Abstract
Some progress has been made in development of methods to regenerate bone from cultured cells, however no method is put to practical use. Here, we developed methods to isolate, purify, and expand mesenchymal stem cells (MSCs) from mouse compact bone that may be used to regenerate bone in vivo. These cells were maintained in long-term culture and were capable of differentiating along multiple lineages, including chondrocyte, osteocyte, and adipocyte trajectories. We used standard cell isolation and culture methods to establish cell cultures from mouse compact bone and bone marrow. Cultures were grown in four distinct media to determine the optimal composition of culture medium for bone-derived MSCs. Putative MSCs were subjected to flow cytometry, alkaline phosphatase assays, immunohistochemical staining, and several differentiation assays to assess cell identity, protein expression, and developmental potential. Finally, we used an in vivo bone formation assay to determine whether putative MSCs were capable of regenerating bone. We found that compact bone of mice was a better source of MCSs than the bone marrow, that growth in plastic flasks served to purify MSCs from hematopoietic cells, and that MSCs grown in basic fibroblast growth factor (bFGF)-conditioned medium were, based on multiple criteria, superior to those grown in leukemia inhibitory factor-conditioned medium. Moreover, we found that the MSCs isolated from compact bone and grown in bFGF-conditioned medium were capable of supporting bone formation in vivo. The methods and results described here have implications for understanding MSC biology and for clinical purpose.
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Amit M, Shariki C, Margulets V, Itskovitz-Eldor J (2004) Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 70(3):837–845. doi:10.1095/biolreprod.103.021147
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(3):301–316
Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP, Schipani E, Divieti P, Bringhurst FR, Milner LA, Kronenberg HM, Scadden DT (2003) Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425(6960):841–846. doi:10.1038/Nature02040
da Silva Meirelles L, Chagastelles PC, Nardi NB (2006) Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 119(Pt 11):2204–2213. doi:10.1242/jcs.02932
Esposito MT, Di Noto R, Mirabelli P, Gorrese M, Parisi S, Montanaro D, Del Vecchio L, Pastore L (2009) Culture conditions allow selection of different mesenchymal progenitors from adult mouse bone marrow. Tissue Eng Part A 15(9):2525–2536. doi:10.1089/ten.tea.2008.0509
Hisha H, Nishino T, Kawamura M, Adachi S, Ikehara S (1995) Successful bone marrow transplantation by bone grafts in chimeric-resistant combination. Exp Hematol 23(4):347–352
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(6893):41–49. doi:10.1038/nature00870
Kitano Y, Radu A, Shaaban A, Flake AW (2000) Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells. Exp Hematol 28(12):1460–1469
Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R (2005) Human embryonic stem cells derived without feeder cells. Lancet 365(9471):1636–1641. doi:10.1016/S0140-6736(05)66473-2
Levenstein ME, Ludwig TE, Xu RH, Llanas RA, VanDenHeuvel-Kramer K, Manning D, Thomson JA (2006) Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24(3):568–574. doi:10.1634/stemcells.2005-0247
Meirelles Lda S, Nardi NB (2003) Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 123(4):702–711
Muraglia A, Cancedda R, Quarto R (2000a) Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 113(Pt 7):1161–1166
Muraglia A, Cancedda R, Quarto R (2000b) Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 113(7):1161–1166
Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ (2004) Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103(5):1662–1668. doi:10.1182/blood-2003-09-3070
Phinney DG, Kopen G, Isaacson RL, Prockop DJ (1999) Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 72(4):570–585
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147
Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10(1):55–63. doi:10.1038/nm979
Short BJ, Brouard N, Simmons PJ (2009) Prospective isolation of mesenchymal stem cells from mouse compact bone. Methods Mol Biol 482:259–268. doi:10.1007/978-1-59745-060-7_16
Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336(6200):688–690. doi:10.1038/336688a0
Sun S, Guo Z, Xiao X, Liu B, Liu X, Tang PH, Mao N (2003) Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 21(5):527–535. doi:10.1634/stemcells.21-5-527
Tropel P, Noel D, Platet N, Legrand P, Benabid AL, Berger F (2004) Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp Cell Res 295(2):395–406. doi:10.1016/j.yexcr.2003.12.030
Wang L, Li L, Menendez P, Cerdan C, Bhatia M (2005) Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 105(12):4598–4603. doi:10.1182/blood-2004-10-4065
Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336(6200):684–687. doi:10.1038/336684a0
Xu C, Rosler E, Jiang J, Lebkowski JS, Gold JD, O’Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK (2005a) Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 23(3):315–323. doi:10.1634/stemcells.2004-0211
Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA (2005b) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2(3):185–190. doi:10.1038/nmeth744
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This work was supported in part by grants-in aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Yamachika, E., Tsujigiwa, H., Matsubara, M. et al. Basic fibroblast growth factor supports expansion of mouse compact bone-derived mesenchymal stem cells (MSCs) and regeneration of bone from MSC in vivo. J Mol Hist 43, 223–233 (2012). https://doi.org/10.1007/s10735-011-9385-8
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DOI: https://doi.org/10.1007/s10735-011-9385-8