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.
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References
Arinzeh TL (2005) Mesenchymal stem cells for bone repair: preclinical studies and potential orthopedic applications. Foot Ankle Clin 10(4):651–665, viii
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):15–29
Bhagavati S, Xu W (2004) Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow. Biochem Biophys Res Commun 318(1):119–124
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):625–634
Bruder SP, Fink DJ, Caplan AI (1994) Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem 56(3):283–294
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 38–29
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):679–685
Cohen Y, Nagler A (2004) Umbilical cord blood transplantation–how, when and for whom? Blood Rev 18(3):167–179
Damsky CH, Ilic D (2002) Integrin signaling: it’s where the action is. Curr Opin Cell Biol 14(5):594–602
Dazzi F, Ramasamy R, Glennie S et al (2005) The role of mesenchymal stem cells in haemopoiesis. Blood Rev 20(3):161–171
Dennis JE, Charbord P (2002) Origin and differentiation of human and murine stroma. Stem Cells 20(3):205–214
Diaz-Flores L Jr, Madrid JF, Gutierrez R et al (2006) Adult stem and transit-amplifying cell location. Histol Histopathol 21(9):995–1027
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):283–290
Ducy P, Zhang R, Geoffroy V et al (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89(5):747–754
Erices A, Conget P, Minguell JJ (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109(1):235–242
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 938:9–17
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):617–624
Ge Z, Goh JC, Lee EH (2005) Selection of cell source for ligament tissue engineering. Cell Transplant 14(8):573–583
Grove JE, Bruscia E, Krause DS (2004) Plasticity of bone marrow-derived stem cells. Stem Cells 22(4):487–500
Herzog EL, Chai L, Krause DS (2003) Plasticity of marrow-derived stem cells. Blood 102(10):3483–3493
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):2010–2019
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):1153–1161
Jeong JA, Gang EJ, Hong SH et al (2004) Rapid neural differentiation of human cord blood-derived mesenchymal stem cells. Neuroreport 15(11):1731–1734
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):2723–2732
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):230–233
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):5953–5957
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):123–135
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):573–583
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):639–644
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):1878–1887
Murohara T, Ikeda H, Duan J et al (2000) Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 105(11):1527–1536
Rogers I, Casper RF (2004) Umbilical cord blood stem cells. Best Pract Res Clin Obstet Gynaecol 18(6):893–908
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):24–34
Shalhoub V, Elliott G, Chiu L et al (2000) Characterization of osteoclast precursors in human blood. Br J Haematol 111(2):501–512
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):150–156
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):196–203
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):705–715
Yin T, Li L (2006) The stem cell niches in bone. J Clin Invest 116(5):1195–1201
Zuk PA, Zhu M, Mizuno H et al (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7(2):211–228
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This research was supported by the Israeli Ministry of Commerce Magnet Bereshit Grant No. 2004473.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s10616-007-9077-0
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Maurice, S., Srouji, S. & Livne, E. Isolation of progenitor cells from cord blood using adhesion matrices. Cytotechnology 52, 125–137 (2006). https://doi.org/10.1007/s10616-007-9043-x
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DOI: https://doi.org/10.1007/s10616-007-9043-x