Isolation and characterization of bone marrow–derived equine mesenchymal stem cells

Stefan J. Arnhold Department of Anatomy, University of Cologne, Josef-Stelzmann Str 9, 50931 Köln, Germany

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Iris Goletz Department of Anatomy, University of Cologne, Josef-Stelzmann Str 9, 50931 Köln, Germany

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Helmut Klein Department of Anatomy, University of Cologne, Josef-Stelzmann Str 9, 50931 Köln, Germany

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Gerald Stumpf Clinic for Horses, Justus-Liebig-University Giessen, Frankfurter Str 108, 35392 Giessen, Germany

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Lisa A. Beluche Clinic for Horses, Beckers Kreuz 25, 53343 Meckenheim, Germany

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Carsten Rohde Clinic for Horses, Beckers Kreuz 25, 53343 Meckenheim, Germany

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Klaus Addicks Department of Anatomy, University of Cologne, Josef-Stelzmann Str 9, 50931 Köln, Germany

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Lutz F. Litzke Clinic for Horses, Justus-Liebig-University Giessen, Frankfurter Str 108, 35392 Giessen, Germany

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Abstract

Objective—To isolate and characterize bone marrow–derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses.

Sample Population—Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses.

Procedures—Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs.

Results—Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation.

Conclusions and Clinical Relevance—Our data indicate that bone marrow–derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.

Abstract

Objective—To isolate and characterize bone marrow–derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses.

Sample Population—Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses.

Procedures—Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs.

Results—Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation.

Conclusions and Clinical Relevance—Our data indicate that bone marrow–derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.

  • 1.

    Rossdale PD, Hopes R, Digby NJ, et al. Epidemiological study of wastage among racehorses 1982 and 1983. Vet Rec 1985;116:6669.

  • 2.

    Silver IA, Brown PN, Goodship AE, et al. A clinical and experimental study of tendon injury, healing and treatment in the horse. Equine Vet J Suppl 1983;1:143.

    • Search Google Scholar
    • Export Citation
  • 3.

    Marr CM, Love S, Boyd JS, et al. Factors affecting the clinical outcome of injuries to the superficial digital flexor tendon in National Hunt and point-to-point racehorses. Vet Rec 1993;132:476479.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Short CR, Beadle RE. Pharmacology of antiarthritic drugs. Vet Clin North Am 1978;8:401417.

  • 5.

    Yovich JV, Trotter GW, McIlwraith CW, et al. Effects of polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage. Am J Vet Res 1987;48:14071414.

    • Search Google Scholar
    • Export Citation
  • 6.

    Johnson LL. Arthroscopic abrasion arthroplasty historical and pathologic perspective: present status. Arthroscopy 1986;2:5469.

  • 7.

    Barnewitz D, Evers A, Zimmermann J, et al. Tissue engineering: new treatment of cartilage alterations in degenerative joint diseases in horses—preliminary results of a long term study [in German]. Berl Munch Tierarztl Wochenschr 2003;116:157161.

    • Search Google Scholar
    • Export Citation
  • 8.

    Litzke LE, Wagner E, Baumgaertner W, et al. Repair of extensive articular cartilage defects in horses by autologous chondrocyte transplantation. Ann Biomed Eng 2004;32:5769.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Stromberg B, Tufvesson G. An experimental study of autologous digital tendon transplants in the horse. Equine Vet J 1977;9:231237.

  • 10.

    Fackelman GE. The nature of tendon damage and its repair. Equine Vet J 1973;5:141149.

  • 11.

    Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood 2003;102:34833493.

  • 12.

    Smith RK, Korda M, Blunn GW, et al. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment. Equine Vet J 2003;35:99102.

    • Search Google Scholar
    • Export Citation
  • 13.

    Noel D, Djouad F, Jorgense C. Regenerative medicine through mesenchymal stem cells for bone and cartilage repair. Curr Opin Investig Drugs 2002;3:10001004.

    • Search Google Scholar
    • Export Citation
  • 14.

    Lee HS, Huang GT, Chiang H, et al. Multipotential mesenchymal stem cells from femoral bone marrow near the site of osteonecrosis. Stem Cells 2003;21:190199.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Lee KD, Kuo TK, Whang-Peng J, et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 2004;40:12751284.

  • 16.

    Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362367.

  • 17.

    Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A 2001;98:1034410349.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Dezawa M, Ishikawa H, Itokazu Y, et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005;309:314317.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364370.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Arnhold S, Klein H, Klinz FJ, et al. Human bone marrow stroma cells display certain neural characteristics and integrate in the subventricular compartment after injection into the liquor system. Eur J Cell Biol 2006;85:551565.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Arnhold S, Absenger Y, Klein H, et al. Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse. Graefes Arch Clin Exp Ophthalmol 2007;245:414422.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Fortier LA, Nixon AJ, Williams J, et al. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res 1998;59:11821187.

    • Search Google Scholar
    • Export Citation
  • 23.

    Worster AA, Nixon AJ, Brower-Toland BD, et al. Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am J Vet Res 2000;61:10031010.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Koerner J, Nesic D, Romero JD, et al. Equine peripheral bloodderived progenitors in comparison to bone marrow-derived mesenchymal stem cells. Stem Cells 2006;24:16131619.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Vidal MA, Kilroy GE, Johnson JR, et al. Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity. Vet Surg 2006;35:601610.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Jaiswal N, Haynesworth SE, Caplan AI, et al. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295312.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Dinser R, Kreppel F, Zaucke F, et al. Comparison of long-term transgene expression after non-viral and adenoviral gene transfer into primary articular chondrocytes. Histochem Cell Biol 2001;116:6977.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Yoshimura H, Muneta T, Nimura A, et al. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007;327:449462.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Gindraux F, Selmani Z, Obert L, et al. Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule. Cell Tissue Res 2007;327:471483.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Izadpanah R, Joswig T, Tsien F, et al. Characterization of multipotent mesenchymal stem cells from the bone marrow of rhesus macaques. Stem Cells Dev 2005;14:440451.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Djouad F, Bony C, Haupl T, et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther 2005;7:R1304R1315.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143147.

  • 33.

    Vescovi A, Gritti A, Cossu G, et al. Neural stem cells: plasticity and their transdifferentiation potential. Cells Tissues Organs 2002;171:6476.

  • 34.

    Milosevic J, Storch A, Schwarz J. Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells. Stem Cells 2005;23:681688.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, selfrenewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997;64:278294.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:7174.

  • 37.

    Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:1125.

  • 38.

    Hynes MA, Poulsen K, Armanini M, et al. Neurotrophin-4/5 is a survival factor for embryonic midbrain dopaminergic neurons in enriched cultures. J Neurosci Res 1994;37:144154.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Giancotti FG, Ruoslahti E. Integrin signaling. Science 1999;285:10281032.

  • 40.

    Chastain SR, Kundu AK, Dhar S, et al. Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM ligands and controls their osteogenic differentiation. J Biomed Mater Res A 2006;78:7385.

    • Search Google Scholar
    • Export Citation
  • 41.

    Arinzeh TL. Mesenchymal stem cells for bone repair: preclinical studies and potential orthopedic applications. Foot Ankle Clin 2005;10:651665.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Ishihara A, Zachos TA, Bartlett JS, et al. Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery. Am J Vet Res 2006;67:11451155.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Schmidt A, Ladage D, Schinkothe T, et al. Basic fibroblast growth factor controls migration in human mesenchymal stem cells. Stem Cells 2006;24:17501758.

    • Crossref
    • Search Google Scholar
    • Export Citation

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