Isolation, Culture, and Characterization of Human Umbilical Cord Blood-Derived Mesenchymal Stromal Cells

  • Karen BiebackEmail author
  • Philipp Netsch
Part of the Methods in Molecular Biology book series (MIMB, volume 1416)


Umbilical cord blood (CB) is considered one of the youngest available sources of adult stem cells. Besides hematopoietic stem cells, CB has been shown to contain endothelial progenitor cells as well as mesenchymal stromal/stem cells (MSC). To isolate MSC from cord blood, CB is collected into a sterile bag containing the anticoagulant citrate-phosphate-dextrose (CPD). The CB is then processed by density-gradient centrifugation to obtain mononuclear cells (MNC). These are cultured until the outgrowth of fibroblastoid cell colonies appears. After reaching a subconfluent stage, cells are harvested, expanded, and characterized as cord blood mesenchymal stromal cells (CB-MSC) according to standard criteria: plastic adherence, fibroblast morphology, CFU-f assay, proliferation potential, immune phenotype, and differentiation potential.

Apparently, the frequency of MSC in CB is extremely low. Thus, not every CB unit will provide adequate MSC isolation yields. Different strategies have been proposed aiming to optimize the isolation success by selecting CB units of optimal quality. It is commonly agreed on that a high CB volume, a high cellular content, and a short time frame between birth and MSC isolation are criteria that will enhance the MSC isolation success.

The procedures in this chapter are standardized protocols that were established and optimized in the authors’ research laboratory; however, various modifications of the protocols are possible.

Key words

Mesenchymal stromal cells Mesenchymal stem cells Cord blood Umbilical cord blood Isolation Culture Characterization Differentiation Immune phenotype 



This work was supported by the German Jose-Carreras Leukemia-Foundation e.V., DJCLS-R03/18, the German Ministry of Education and Research (BMBF) 01GN0531 and 01GN0939, and by the European Community, LSHB-CT-2005-018999.

We greatly acknowledge the work of Susanne Pilopp née Kern, Andrea Hecker, Cora Ecker, Susanne Elvers-Hornung, and Stephanie Uhlig. P.N. was supported by the “Stiftung Transfusionsmedizin und Immunhämatologie.”


  1. 1.
    Bieback K, Wuchter P, Besser D et al (2012) Mesenchymal stromal cells (MSCs): science and F(R)Iction. J Mol Med (Berl) 90:773–782CrossRefGoogle Scholar
  2. 2.
    Keating A (2012) Mesenchymal stromal cells: new directions. Cell Stem Cell 10:709–716CrossRefPubMedGoogle Scholar
  3. 3.
    Prockop DJ, Oh JY (2012) Medical therapies with adult stem/progenitor cells (MSCs): a backward journey from dramatic results in vivo to the cellular and molecular explanations. J Cell Biochem 113:1460–1469PubMedPubMedCentralGoogle Scholar
  4. 4.
    Phinney DG, Sensebe L (2013) Mesenchymal stromal cells: misconceptions and evolving concepts. Cytotherapy 15:140–145CrossRefPubMedGoogle Scholar
  5. 5.
    Sherman LS, Munoz J, Patel SA et al (2011) Moving from the laboratory bench to patients’ bedside: considerations for effective therapy with stem cells. Clin Transl Sci 4:380–386CrossRefPubMedGoogle Scholar
  6. 6.
    Lee OK, Kuo TK, Chen WM et al (2004) Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103:1669–1675CrossRefPubMedGoogle Scholar
  7. 7.
    Kern S, Eichler H, Stoeve J et al (2006) Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24:1294–1301CrossRefPubMedGoogle Scholar
  8. 8.
    Bieback K, Brinkmann I (2010) Mesenchymal stromal cells from human perinatal tissues: from biology to cell therapy. World J Stem Cells 2:81–92CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Crisan M, Yap S, Casteilla L et al (2008) A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3:301–313CrossRefPubMedGoogle Scholar
  10. 10.
    Roufosse CA, Direkze NC, Otto WR et al (2004) Circulating mesenchymal stem cells. Int J Biochem Cell Biol 36:585–597CrossRefPubMedGoogle Scholar
  11. 11.
    Bieback K, Kern S, Klueter H et al (2004) Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22:625–634CrossRefPubMedGoogle Scholar
  12. 12.
    Bieback K, Klueter H (2007) Mesenchymal stromal cells from umbilical cord blood. Curr Stem Cell Res Ther 2:310–323CrossRefPubMedGoogle Scholar
  13. 13.
    Wexler SA, Donaldson C, Denning-Kendall P et al (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 121:368–374CrossRefPubMedGoogle Scholar
  14. 14.
    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 21:105–110CrossRefPubMedGoogle Scholar
  15. 15.
    Javed MJ, Mead LE, Prater D et al (2008) Endothelial colony forming cells and mesenchymal stem cells are enriched at different gestational ages in human umbilical cord blood. Pediatr Res 64:68–73CrossRefPubMedGoogle Scholar
  16. 16.
    Perdikogianni C, Dimitriou H, Stiakaki E et al (2008) Could cord blood be a source of mesenchymal stromal cells for clinical use? Cytotherapy 10:452–459CrossRefPubMedGoogle Scholar
  17. 17.
    Zhang X, Hirai M, Cantero S et al (2011) Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. J Cell Biochem 112:1206–1218CrossRefPubMedGoogle Scholar
  18. 18.
    Lee M, Jeong SY, Ha J et al (2014) Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo. Biochem Biophys Res Commun 446:983–989CrossRefPubMedGoogle Scholar
  19. 19.
    Hare JM, Fishman JE, Gerstenblith G et al (2012) Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the Poseidon randomized trial. JAMA 308:2369–2379CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Alagesan S, Griffin MD (2014) Autologous and allogeneic mesenchymal stem cells in organ transplantation: what do we know about their safety and efficacy? Curr Opin Organ Transplant 19:65–72CrossRefPubMedGoogle Scholar
  21. 21.
    Karagianni M, Brinkmann I, Kinzebach S et al (2013) A comparative analysis of the adipogenic potential in human mesenchymal stromal cells from cord blood and other sources. Cytotherapy 15:76–88CrossRefPubMedGoogle Scholar
  22. 22.
    Ragni E, Vigano M, Parazzi V et al (2013) Adipogenic potential in human mesenchymal stem cells strictly depends on adult or foetal tissue harvest. Int J Biochem Cell Biol 45:2456–2466CrossRefPubMedGoogle Scholar
  23. 23.
    Kluth SM, Buchheiser A, Houben AP et al (2010) Dlk-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood. Stem Cells Dev 19:1471–1483CrossRefPubMedGoogle Scholar
  24. 24.
    Kim J, Shin JM, Jeon YJ et al (2012) Proteomic validation of multifunctional molecules in mesenchymal stem cells derived from human bone marrow, umbilical cord blood and peripheral blood. PLoS One 7:e32350CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Castro-Manrreza ME, Mayani H, Monroy-Garcia A et al (2014) Human mesenchymal stromal cells from adult and neonatal sources: a comparative in vitro analysis of their immunosuppressive properties against T cells. Stem Cells Dev 23:1217–1232CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Pievani A, Scagliotti V, Russo FM et al (2014) Comparative analysis of multilineage properties of mesenchymal stromal cells derived from fetal sources shows an advantage of mesenchymal stromal cells isolated from cord blood in chondrogenic differentiation potential. Cytotherapy 16:893–905CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Stubbendorff M, Deuse T, Hua X et al (2013) Immunological properties of extraembryonic human mesenchymal stromal cells derived from gestational tissue. Stem Cells Dev 22:2619–2629CrossRefPubMedGoogle Scholar
  28. 28.
    Ragni E, Montemurro T, Montelatici E et al (2013) Differential microrna signature of human mesenchymal stem cells from different sources reveals an “environmental-niche memory” for bone marrow stem cells. Exp Cell Res 319:1562–1574CrossRefPubMedGoogle Scholar
  29. 29.
    Lv F, Lu M, Cheung KM et al (2012) Intrinsic properties of mesenchymal stem cells from human bone marrow, umbilical cord and umbilical cord blood comparing the different sources of Msc. Curr Stem Cell Res Ther 7:389–399CrossRefPubMedGoogle Scholar
  30. 30.
    Kang BJ, Ryu HH, Park SS et al (2012) Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton’s Jelly for treating bone defects. J Vet Sci 13:299CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Jin HJ, Bae YK, Kim M et al (2013) Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. Int J Mol Sci 14:17986–18001CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Bosch J, Houben AP, Hennicke T et al (2013) Comparing the gene expression profile of stromal cells from human cord blood and bone marrow: lack of the typical “bone” signature in cord blood cells. Stem Cells Int 2013:631984CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Akimoto K, Kimura K, Nagano M et al (2013) Umbilical cord blood-derived mesenchymal stem cells inhibit, but adipose tissue-derived mesenchymal stem cells promote, glioblastoma multiforme proliferation. Stem Cells Dev 22:1370–1386CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Secunda R, Vennila R, Mohanashankar AM et al (2014) Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study. Cytotechnology 2014 May 6. [Epub ahead of print]Google Scholar
  35. 35.
    Martins AA, Paiva A, Morgado JM et al (2009) Quantification and immunophenotypic characterization of bone marrow and umbilical cord blood mesenchymal stem cells by multicolor flow cytometry. Transplant Proc 41:943–946CrossRefPubMedGoogle Scholar
  36. 36.
    Yoo KH, Jang IK, Lee MW et al (2009) Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol 259:150–156CrossRefPubMedGoogle Scholar
  37. 37.
    Hutson EL, Boyer S, Genever PG (2005) Rapid isolation, expansion, and differentiation of osteoprogenitors from full-term umbilical cord blood. Tissue Eng 11:1407–1420CrossRefPubMedGoogle Scholar
  38. 38.
    Attar A, Ghalyanchi Langeroudi A, Vassaghi A et al (2013) Role of Cd271 enrichment in the isolation of mesenchymal stromal cells from umbilical cord blood. Cell Biol Int 37:1010–1015CrossRefPubMedGoogle Scholar
  39. 39.
    Reinisch A, Bartmann C, Rohde E et al (2007) Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application. Regen Med 2:371–382CrossRefPubMedGoogle Scholar
  40. 40.
    Aktas M, Buchheiser A, Houben A et al (2010) Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood. Cytotherapy 12:338–348CrossRefPubMedGoogle Scholar
  41. 41.
    Pham PV, Vu NB, Pham VM et al (2014) Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells. J Transl Med 12:56CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Lee MW, Choi J, Yang MS et al (2004) Mesenchymal stem cells from cryopreserved human umbilical cord blood. Biochem Biophys Res Commun 320:273–278CrossRefPubMedGoogle Scholar
  43. 43.
    Rojewski MT, Weber BM, Schrezenmeier H (2008) Phenotypic characterization of mesenchymal stem cells from various tissues. Transfus Med Hemother 35:168–184CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Dominici M, Le Blanc K, Mueller I et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317CrossRefPubMedGoogle Scholar
  45. 45.
    Sekiya I, Larson BL, Smith JR et al (2002) Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 20:530–541CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Medical Faculty Mannheim, Institute of Transfusion Medicine and ImmunologyHeidelberg UniversityHeidelbergGermany
  2. 2.German Red Cross Blood Service Baden-Württemberg – HessenMannheimGermany

Personalised recommendations