Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan.
In Vitro Cell Dev Biol Anim. 2012 Apr;48(4):203-15. doi: 10.1007/s11626-012-9488-x. Epub 2012 Mar 7.
The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.
本研究旨在评估从脂肪结缔组织（AD-MSCs）、骨髓（BM-MSCs）和骨骼肌组织（MC-MSCs）中分离的间充质干细胞（MSCs）的体外分离和肌向分化效率。从两只 6 周龄成年大鼠的脂肪结缔组织、骨髓和骨骼肌组织中分离 MSCs。培养的 MSCs 用 5-氮杂胞苷（AZA）处理以诱导肌向分化。通过免疫细胞化学（ICC）、荧光激活细胞分选（FACS）、PCR 和 RT-PCR 评估分离的 MSCs 和分化细胞。AD-MSCs 显示出最高的增殖率，而 BM-MSCs 则具有最低的增殖率。在 ICC 中，分离的 MSCs 强烈表达 CD90 和 CD44，而 CD45、CD31 和 CD34 表达阴性，而 AZA 处理的 MSCs 强烈表达结蛋白。在 FACS 分析中，AD-MSCs 具有最高比例的 CD90 和 CD44 阳性表达细胞（99%和 96%），其次是 BM-MSCs（97%和 94%）和 MC-MSCs（92%和 91%）。在用 AZA 处理孵育 1 周后，分化的 MC-MSCs 中肌球蛋白基因表达峰值达到 93%，BM-MSCs 中达到 83.3%，AD-MSCs 中达到 77%。从脂肪结缔组织、骨髓和骨骼肌组织中分离的 MSCs 具有相同的形态和表型，但 AD-MSCs 最容易获得，在培养中生长速度最快，干细胞标志物表达比例最高。此外，尽管 MC-MSCs 显示出最高的成肌分化潜能和肌母细胞标志物表达，但 AD-MSCs 和 BM-MSCs 仍然是有价值的替代品。分化的成肌细胞可能是再生医学中肌母细胞自体移植的一种新的可行选择。
