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Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

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Abstract

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.

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References

  • Bieback K, Kern S, Kluter H, Eichler H (2004) Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22:625–634

    Article  Google Scholar 

  • Campagnoli C, Roberts I, Kumar S, Bennett PR, Fisk NM (2001) Clonal culture of fetal cells from maternal blood. Lancet 357:962

    Article  CAS  Google Scholar 

  • Chang YJ, Tseng CP, Hsu LF, Hsieh TB, Hwang SM (2006) Characterization of two populations of mesenchymal progenitor cells in umbilical cord blood. Cell Biol Int 30:495–499

    Article  CAS  Google Scholar 

  • Delorme B, Ringe J, Gallay N, Le Vern Y, Kerboeuf D, Jorgensen C, Rosset P, Sensebé L, Layrolle P, Häupl T, Charbord P (2008) Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. Blood 111:2631–2635

    Article  CAS  Google Scholar 

  • Duff SE, Li C, Garland JM, Kumar S (2003) CD105 is important for angiogenesis: evidence and potential applications. Faseb J 17:984–992

    Article  CAS  Google Scholar 

  • Erices A, Conget P, Minguell JJ (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109:235–242

    Article  CAS  Google Scholar 

  • 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 114:1752–1761

    CAS  Google Scholar 

  • Gang EJ, Hong SH, Jeong JA, Hwang SH, Kim SW, Yang IH, Ahn C, Han H, Kim H (2004) In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells. Biochem Biophys Res Commun 321:102–108

    Article  CAS  Google Scholar 

  • Gotherstrom C, Ringden O, Tammik C, Zetterberg E, Westgren M, Le Blanc K (2004) Immunologic properties of human fetal mesenchymal stem cells. Am J Obstet Gynecol 190:239–245

    Article  CAS  Google Scholar 

  • Gregoire FM (2001) Adipocyte differentiation: from fibroblast to endocrine cell. Exp Biol Med (Maywood) 226:997–1002

    CAS  Google Scholar 

  • Grimaldi PA (2001) The roles of PPARs in adipocyte differentiation. Prog Lipid Res 40:269–281

    Article  CAS  Google Scholar 

  • Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41–49

    Article  CAS  Google Scholar 

  • Jin HJ, Park SK, Oh W, Yang YS, Kim SW, Choi SJ (2009) Down-regulation of CD105 is associated with multi-lineage differentiation in human umbilical cord blood-derived mesenchymal stem cells. Biochem Biophys Res Commun 381:676–681

    Article  CAS  Google Scholar 

  • Kang XQ, Zang WJ, Bao LJ, Li DL, Xu XL, Yu XJ (2006) Differentiating characterization of human umbilical cord blood-derived mesenchymal stem cells in vitro. Cell Biol Int 30:569–575

    Article  CAS  Google Scholar 

  • Lee MW, Choi J, Yang MS, Moon YJ, Park JS, Kim HC, Kim YJ (2004) Mesenchymal stem cells from cryopreserved human umbilical cord blood. Biochem Biophys Res Commun 320:273–278

    Article  CAS  Google Scholar 

  • Linhart HG, Ishimura-Oka K, DeMayo F, Kibe T, Repka D, Poindexter B, Bick RJ, Darlington GJ (2001) C/EBPalpha is required for differentiation of white, but not brown, adipose tissue. Proc Natl Acad Sci USA 98:12532–12537

    Article  CAS  Google Scholar 

  • MacDougald OA, Mandrup S (2002) Adipogenesis: forces that tip the scales. Trends Endocrinol Metab 13:5–11

    Article  CAS  Google 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 112:295–307

    Article  CAS  Google Scholar 

  • Park KS, Lee YS, Kang KS (2006) In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood. J Vet Sci 7:343–348

    Article  Google 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 4:590–593

    CAS  Google 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 19:1205–1207

    CAS  Google Scholar 

  • Roelen BA, Dijke P (2003) Controlling mesenchymal stem cell differentiation by TGFBeta family members. J Orthop Sci 8:740–748

    Article  Google Scholar 

  • Romanov YA, Svintsitskaya VA, Smirnov VN (2003) Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 21:105–110

    Article  Google Scholar 

  • Rosen ED, Spiegelman BM (2000) Molecular regulation of adipogenesis. Annu Rev Cell Dev Biol 16:145–171

    Article  CAS  Google Scholar 

  • Schilling T, Noth U, Klein-Hitpass L, Jakob F, Schutze N (2007) Plasticity in adipogenesis and osteogenesis of human mesenchymal stem cells. Mol Cell Endocrinol 271:1–17

    Article  CAS  Google Scholar 

  • Secco M, Zucconi E, Vieira NM, Fogaca LL, Cerqueira A, Carvalho MD, Jazedje T, Okamoto OK, Muotri AR, Zatz M (2008) Mesenchymal stem cells from umbilical cord: do not discard the cord! Neuromuscul Disord 18:17–18

    Article  Google 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 134:762–770

    CAS  Google Scholar 

  • Vencio RZ, Koide T (2005) HTself: self-self based statistical test for low replication microarray studies. DNA Res 12:211–214

    Article  CAS  Google Scholar 

  • Wexler SA, Donaldson C, Denning-Kendall P, Rice C, Bradley B, Hows JM (2003) Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not. Br J Haematol 121:368–374

    Article  Google 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 43:489–496

    Google Scholar 

Download references

Acknowledgments

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).

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. 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

    Tatiana Taís Sibov, L. F. Pavon, E. Amaro Jr. & L. F Gamarra

  2. Universidade de Brasília, Brasília, DF, Brazil

    D. M. Oliveira

  3. Centro de Pesquisa Experimental, Instituto Israelita de Ensino e Pesquisa Albert Einstein-IIEPAE, São Paulo, SP, Brazil

    P. Severino, L. C. Marti, P. R. Tobo, A. H. Campos & A. T. Paes

  4. Departamento de Pediatria, Faculdade de Medicina da Universidade de São Paulo-USP, São Paulo, SP, Brazil

    C. A. Moreira-Filho

Authors
  1. Tatiana Taís Sibov
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  2. P. Severino
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  3. L. C. Marti
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  4. L. F. Pavon
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  5. D. M. Oliveira
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  6. P. R. Tobo
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  7. A. H. Campos
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  8. A. T. Paes
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  9. E. Amaro Jr.
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  10. L. F Gamarra
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  11. C. A. Moreira-Filho
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Correspondence to Tatiana Taís Sibov.

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Sibov, T.T., Severino, P., Marti, L.C. et al. Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation. Cytotechnology 64, 511–521 (2012). https://doi.org/10.1007/s10616-012-9428-3

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  • DOI: https://doi.org/10.1007/s10616-012-9428-3

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