Histochemistry and Cell Biology

, 132:547

Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells

  • Elisa Leonardi
  • Gabriela Ciapetti
  • Serena Rubina Baglìo
  • Valentina Devescovi
  • Nicola Baldini
  • Donatella Granchi
Original Paper

Abstract

The nonadherent (NA) population of bone-marrow-derived mononuclear cells (MNC) has been demonstrated to be a source of osteogenic precursors in addition to the plastic-adherent mesenchymal stromal cells (MSC). In the current study, two subpopulations of late adherent (LA) osteoprogenitors were obtained by subsequent replating of NA cells, and their phenotypic, functional, and molecular properties were compared with those of early adherent (EA) MSC. Approximately 35% of MNC were LA cells, and they acquired a homogeneous expression of MSC antigens later than EA cells. In EA-MSC, the alkaline phosphatase (ALP) activity increased significantly from time of seeding to the first confluence, whereas in LA cells it raised later, after the addition of mineralization medium. All subpopulations were able to produce type I collagen and to deposit extracellular matrix with organized collagen fibrils. The proportion of large colonies with more than 50% of ALP positive cells as well as the calcium content was higher in LA than in EA cells. Molecular analysis highlighted the upregulation of bone-related genes in LA-MSC, especially after the addition of mineralization medium. Our results confirm that bone marrow contains LA osteoprogenitors which exhibit a delay in the differentiation process, despite an osteogenic potential similar to or better than EA-MSC. LA cells represent a reservoir of osteoprogenitors to be recruited to gain an adequate bone tissue repair and regeneration when a depletion of the most differentiated component occurs. Bone tissue engineering and cell therapy strategies could take advantage of LA cells, since an adequate amount of osteogenic MSCs may be obtained while avoiding bone marrow manipulation and cell culture expansion.

Keywords

Bone marrow stromal cells Nonadherent cells Osteoprogenitors Bone repair Gene expression 

References

  1. Baksh D, Song L, Tuan RS (2004) Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 8:301–316CrossRefPubMedGoogle Scholar
  2. Bianco P, Riminucci M, Gronthos S, Robey PG (2001) Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19:180–192CrossRefPubMedGoogle Scholar
  3. Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P, Karsenty G (1998) Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone 23:187–196CrossRefPubMedGoogle Scholar
  4. Brooke G, Cook M, Blair C, Han R, Heazlewood C, Jones B, Kambouris M, Kollar K, McTaggart S, Pelekanos R, Rice A, Rossetti T, Atkinson K (2007) Therapeutic applications of mesenchymal stromal cells. Semin Cell Dev Biol 18:846–858CrossRefPubMedGoogle Scholar
  5. Ciapetti G, Ambrosio L, Marletta G, Baldini N, Giunti A (2006) Human bone marrow stromal cells: in vitro expansion and differentiation for bone engineering. Biomaterials 27:6150–6160CrossRefPubMedGoogle Scholar
  6. Dallari D, Savarino L, Stagni C, Cenni E, Cenacchi A, Fornasari PM, Albisinni U, Rimondi E, Baldini N, Giunti A (2007) Enhanced tibial osteotomy healing with use of bone grafts supplemented with platelet gel or platelet gel and bone marrow and stromal cells. J Bone Joint Surg Am 89:2413–2420CrossRefPubMedGoogle Scholar
  7. Di Cesare PE, Fang C, Leslie MP, Tulli H, Perris R, Carlson CS (2000) Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts. J Orthop Res 18:713–720CrossRefPubMedGoogle Scholar
  8. Dominici M, Pritchard C, Garlits JE, Hofmann TJ, Persons DA, Horwitz EM (2004) Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation. Proc Natl Acad Sci USA 101:11761–11766CrossRefPubMedGoogle Scholar
  9. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop DJ, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement. Cytotherapy 8:315–317CrossRefPubMedGoogle Scholar
  10. Eghbali-Fatourechi GZ, Modder UI, Charatcharoenwitthaya N, Sanyal A, Undale AH, Clowes JA, Tarara JE, Khosla S (2007) Characterization of circulating osteoblast lineage cells in humans. Bone 40:1370–1377CrossRefPubMedGoogle Scholar
  11. Eipers PG, Kale S, Taichman RS, Pipia GG, Swords NA, Mann KG, Long MW (2000) Bone marrow accessory cells regulate human bone precursor cell development. Exp Hematol 28:815–825CrossRefPubMedGoogle Scholar
  12. Falla N, Van Vlasselaer P, Bierkens J, Borremans B, Schoeters G, Van Gorp U (1993) Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow. Blood 82:3580–3591PubMedGoogle Scholar
  13. Gori F, Hofbauer LC, Dunstan CR, Spelsberg TC, Khosla S, Riggs BL (2000) The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal–osteoblast lineage cells is developmentally regulated. Endocrinology 41:4768–4776CrossRefGoogle Scholar
  14. Granero-Molto F, Weis JA, Longobardi L, Spagnoli A (2008) Role of mesenchymal stem cells in regenerative medicine: application to bone and cartilage repair. Expert Opin Biol Ther 8(3):255–268CrossRefPubMedGoogle Scholar
  15. Hernigou P, Mathieu G, Poignard A, Manicom O, Beaujean F, Rouard H (2006) Percutaneous autologous bone-marrow grafting for nonunions: surgical technique. J Bone Joint Surg Am 88(S1):322–327CrossRefPubMedGoogle Scholar
  16. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A (2005) The International Society for Cellular Therapy Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7:393–395CrossRefPubMedGoogle Scholar
  17. Huang W, Yang S, Shao J, Li YP (2007) Signaling and transcriptional regulation in osteoblast commitment and differentiation. Front Biosci 12:3068–3092CrossRefPubMedGoogle Scholar
  18. Iba K, Chiba H, Yamashita T, Ishii S, Sawada N (2001) Phase-independent inhibition by retinoic acid of mineralization correlated with loss of tetranectin expression in a human osteoblastic cell line. Cell Struct Funct 26:227–233CrossRefPubMedGoogle Scholar
  19. Karahuseyinoglu S, Cinar O, Kilic E, Kara F, Akay GG, Demiralp DO, Tukun A, Uckan D, Can A (2007) Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells 25:319–331CrossRefPubMedGoogle Scholar
  20. Katoh M (2008) WNT signaling in stem cell biology and regenerative medicine. Curr Drug Targets 9:565–570CrossRefPubMedGoogle Scholar
  21. Kumagai K, Vasanji A, Drazba JA, Butler RS, Muschler GF (2008) Circulating cells with osteogenic potential are physiologically mobilized into the fracture healing site in the parabiotic mice model. J Orthop Res 26(2):165–175CrossRefPubMedGoogle Scholar
  22. Litvin J, Selim AH, Montgomery MO, Lehmann K, Rico MC, Devlin H, Bednarik DP, Safadi FF (2004) Expression and function of periostin-isoforms in bone. J Cell Biochem 92:1044–1061CrossRefPubMedGoogle Scholar
  23. Long MW, Williams JL, Mann KG (1990) Expression of human bone-related proteins in the hematopoietic microenvironment. J Clin Invest 86:1387–1395CrossRefPubMedGoogle Scholar
  24. Marcacci M, Kon E, Moukhachev V, Lavroukov A, Kutepov S, Quarto R, Mastrogiacomo M, Cancedda R (2007) Stem cells associated with macroporous bioceramics for long bone repair: 6- to 7-year outcome of a pilot clinical study. Tissue Eng 13:947–955CrossRefPubMedGoogle Scholar
  25. Mödder UI, Khosla S (2008) Skeletal stem/osteoprogenitor cells: current concepts, alternate hypotheses, and relationship to the bone remodeling compartment. J Cell Biochem 103:393–400CrossRefPubMedGoogle Scholar
  26. Motamed K (1999) SPARC (osteonectin/BM-40). Int J Biochem Cell Biol 31:1363–1366CrossRefPubMedGoogle Scholar
  27. Mouritzen P, Noerholm M, Nielsen PS (2005) ProbeLibrary: a new method for faster design and execution of quantitative real-time PCR. Nat Methods 4:313–316CrossRefGoogle Scholar
  28. Muschler GF, Nakamoto C, Griffith LG (2004) Engineering principles of clinical cell-based tissue engineering. J Bone Joint Surg Am 86:1541–1558PubMedGoogle Scholar
  29. Neuhuber B, Swanger SA, Howard L, Mackay A, Fischer I (2008) Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol 36:1176–1185CrossRefPubMedGoogle Scholar
  30. Niemeyer P, Krause U, Kasten P, Kreuz PC, Henle P, Südkam NP, Mehlhorn A (2006) Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage. Curr Stem Cell Res Ther 1(1):21–27CrossRefPubMedGoogle Scholar
  31. Ogata Y (2008) Bone sialoprotein and its transcriptional regulatory mechanism. J Periodontal Res 43:127–135CrossRefPubMedGoogle Scholar
  32. Tseng SS, Lee MA, Reddi AH (2008) Nonunions and the potential of stem cells in fracture-healing. J Bone Joint Surg Am 90((S1)):92–98CrossRefPubMedGoogle Scholar
  33. Valtieri M, Sorrentino A (2008) The mesenchymal stromal cell contribution to homeostasis. J Cell Physiol 217(2):296–300CrossRefPubMedGoogle Scholar
  34. Veyrat-Masson R, Boiret-Dupré N, Rapatel C, Descamps S, Guillouard L, Guérin JJ, Pigeon P, Boisgard S, Chassagne J, Berger MG (2007) Mesenchymal content of fresh bone marrow: a proposed quality control method for cell therapy. Br J Haematol 139(2):312–320CrossRefPubMedGoogle Scholar
  35. Wälchli C, Koch M, Chiquet M, Odermatt BF, Trueb B (1994) Tissue-specific expression of the fibril-associated collagens XII and XIV. J Cell Sci 107:669–681PubMedGoogle Scholar
  36. Wan C, He Q, McCaigue M, Marsh D, Li G (2006) Nonadherent cell population of human marrow culture is a complementary source of mesenchymal stem cells (MSCs). J Orthop Res 24:21–28CrossRefPubMedGoogle Scholar
  37. Wiesmann A, Bühring HJ, Mentrup C, Wiesmann HP (2006) Decreased CD90 expression in human mesenchymal stem cells by applying mechanical stimulation. Head Face Med 2:8–13CrossRefPubMedGoogle Scholar
  38. Wlodarski KH, Galus R, Włodarski P (2004) Non-adherent bone marrow cells are a rich source of cells forming bone in vivo. Folia Biol (Praha) 50(5):167–173Google Scholar
  39. Zhang ZL, Tong J, Lu RN, Scutt AM, Goltzman D, Miao DS (2009) Therapeutic potential of non-adherent BM-derived mesenchymal stem cells in tissue regeneration. Bone Marrow Transplant 43(1):69–81CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Elisa Leonardi
    • 1
  • Gabriela Ciapetti
    • 1
  • Serena Rubina Baglìo
    • 1
  • Valentina Devescovi
    • 1
  • Nicola Baldini
    • 1
  • Donatella Granchi
    • 1
  1. 1.Laboratory for Orthopaedic Pathophysiology and Regenerative MedicineIstituto Ortopedico RizzoliBolognaItaly

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