Carvalho K A T C, Oliveira L, Malvezzi M, Simeoni R B, Francisco J C, Olandoski M, Guarita-Souza L C
Cellular Culture Experimental Laboratory, PUCPR, Curitiba, Brazil.
Transplant Proc. 2008 Apr;40(3):842-4. doi: 10.1016/j.transproceed.2008.02.053.
The product generated by skeletal muscle and bone marrow mesenchymal stem cell cocultures has been demonstrated to improve the functional outcomes after cell therapy in postinfarction or Chagas myocardiopathy. This coculture method allows cell interactions in vitro, diminishing the operational costs of the culture/expansion as well as leading to angiogenesis and myogenesis for regeneration of the injured heart. Flow cytometric analysis may better characterize the cellular types in this model. Our objective was to use flow cytometry to analyze the immunophenotype expressed in this coculture model. The coculture was performed in accordance with Carvalho for 21 days. Flow cytometry was performed before and after coculture to characterize the immunophenotypic profile of cellular subsets, namely, the surface markers CD31, CD34, CD44H, CD45, CD49d, CD54, CD73, CD90, CD105, CD106, Myo-D, M-cadherin, and Connexin-43. Statistics were performed by the nonparametric Friedman test (P < .05) with post-hoc analysis by the nonparametric Wilcoxon test (P < or = .017, Bonferroni correction). The results demonstrated statistical significance for CD45(+) in 89.49% of mononuclear cells, 3.58% in skeletal muscle cells, and 4.74% among cocultured cells (P = .0094); and CD90(+) in 36.18% of mononuclear cells, 6.01% in skeletal muscle cells, and 48.94% among cocultured cells (P = .0420). The cocultured cells expressed the markers CD73(++), CD90(+++), CD45(-), CD34(+), CD105(-/+), CD106(-/+), M-cadherin(-/+), and Connexin-43(-/+). In conclusion, flow cytometric analysis showed a heterogeneous adherent cell population in this coculture model.
骨骼肌与骨髓间充质干细胞共培养所产生的产物已被证明可改善心肌梗死后或恰加斯心肌病细胞治疗后的功能结局。这种共培养方法允许细胞在体外相互作用，降低培养/扩增的操作成本，并导致血管生成和肌生成，以促进受损心脏的再生。流式细胞术分析可更好地表征该模型中的细胞类型。我们的目的是使用流式细胞术分析此共培养模型中表达的免疫表型。按照卡瓦略的方法进行共培养21天。在共培养前后进行流式细胞术，以表征细胞亚群的免疫表型特征，即表面标志物CD31、CD34、CD44H、CD45、CD49d、CD54、CD73、CD90、CD105、CD106、Myo-D、M-钙黏蛋白和连接蛋白-43。采用非参数弗里德曼检验进行统计学分析（P <.05）并通过非参数威尔科克森检验进行事后分析（P <或 =.017，邦费罗尼校正）。结果显示，在89.49%的单核细胞、3.58%的骨骼肌细胞和4.74%的共培养细胞中，CD45(+)具有统计学意义（P =.0094）；在36.18%的单核细胞、6.01%的骨骼肌细胞和48.94%的共培养细胞中，CD90(+)具有统计学意义（P =.0420）。共培养细胞表达标志物CD73(++)、CD90(+++)、CD45(-)、CD34(+)、CD105(-/+)、CD106(-/+)、M-钙黏蛋白(-/+)和连接蛋白-43(-/+)。总之，流式细胞术分析显示该共培养模型中存在异质性贴壁细胞群体。
