Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation
- 510 Downloads
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.
KeywordsMesenchymal stem cells Umbilical cord blood Cell isolation Cell characterization and adipogenesis
We thank all members from the Banco Público de Sangue de Cordão Umbilical do Hospital Israelita Albert Einstein who assisted us in the umbilical cord blood sample collection. We are also grateful to Laboratório de Microscopia Eletrônica, Departamento de Biologia, UNESP de Rio Claro, SP, Brazil and to Antonio T. Yabuki and Morita Iamonte for technical support. This work was financed by Instituto de Ensino e Pesquisa Albert Einstein, Sociedade Beneficente Israelita Hospital Albert Einstein (SBIBHAE) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).
Conflict of interest
The authors declare no conflict of interest.
- Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I (2004) Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 114:1752–1761Google Scholar
- Gregoire FM (2001) Adipocyte differentiation: from fibroblast to endocrine cell. Exp Biol Med (Maywood) 226:997–1002Google Scholar
- Ng F, Boucher S, Koh S, Sastry KS, Chase L, Lakshmipathy U, Choong C, Yang Z, Vemuri MC, Rao MS, Tanavde V(2008) PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood 112:295–307CrossRefGoogle Scholar
- Reitman A, Friedrich I, Ben-Amotz A, Levy Y (2002) Low plasma antioxidants and normal plasma B vitamins and homocysteine in patients with severe obesity. Isr Med Assoc J 4:590–593Google Scholar
- Rim JS, Mynatt RL, Gawronska-Kozak B (2005) Mesenchymal stem cells from the outer ear: a novel adult stem cell model system for the study of adipogenesis. Faseb J 19:1205–1207Google Scholar
- Urs S, Smith C, Campbell B, Saxton AM, Taylor J, Zhang B, Snoddy J, Jones Voy B, Moustaid-Moussa N (2004) Gene expression profiling in human preadipocytes and adipocytes by microarray analysis. J Nutr 134:762–770Google Scholar
- Yang JW, de Isla N, Huselstein C, Sarda-Kolopp MN, Li N, Li YP, Jing-Ping OY, Stoltz JF, Eljaafari A (2006) Evaluation of human MSCs cell cycle, viability and differentiation in micromass culture. Biorheology 43:489–496Google Scholar