Advertisement

Cytotechnology

, Volume 52, Issue 2, pp 125–137 | Cite as

Isolation of progenitor cells from cord blood using adhesion matrices

  • Sarah MauriceEmail author
  • Samer Srouji
  • Erella Livne
Original Research

Abstract

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (PCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in αMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue® assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45+, CD105+, CD14+, CD49a+, CD49f+, CD44+ and CD34. The highest incidence of PCs was observed on fibronectin and polystyrene. Passages were CD45, CD14, CD34 and weakly CD105+. Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing Vit D3 and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.

Keywords

Adhesion molecules Cord blood Progenitor cells Osteogenesis 

Notes

Acknowledgments

This research was supported by the Israeli Ministry of Commerce Magnet Bereshit Grant No. 2004473.

References

  1. Arinzeh TL (2005) Mesenchymal stem cells for bone repair: preclinical studies and potential orthopedic applications. Foot Ankle Clin 10(4):651–665, viiiCrossRefGoogle Scholar
  2. Bellows CG, Heersche JN, Aubin JE (1992) Inorganic phosphate added exogenously or released from beta-glycerophosphate initiates mineralization of osteoid nodules in vitro. Bone Miner 17(1):15–29CrossRefGoogle Scholar
  3. Bhagavati S, Xu W (2004) Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow. Biochem Biophys Res Commun 318(1):119–124CrossRefGoogle Scholar
  4. Bieback K, Kern S, Kluter H et al (2004) Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 22(4):625–634CrossRefGoogle Scholar
  5. Bruder SP, Fink DJ, Caplan AI (1994) Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem 56(3):283–294CrossRefGoogle Scholar
  6. Buhring HJ, Seiffert M, Bock TA et al (1999) Expression of novel surface antigens on early hematopoietic cells. Ann N Y Acad Sci 872:25–38; discussion 38–29Google Scholar
  7. Chang YJ, Shih DT, Tseng CP et al (2006) Disparate mesenchyme-lineage tendencies in mesenchymal stem cells from human bone marrow and umbilical cord blood. Stem Cells 24(3):679–685CrossRefGoogle Scholar
  8. Cohen Y, Nagler A (2004) Umbilical cord blood transplantation–how, when and for whom? Blood Rev 18(3):167–179CrossRefGoogle Scholar
  9. Damsky CH, Ilic D (2002) Integrin signaling: it’s where the action is. Curr Opin Cell Biol 14(5):594–602CrossRefGoogle Scholar
  10. Dazzi F, Ramasamy R, Glennie S et al (2005) The role of mesenchymal stem cells in haemopoiesis. Blood Rev 20(3):161–171CrossRefGoogle Scholar
  11. Dennis JE, Charbord P (2002) Origin and differentiation of human and murine stroma. Stem Cells 20(3):205–214CrossRefGoogle Scholar
  12. Diaz-Flores L Jr, Madrid JF, Gutierrez R et al (2006) Adult stem and transit-amplifying cell location. Histol Histopathol 21(9):995–1027Google Scholar
  13. Dicker A, Le Blanc K, Astrom G et al (2005) Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 308(2):283–290CrossRefGoogle Scholar
  14. Ducy P, Zhang R, Geoffroy V et al (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89(5):747–754CrossRefGoogle Scholar
  15. Erices A, Conget P, Minguell JJ (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109(1):235–242CrossRefGoogle Scholar
  16. Fibbe WE, Noort WA, Schipper F et al (2001) Ex vivo expansion and engraftment potential of cord blood-derived CD34+ cells in NOD/SCID mice. Ann N Y Acad Sci 938:9–17CrossRefGoogle Scholar
  17. Gang EJ, Jeong JA, Hong SH et al (2004) Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. Stem Cells 22(4):617–624CrossRefGoogle Scholar
  18. Ge Z, Goh JC, Lee EH (2005) Selection of cell source for ligament tissue engineering. Cell Transplant 14(8):573–583Google Scholar
  19. Grove JE, Bruscia E, Krause DS (2004) Plasticity of bone marrow-derived stem cells. Stem Cells 22(4):487–500CrossRefGoogle Scholar
  20. Herzog EL, Chai L, Krause DS (2003) Plasticity of marrow-derived stem cells. Blood 102(10):3483–3493CrossRefGoogle Scholar
  21. Hildbrand P, Cirulli V, Prinsen RC et al (2004) The role of angiopoietins in the development of endothelial cells from cord blood CD34+ progenitors. Blood 104(7):2010–2019CrossRefGoogle Scholar
  22. Hong SH, Gang EJ, Jeong JA et al (2005) In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells. Biochem Biophys Res Commun 330(4):1153–1161CrossRefGoogle Scholar
  23. Jeong JA, Gang EJ, Hong SH et al (2004) Rapid neural differentiation of human cord blood-derived mesenchymal stem cells. Neuroreport 15(11):1731–1734CrossRefGoogle Scholar
  24. Jiang XS, Chai C, Zhang Y et al (2006) Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34+ cells. Biomaterials 27(13):2723–2732CrossRefGoogle Scholar
  25. Kang TJ, Yeom JE, Lee HJ et al (2004) Growth kinetics of human mesenchymal stem cells from bone marrow and umbilical cord blood. Acta Haematol 112(4):230–233CrossRefGoogle Scholar
  26. Keselowsky BG, Collard DM, Garcia AJ (2005) Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation. Proc Natl Acad Sci USA 102(17):5953–5957CrossRefGoogle Scholar
  27. Kogler G, Sensken S, Airey JA et al (2004) A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 200(2):123–135CrossRefGoogle Scholar
  28. Kogler G, Radke TF, Lefort A et al (2005) Cytokine production and hematopoiesis supporting activity of cord blood-derived unrestricted somatic stem cells. Exp Hematol 33(5):573–583CrossRefGoogle Scholar
  29. Kundu M, Javed A, Jeon JP et al (2002) Cbfbeta interacts with Runx2 and has a critical role in bone development. Nat Genet 32(4):639–644CrossRefGoogle Scholar
  30. Muguruma Y, Yahata T, Miyatake H et al (2006) Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood 107(5):1878–1887CrossRefGoogle Scholar
  31. Murohara T, Ikeda H, Duan J et al (2000) Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 105(11):1527–1536CrossRefGoogle Scholar
  32. Rogers I, Casper RF (2004) Umbilical cord blood stem cells. Best Pract Res Clin Obstet Gynaecol 18(6):893–908CrossRefGoogle Scholar
  33. Salasznyk RM, Williams WA, Boskey A et al (2004) Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. J Biomed Biotechnol 2004(1):24–34CrossRefGoogle Scholar
  34. Shalhoub V, Elliott G, Chiu L et al (2000) Characterization of osteoclast precursors in human blood. Br J Haematol 111(2):501–512CrossRefGoogle Scholar
  35. Silva GV, Litovsky S, Assad JA et al (2005) Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 111(2):150–156CrossRefGoogle Scholar
  36. Tsuboi H, Matsui Y, Hayashida K et al (2003) Tartrate resistant acid phosphatase (TRAP) positive cells in rheumatoid synovium may induce the destruction of articular cartilage. Ann Rheum Dis 62(3):196–203CrossRefGoogle Scholar
  37. Yang S, Wei D, Wang D et al (2003) In vitro and in vivo synergistic interactions between the Runx2/Cbfa1 transcription factor and bone morphogenetic protein-2 in stimulating osteoblast differentiation. J Bone Miner Res 18(4):705–715CrossRefGoogle Scholar
  38. Yin T, Li L (2006) The stem cell niches in bone. J Clin Invest 116(5):1195–1201CrossRefGoogle Scholar
  39. Zuk PA, Zhu M, Mizuno H et al (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7(2):211–228CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2007

Authors and Affiliations

  1. 1.Department of Anatomy and Cell Biology, Faculty of MedicineTechnionHaifaIsrael

Personalised recommendations