A simple and reliable protocol for long-term culture of murine bone marrow stromal (mesenchymal) stem cells that retained their in vitro and in vivo stemness in long-term culture
Tóm tắt
Bone marrow derived stromal stem cells (BMSCs) are a clonogenic cell population that is characterized by self-renewal capacity and differentiation potential into osteoblasts, and other mesenchymal cell types. Mouse BMSCs (mBMSCs) are difficult to be cultured and propagated in vitro due to their replicative senescent phenotype, heterogeneity and high contamination with plastic adherent hematopoietic progenitors (HPCs). In this study, we described long-term culture of homogenous population of mBMSCs using simple and highly reproducible approach based on frequent subculturing (FS) at fixed split ratio in the presence of basic fibroblast growth factor (bFGF). Cultured mBMSCs using this protocol (mBMSCs-FS) showed long-term survival in culture > 70 population doubling (PD) and retained their characteristic surface markers and differentiation capacity into osteoblast and adipocyte lineages. When compared to the clonal bone marrow-derived cell line ST2, mBMSCs-FS displayed more enhanced osteoblast differentiation potential and responsiveness to osteogenic factors including BMPs, IGF-1, PDGF, TGFβ1,3, FGF, cAMP, Wnt3a and VEGF. In addition, unlike ST2 cells, mBMSCs-FS maintained capacity to form ectopic bone and bone marrow stroma upon in vivo transplantation in immune-compromising mice, even at high PD levels. Interestingly, by applying the same FS + bFGF protocol, we succeeded to obtain long-term cultures of primary neonatal calvarial osteoprogenitor cells (OBs) that were cultured for more than 70 PD and maintained in vitro and in vivo osteoblast differentiation capacities. Our data provide a simple and reliable protocol for generating long-term cultures of mBMSCs and OBs with retained high in vitro and in vivo osteoblast differentiation capacities for use in pre-clinical and molecular mechanism studies.
Tài liệu tham khảo
Abdallah BM, Jafari A, Zaher W, Qiu W, Kassem M. Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation. Bone. 2015;70:28–36.
Bianco P, Robey PG. Skeletal stem cells. Development. 2015;142(6):1023–7.
Baddoo M, Hill K, Wilkinson R, Gaupp D, Hughes C, Kopen GC, Phinney DG. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem. 2003;89(6):1235–49.
Tropel P, Noël D, Platet N, Legrand P, Benabid A-L, Berger F. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp Cell Res. 2004;295(2):395–406.
Phinney DG, Kopen G, Isaacson RL, Prockop DJ. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem. 1999;72(4):570–85.
Houlihan DD, Mabuchi Y, Morikawa S, Niibe K, Araki D, Suzuki S, Okano H, Matsuzaki Y. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α. Nat Protoc. 2012;7:2103.
Mahara A, Yamaoka T. Antibody-immobilized column for quick cell separation based on cell rolling. Biotechnol Prog. 2010;26(2):441–7.
Xu S, De Becker A, Van Camp B, Vanderkerken K, Van Riet I. An improved harvest and in vitro expansion protocol for murine bone marrow-derived mesenchymal stem cells. J Biomed Biotechnol. 2010;2010:105940.
Soleimani M, Nadri S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc. 2009;4:102.
Sreejit P, Dilip KB, Verma RS. Generation of mesenchymal stem cell lines from murine bone marrow. Cell Tissue Res. 2012;350(1):55–68.
Rosca AM, Burlacu A. Isolation of a mouse bone marrow population enriched in stem and progenitor cells by centrifugation on a Percoll gradient. Biotechnol Appl Biochem. 2010;55(4):199–208.
Suto EG, Mabuchi Y, Suzuki N, Suzuki K, Ogata Y, Taguchi M, Muneta T, Sekiya I, Akazawa C. Prospectively isolated mesenchymal stem/stromal cells are enriched in the CD73(+) population and exhibit efficacy after transplantation. Sci Rep. 2017;7:4838.
Ogawa M, Nishikawa S, Ikuta K, Yamamura F, Naito M, Takahashi K, Nishikawa S. B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST2: B cell progenitor develops first in the embryonal body rather than in the yolk sac. EMBO J. 1988;7(5):1337–43.
Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen V, Wang EA, Tanaka H, Omura S, Suda T. The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem Biophys Res Commun. 1990;172(1):295–9.
Negishi Y, Kudo A, Obinata A, Kawashima K, Hirano H, Yanai N, Obinata M, Endo H. Multipotency of a bone marrow stromal cell line, TBR31-2, established from ts-SV40 T antigen gene transgenic mice. Biochem Biophys Res Commun. 2000;268(2):450–5.
Yamaguchi A, Ishizuya T, Kintou N, Wada Y, Katagiri T, Wozney JM, Rosen V, Yoshiki S. Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochem Biophys Res Commun. 1996;220(2):366–71.
Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ. 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. 2004;103(5):1662–8.
Abdallah BM, Ditzel N, Kassem M. Assessment of bone formation capacity using in vivo transplantation assays: procedure and tissue analysis. Methods Mol Biol. 2008;455:89–100.
Andersen RK, Zaher W, Larsen KH, Ditzel N, Drews K, Wruck W, Adjaye J, Abdallah BM, Kassem M. Association between in vivo bone formation and ex vivo migratory capacity of human bone marrow stromal cells. Stem Cell Res Ther. 2015;6:196.
Larsen KH, Frederiksen CM, Burns JS, Abdallah BM, Kassem M. Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone forming capacity. J Bone Miner Res. 2010;25(4):796–808.
Post S, Abdallah BM, Bentzon JF, Kassem M. Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells. Bone. 2008;43(1):32–9.
Phinney DG. Isolation of Mesenchymal Stem Cells from Murine Bone Marrow by Immunodepletion. In: Prockop DJ, Bunnell BA, Phinney DG, editors. Mesenchymal Stem Cells: Methods and Protocols. Totowa, NJ: Humana Press; 2008. p. 171–86.
Coutu DL, Galipeau J. Roles of FGF signaling in stem cell self-renewal, senescence and aging. Aging (Albany NY). 2011;3(10):920–33.
Ng F, Boucher S, Koh S, Sastry KSR, Chase L, Lakshmipathy U, Choong C, Yang Z, Vemuri MC, Rao MS, et al. PDGF, TGF-β, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood. 2008;112(2):295–307.
Sun S, Guo Z, Xiao X, Liu B, Liu X, Tang PH, Mao N. Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells. 2003;21(5):527–35.
Caroti CM, Ahn H, Salazar HF, Joseph G, Sankar SB, Willett NJ, Wood LB, Taylor WR, Lyle AN. A novel technique for accelerated culture of murine mesenchymal stem cells that allows for sustained multipotency. Sci Rep. 2017;7:13334.
Coutu DL, François M, Galipeau J. Inhibition of cellular senescence by developmentally regulated FGF receptors in mesenchymal stem cells. Blood. 2011;117(25):6801–12.
Fábián Z, Ramadurai S, Shaw G, Nasheuer H-P, Kolch W, Taylor C, Barry F. Basic fibroblast growth factor modifies the hypoxic response of human bone marrow stromal cells by ERK-mediated enhancement of HIF-1α activity. Stem Cell Res. 2014;12(3):646–58.
Lisignoli G, Fini M, Giavaresi G, Nicoli Aldini N, Toneguzzi S, Facchini A. Osteogenesis of large segmental radius defects enhanced by basic fibroblast growth factor activated bone marrow stromal cells grown on non-woven hyaluronic acid-based polymer scaffold. Biomaterials. 2002;23(4):1043–51.
Henderson B, Nair SP. Hard Labour: bacterial infection of the skeleton. Trends Microbiol. 2003;11(12):570–7.
House SL, Bolte C, Zhou M, Doetschman T, Klevitsky R, Newman G, Schultz JEJ. Cardiac-specific overexpression of fibroblast growth Factor-2 protects against myocardial dysfunction and infarction in a murine model of low-flow ischemia. Circulation. 2003;108(25):3140–8.
Meirelles LS, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol. 2003;123(4):702–11.
Armesilla-Diaz A, Elvira G, Silva A. p53 regulates the proliferation, differentiation and spontaneous transformation of mesenchymal stem cells. Exp Cell Res. 2009;315(20):3598–610.
Li H, Fan X, Kovi RC, Jo Y, Moquin B, Konz R, Stoicov C, Kurt-Jones E, Grossman SR, Lyle S, et al. Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice. Cancer Res. 2007;67(22):10889–98.
Miura M, Miura Y, Padilla-Nash HM, Molinolo AA, Fu B, Patel V, Seo BM, Sonoyama W, Zheng JJ, Baker CC, et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells. 2006;24(4):1095–103.
Tolar J, Nauta AJ, Osborn MJ, Mortari AP, McElmurry RT, Bell S, Xia L, Zhou N, Riddle M, Schroeder TM, et al. Sarcoma derived from cultured mesenchymal stem cells. Stem Cells. 2007;25(2):371–9.
Boregowda SV, Krishnappa V, Phinney DG. Isolation of Mouse Bone Marrow Mesenchymal Stem Cells. In: Gnecchi M, editor. Mesenchymal Stem Cells: Methods and Protocols. New York, NY: Springer New York; 2016. p. 205–23.
Eslaminejad MB, Nikmahzar A, Taghiyar L, Nadri S, Massumi M. Murine mesenchymal stem cells isolated by low density primary culture system. Develop Growth Differ. 2006;48(6):361–70.
Abdallah BM, Figeac F, Larsen KH, Ditzel N, Keshari P, Isa A, Jafari A, Andersen TL, Delaisse J-M, Goshima Y et al: CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling. Journal of Bone and Mineral Research 2017:n/a-n/a.
Abdallah BM. Marrow adipocytes inhibit the differentiation of mesenchymal stem cells into osteoblasts via suppressing BMP-signaling. J Biomed Sci. 2017;24:11.
Abdallah BM, Al-Shammary A, Skagen P, Abu Dawud R, Adjaye J, Aldahmash A, Kassem M. CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells. Stem Cell Res. 2015;15(3):449–58.