞扩增用的无血清Knockout-DMEM培养基在明胶包被过的培养瓶中培养人骨髓单个核细胞以分离ELSCs,传统方法从相同骨髓中分离MSCs,倒置相差显微镜下观察细胞形态特征,采用免疫荧光染色鉴定多潜能抗原标志的表达。成肌分化液分别培养ELSCs和MSCs,采用免疫染色法检测肌纤维特异性抗原标志肌球蛋白重链(MHC)、成肌素(myogenin)和MyoD蛋白的表达,RT-PCR检测MHC、myogenin和MyoD mRNA的表达,计算MHC阳性肌纤维的比例以比较ELSCs与MSCs的体外成肌分化能力。结果:无血清培养基可从骨髓中分离到弱表达多潜能抗原标志Oct-4、Nanog-3和Sox-2的ELSCs,体积较小,形态纤细均一,在形态方面不同于相同骨髓来源的MSCs,后者不表达多潜能抗原标志。在成肌分化液中培养,ELSCs和MSCs均可被诱导为在蛋白和mRNA水平表达MHC和myogenin的多核肌纤维,但诱导培养10 d时,ELSCs的MHC蛋白阳性肌纤维的比例为(25.7±4.1)%,MSCs为(15.8±7.6)%,ELSCs的成肌分化能力明显高于MSCs(P<0.05)。结论:骨髓ELSCs能被诱导为多核肌纤维,并具有比来自相同骨髓的MSCs更强的成肌分化能力,ELSCs是肌病治疗更理想的种子细胞。
Objective: To compare the myogenic differentiation ability of bone marrow-derived embryonic-like stem cells (ELSCs) and mesenchymal stem cells (MSCs) in vitro. METHODS: Human bone marrow mononuclear cells were cultured in serum-free Knockout-DMEM medium for embryonic stem cell expansion to separate ELSCs. MSCs were separated from the same bone marrow by conventional methods and observed under inverted phase contrast microscope Morphological characteristics, the use of immunofluorescence staining to identify signs of pluripotency antigen expression. Myofibroblasts were cultured in myogenic differentiation solution and MSCs respectively. The expression of myosin heavy chain (MHC), myogenin and MyoD protein were detected by immunostaining. The expression of MHC, myogenin and MyoD mRNA expression, the proportion of MHC-positive muscle fibers was calculated to compare the myogenic differentiation ability of ELSCs and MSCs in vitro. Results: ELSCs with weakly expressed pluripotent antigen markers Oct-4, Nanog-3 and Sox-2 could be isolated from bone marrow by serum-free medium. The ELSCs were smaller in size and homogeneous in morphology and different in morphological characteristics from MSCs derived from the same bone marrow , The latter does not express pluripotency antigen markers. When cultured in myogenic differentiation solution, both ELSCs and MSCs could be induced to express multi-core muscle fibers of MHC and myogenin at the protein and mRNA levels. However, the proportion of MHC protein-positive muscle fibers in ELSCs was (25.7 ± 4.1) %, MSCs was (15.8 ± 7.6)%. The myogenic differentiation ability of ELSCs was significantly higher than that of MSCs (P <0.05). CONCLUSIONS: Bone marrow ELSCs can be induced into multi-nuclear muscle fibers and have stronger myogenic differentiation ability than MSCs from the same bone marrow. ELSCs are more ideal seed cells for myopathy treatment.