Sibov Tatiana Taís, Severino P, Marti L C, Pavon L F, Oliveira D M, Tobo P R, Campos A H, Paes A T, Amaro E, F Gamarra L, Moreira-Filho C A
Instituto do Cérebro, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, Hospital Israelita Albert Einstein, Avenida Albert Einstein, no 627/701, Morumbi, São Paulo, SP, CEP: 05651-901, Brazil,
Cytotechnology. 2012 Oct;64(5):511-21. doi: 10.1007/s10616-012-9428-3. Epub 2012 Feb 12.
Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs' protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.
从足月分娩的脐带血（UCB）中分离间充质干细胞（MSC）是一个费力、耗时的过程，且细胞产量低。在本研究中，我们确定了有助于成功分离UCB-MSC的参数。根据我们的研究结果，当从妊娠时长不超过37周的正常足月妊娠所采集的UCB中分离MSC时，成功分离这些细胞的几率更高。除了妊娠时长外，为成功分离这些细胞，还应考虑UCB的血量和储存时间。在此，我们发现采集的理想血量应超过80 mL，储存时间不应超过6小时。我们通过形态学、免疫表型、蛋白质/基因表达以及成脂分化潜能对UCB-MSC进行了表征。分离出的UCB-MSC呈现成纤维细胞样形态，并具有分化为脂肪细胞样细胞的能力。为寻找UCB-MSC未分化状态的标志物，我们分析了UCB-MSC在分化为脂肪细胞样细胞的不同时间段的蛋白质表达谱。我们的结果表明，标志物CD73、CD90和CD105的表达下降，这与UCB-MSC的分化程度相关。我们建议CD90可作为追踪MSC分化承诺程度的标志物。微阵列结果显示，在脂肪细胞样分化的MSC中，与脂肪生成过程和氧化还原代谢相关的基因上调。我们的研究提供了一组参数信息，这些参数可能有助于成功分离UCB-MSC，并进而有助于表征其分化/未分化状态，这将有助于监测UCB-MSC的分化承诺，并进一步促进这些细胞在干细胞治疗中的应用。
