Isolation and multilineage differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses

BMC Veterinary Research

Oscar A Peralta²  María S Fernández¹  Fernando Dueñas³  Diego Araya³  Margarita Ojeda⁴  Yennifer Cortes³ 

[1] Departamento de Ciencias Biológicas Animales, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile;Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, 24061-0442, Virginia, USA;Departamento de Fomento de la Producción Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile;Instituto de Ciencia Animal, Facultad de Medicina Veterinaria, Universidad Austral de Chile, Valdivia, Chile

关键词: Multipotency;    Differentiation potential;    Bovine fetuses;    Mesenchymal stem cell;   

Others  :  1119505
DOI  :  10.1186/1746-6148-9-133

 received in 2013-03-22, accepted in 2013-06-28,  发布年份 2013

【 摘 要 】

Background

Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses.

Results

Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively.

Conclusions

Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.

【 授权许可】

   
2013 Cortes et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]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: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002, 418:41-49.
• [2]Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV: Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974, 17:331-340.
• [3]Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284:143-147.
• [4]Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM: Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001, 98:2615-2625.
• [5]Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM: Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 2006, 8:315-317.
• [6]Qi H, Aguiar DJ, Williams SM, La Pean A, Pan W, Verfaillie CM: Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci USA 2003, 100:3305-3310.
• [7]Clabaut A, Delplace S, Chauveau C, Hardouin P, Broux O: Human osteoblasts derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipocytes. Differentiation 2010, 80:40-45.
• [8]Lee HJ, Choi BH, Min BH, Park SR: Changes in surface markers of human mesenchymal stem cells during the chondrogenic differentiation and dedifferentiation processes in vitro. Arthritis Rheum 2009, 60:2325-2332.
• [9]Ren H, Cao Y, Zhao Q, Li J, Zhou C, Liao L, Jia M, Zhao Q, Cai H, Han ZC, Yang R, Chen G, Zhao RC: Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions. Biochem Biophys Res Commun 2006, 347:12-21.
• [10]Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A: Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998, 95:379-391.
• [11]Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A: Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003, 113:643-655.
• [12]Lengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S, Gokhale S, Scholer HR, Tomilin A, Jaenisch R: Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 2007, 1:403-415.
• [13]Pierantozzi E, Gava B, Manini I, Roviello F, Marotta G, Chiavarelli M, Sorrentino V: Pluripotency regulators in human mesenchymal stem cells: Expression of nanog but not of oct-4 and sox-2. Stem Cells Dev 2011, 20:915-923.
• [14]Go MJ, Takenaka C, Ohqushi H: Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities. Exp Cell Res 2008, 314:1147-1154.
• [15]Martin DR, Cox NR, Hathcock TL, Niemeyer GP, Baker HJ: Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp Hematol 2002, 30:879-886.
• [16]Huss R, Hoy CA, Deeg HJ: Contact- and growth factor-dependent survival in a canine marrow-derived stromal cell line. Blood 1995, 85:2414-2421.
• [17]Ringe J, Kaps C, Schmitt B, Buscher K, Bartel J, Smolian H, Schultz O, Burmester GR, Haupl T, Sittinger M: Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res 2002, 307:321-327.
• [18]Bosch P, Pratt SL, Stice SL: Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells. Biol Reprod 2006, 74:46-57.
• [19]Patterson–Kane JC, Becker DL, Rich T: The pathogenesis of tendon microdamage in athletes: the horse as a natural model for basic cellular research. J Comp Pathol 2012, 147:227-247.
• [20]Riond JL, Kocabagli N, Spichiger UE, Wanner M: Postparturient hypocalcemia of dairy cows: a model for the study of the interdependence of Ca, Pi, and Mg homeostasis. Bone 1995, 17(4 Suppl):429S-434S.
• [21]Aerssens J, Boonen S, Lowet G, Dequeker J: Interspecies differences in bone composition, density, and quality: potential implications for in vivo bone research. Endocrinology 1998, 139:663-670.
• [22]Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T: Isolation and multilineage differentiation of bovine marrow mesenchymal stem cells. Cell Tissue Res 2005, 319:243-253.
• [23]Colleoni S, Donofrio G, Lagutine I, Duchi R, Galli C, Lazzari G: Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells. Cloning Stem Cells 2005, 7:154-166.
• [24]Dmitrieva RI, Minullina IR, Bilibina AA, Tarasova OV, Anisimov SV, Zaritskey AY: Bone marrow- and subcutaneous adipose tissue-derived mesenchymal stem cells, differences and similarities. Cell Cycle 2012, 11:377-383.
• [25]Raoufi MF, Tajik P, Dehghan MM, Eini F, Barin A: Isolation and differentiation of mesenchymal stem cells from bovine umbilical cord blood. Reprod Dom Anim 2010, 46:95-99.
• [26]Cardoso TC, Ferrari HF, Garcia AF, Novais JB, Silva-Frade CS, Ferrarezi MC, Andrade AL, Gameiro R: Isolation and characterization of wharton’s jellyderived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system. BMC Biotechnol 2012, 12:18. BioMed Central Full Text
• [27]Zhang ZY, Teoh SH, Chong MS, Schantz JT, Fisk NM, Choolani MA, Chan J: Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells. Stem Cells 2009, 27:126-137.
• [28]Zhang ZY, Teoh SH, Hui JHP, Fisk NM, Choolani M, Chan JKY: The potential of human fetal mesenchymal stem cells for off-the-shelf bone tissue engineering application. Biomaterials 2012, 33:2656-2672.
• [29]Kon E, Muraglia A, Corsi A, Bianco P, Marcacci M, Martin I, Boyde A, Ruspantini I, Chistolini P, Rocca M, Giardino R, Cancedda R, Quarto R: Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res 2000, 49:328-337.
• [30]Russell KC, Phinney DG, Lacey MR, Barrilleaux BL, Meyertholen KE, O’Connor KC: In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells 2010, 28:788-798.
• [31]Heng BC, Cao T, Haider HK, Wang DZ, Sim EK, Ng SC: An overview and synopsis of techniques for directing stem cell differentiation in vitro. Cell Tissue Res 2004, 315:291-303.
• [32]Colleoni S, Bottani E, Tessaro I, Mari G, Merlo B, Romagnoli N, Spadari A, Galli C, Lazzari G: Isolation, growth and differentiation of equine mesenchymal stem cells: effect of donor, source, amount of tissue and supplementation with basic fibroblast growth factor. Vet Res Commun 2009, 33:811-821.
• [33]McCarty RC, Gronthos S, Zannettino AC, Foster BK, Xian CJ: Characterisation and developmental potential of ovine bone marrow derived mesenchymal stem cells. J Cell Physiol 2008, 219:324-333.
• [34]Takedachi M, Oohara H, Smith BJ, Iyama M, Kobashi M, Maeda K, Long C, Humphrey MB, Stoecker BJ, Toyosawa S, Thompson LF, Murakami S: CD73-generated adenosine promotes osteoblast differentiation. J Cell Physiol 2012, 227:2622-2631.
• [35]Delorme B, Ringe J, Gallay N, Le Vern Y, Kerboeuf D, Jorgensen C, Rosset P, Sensebé L, Layrolle P, Haupl T, Charbord P: Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. Blood 2008, 111:2631-2635.
• [36]Geesin JC, Hendricks LJ, Gordon JS, Berg RA: Modulation of collagen synthesis by growth factors: the role of ascorbate-stimulated lipid peroxidation. Arch Biochem Biophys 1991, 289:6-11.
• [37]Alcain FJ, Buron MI: Ascorbate on cell growth and differentiation. J Bioenerg Biomembr 1994, 26:393-398.
• [38]Deorosan B, Nauman EA: The role of glucose, serum, and three-dimensional cell culture on the metabolism of bone marrow-derived mesenchymal stem cells. Stem Cells Int 2011, 42:9187.
• [39]Bosnakovski D, Mizuno M, Kim G, Ishiguro T, Okumura M, Iwanaga T, Kadosawa T, Fujinaga T: Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system. Exp Hematol 2004, 32:502-509.
• [40]Hausman GJ: Dexamethasone induced preadipocyte recruitment and expression of CAAT/enhancing binding protein α and peroxisome proliferator activated receptor-γ proteins in porcine stromal-vascular (S-V) cell cultures obtained before and after onset of fetal adipogenesis. Gen Comp Endocinol 2003, 133:61-70.
• [41]Harris SG, Phipps RP: The nuclear receptor PPAR γ is expressed by mouse T lymphocytes and PPAR γ agonists induce apoptosis. Eur J Immunol 2001, 31:1098-1105.
• [42]Inoue K, Kawahito Y, Tsubouchi Y, Kohno M, Yoshimura R, Yoshikawa T, Sano H: Expression of peroxisome proliferator-activated receptor gamma in renal cell carcinoma and growth inhibition by its agonists. Biochem Biophys Res Commun 2001, 287:727-732.
• [43]Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA: Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005, 122:947-956.
• [44]Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, Bourque G, George J, Leong B, Liu J, Wong KY, Sung KW, Lee CW, Zhao XD, Chiu KP, Lipovich L, Kuznetsov VA, Robson P, Stanton LW, Wei CL, Ruan Y, Lim B, Ng HH: The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006, 38:431-440.
• [45]Riekstina U, Cakstina I, Parfejevs V, Hoogduijn M, Jankovskis G, Muiznieks I, Muceniece R, Ancans J: Embryonic stem cell marker expression pattern in human mesenchymal stem cells derived from bone marrow, adipose tissue, heart and dermis. Stem Cell Rev 2009, 5:378-386.
• [46]Berg JS, Goodell MA: An argument against a role for Oct4 in somatic stem cells. Cell Stem Cell 2007, 1:359-360.