Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue

BMC Veterinary Research

Clementina Rodellar²  Inmaculada Martín-Burriel³  Pilar Zaragoza³  Francisco José Vázquez¹  Antonio Romero¹  Samuel Álvarez-Arguedas³  Ana Rosa Remacha³  Beatriz Ranera³ 

[1] Hospital Veterinario, Facultad de Veterinaria, Universidad de Zaragoza, 50013, Zaragoza, Spain;Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, 50009, Spain;Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013, Zaragoza, Spain

关键词: Characterisation;    BM-MSC;    AT-MSC;    Horse;    Hypoxia;   

Others  :  1119745
DOI  :  10.1186/1746-6148-8-142

 received in 2012-02-03, accepted in 2012-08-13,  发布年份 2012

【 摘 要 】

Background

Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O₂). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O₂.

Results

At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O₂ for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state.

Conclusions

Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

【 授权许可】

   
2012 Ranera et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Koch TG, Berg LC, Betts DH: Current and future regenerative medicine - principles, concepts, and therapeutic use of stem cell therapy and tissue engineering in equine medicine. Can Vet J 2009, 50(2):155-165.
• [2]Harrison JS, Rameshwar P, Chang V, Bandari P: Oxygen saturation in the bone marrow of healthy volunteers. Blood 2002, 99(1):394.
• [3]Pasarica M, Sereda OR, Redman LM, Albarado DC, Hymel DT, Roan LE, Rood JC, Burk DH, Smith SR: Reduced adipose tissue oxygenation in human obesity: evidence for rarefaction, macrophage chemotaxis, and inflammation without an angiogenic response. Diabetes 2009, 58(3):718-725.
• [4]Semenza GL: HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell 2001, 107(1):1-3.
• [5]Ohnishi S, Yasuda T, Kitamura S, Nagaya N: Effect of hypoxia on gene expression of bone marrow-derived mesenchymal stem cells and mononuclear cells. Stem Cells 2007, 25(5):1166-1177.
• [6]Chacko SM, Ahmed S, Selvendiran K, Kuppusamy ML, Khan M, Kuppusamy P: Hypoxic preconditioning induces the expression of prosurvival and proangiogenic markers in mesenchymal stem cells. Am J Physiol Cell Physiol 2010, 299(6):C1562-C1570.
• [7]Zhu W, Chen J, Cong X, Hu S, Chen X: Hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells. Stem Cells 2006, 24(2):416-425.
• [8]Ren H, Cao Y, Zhao Q, Li J, Zhou C, Liao L, Jia M, Cai H, Han ZC, Yang R, et al.: Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions. Biochem Biophys Res Commun 2006, 347(1):12-21.
• [9]Holzwarth C, Vaegler M, Gieseke F, Pfister SM, Handgretinger R, Kerst G, Muller I: Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells. BMC Cell Biol 2010, 11:11. BioMed Central Full Text
• [10]Lennon DP, Edmison JM, Caplan AI: Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. J Cell Physiol 2001, 187(3):345-355.
• [11]D'Ippolito G, Diabira S, Howard GA, Roos BA, Schiller PC: Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone 2006, 39(3):513-522.
• [12]Godwin EE, Young NJ, Dudhia J, Beamish IC, Smith RK: Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon. Equine Vet J 2011, 44:25-32.
• [13]Frisbie DD, Kisiday JD, Kawcak CE, Werpy NM, McIlwraith CW: Evaluation of adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis. J Orthop Res 2009, 27(12):1675-1680.
• [14]Grimshaw MJ, Mason RM: Bovine articular chondrocyte function in vitro depends upon oxygen tension. Osteoarthr Cartil 2000, 8(5):386-392.
• [15]Zhao J, Zhang P, Qin L, Pan XH: Hypoxia is essential for bone-tendon junction healing: the molecular biological evidence. Int Orthop 2010, 35(6):925-928.
• [16]Rosova I, Dao M, Capoccia B, Link D, Nolta JA: Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells 2008, 26(8):2173-2182.
• [17]Berg L, Koch T, Heerkens T, Bessonov K, Thomsen P, Betts D: Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood. Vet Comp Orthop Traumatol 2009, 22(5):363-370.
• [18]Schnabel LV, Lynch ME, van der Meulen MC, Yeager AE, Kornatowski MA, Nixon AJ: Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J Orthop Res 2009, 27(10):1392-1398.
• [19]Carrade DD, Owens SD, Galuppo LD, Vidal MA, Ferraro GL, Librach F, Buerchler S, Friedman MS, Walker NJ, Borjesson DL: Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses. Cytotherapy 2010, 13(4):419-430.
• [20]Guest DJ, Smith MR, Allen WR: Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon. Equine Vet J 2010, 42(7):636-642.
• [21]Watts AE, Yeager AE, Kopyov OV, Nixon AJ: Fetal derived embryonic-like stem cells improve healing in a large animal flexor tendonitis model. Stem Cell Res Ther 2011, 2(1):4. BioMed Central Full Text
• [22]Vidal MA, Kilroy GE, Johnson JR, Lopez MJ, Moore RM, Gimble JM: Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity. Vet Surg 2006, 35(7):601-610.
• [23]Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Addicks K, Litzke LF: Isolation and characterization of bone marrow-derived equine mesenchymal stem cells. Am J Vet Res 2007, 68(10):1095-1105.
• [24]Mambelli LI, Santos EJ, Frazao PJ, Chaparro MB, Kerkis A, Zoppa AL, Kerkis I: Characterization of equine adipose tissue-derived progenitor cells before and after cryopreservation. Tissue Eng Part C Methods 2009, 15(1):87-94.
• [25]Toupadakis CA, Wong A, Genetos DC, Cheung WK, Borjesson DL, Ferraro GL, Galuppo LD, Leach JK, Owens SD, Yellowley CE: Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue. Am J Vet Res 2010, 71(10):1237-1245.
• [26]Ranera B, Ordovas L, Lyahyai J, Bernal ML, Fernandes F, Remacha AR, Romero A, Vazquez FJ, Osta R, Cons C, et al.: Comparative study of equine bone marrow and adipose tissue-derived mesenchymal stromal cells. Equine Vet J 2011, 44(1):33-42.
• [27]Dos Santos F, Andrade PZ, Boura JS, Abecasis MM, da Silva CL, Cabral JM: Ex vivo expansion of human mesenchymal stem cells: a more effective cell proliferation kinetics and metabolism under hypoxia. J Cell Physiol 2010, 223(1):27-35.
• [28]Chung DJ, Hayashi K, Toupadakis CA, Wong A, Yellowley CE: Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia. Res Vet Sci 2010, 92(1):66-75.
• [29]Zeng HL, Zhong Q, Qin YL, Bu QQ, Han XA, Jia HT, Liu HW: Hypoxia-mimetic agents inhibit proliferation and alter the morphology of human umbilical cord-derived mesenchymal stem cells. BMC Cell Biol 2011, 12:32. BioMed Central Full Text
• [30]Gardner LB, Li Q, Park MS, Flanagan WM, Semenza GL, Dang CV: Hypoxia inhibits G1/S transition through regulation of p27 expression. J Biol Chem 2001, 276(11):7919-7926.
• [31]Iida T, Mine S, Fujimoto H, Suzuki K, Minami Y, Tanaka Y: Hypoxia-inducible factor-1alpha induces cell cycle arrest of endothelial cells. Genes Cells 2002, 7(2):143-149.
• [32]Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett JC, Huang LE: HIF-1alpha induces cell cycle arrest by functionally counteracting Myc. EMBO J 2004, 23(9):1949-1956.
• [33]Peterson KM, Aly A, Lerman A, Lerman LO, Rodriguez-Porcel M: Improved survival of mesenchymal stromal cell after hypoxia preconditioning: role of oxidative stress. Life Sci 2010, 88(1–2):65-73.
• [34]Volkmer E, Kallukalam BC, Maertz J, Otto S, Drosse I, Polzer H, Bocker W, Stengele M, Docheva D, Mutschler W, et al.: Hypoxic preconditioning of human mesenchymal stem cells overcomes hypoxia-induced inhibition of osteogenic differentiation. Tissue Eng Part A 2010, 16(1):153-164.
• [35]Lavrentieva A, Majore I, Kasper C, Hass R: Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells. Cell Commun Signal 2010, 8:18. BioMed Central Full Text
• [36]Nakanishi C, Nagaya N, Ohnishi S, Yamahara K, Takabatake S, Konno T, Hayashi K, Kawashiri MA, Tsubokawa T, Yamagishi M: Gene and protein expression analysis of mesenchymal stem cells derived from rat adipose tissue and bone marrow. Circ J 2011, 75(9):2260-2268.
• [37]Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, Bae YC, Jung JS: Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004, 14(4–6):311-324.
• [38]Pascucci L, Curina G, Mercati F, Marini C, Dall'aglio C, Paternesi B, Ceccarelli P: Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: Towards the definition of minimal stemness criteria. Vet Immunol Immunopathol 2011, 144(3-4):499-506.
• [39]Braun J, Hack A, Weis-Klemm M, Conrad S, Treml S, Kohler K, Walliser U, Skutella T, Aicher WK: Evaluation of the osteogenic and chondrogenic differentiation capacities of equine adipose tissue-derived mesenchymal stem cells. Am J Vet Res 2010, 71(10):1228-1236.
• [40]Raabe O, Shell K, Wurtz A, Reich CM, Wenisch S, Arnhold S: Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells. Vet Res Commun 2011, 35(6):355-365.
• [41]de Mattos Carvalho A, Alves AL, Golim MA, Moroz A, Hussni CA, de Oliveira PG, Deffune E: Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue. Vet Immunol Immunopathol 2009, 132(2–4):303-306.
• [42]Radcliffe CH, Flaminio MJ, Fortier LA: Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations. Stem Cells Dev 2010, 19(2):269-282.
• [43]Ranera B, Lyahyai J, Romero A, Vazquez FJ, Remacha AR, Bernal ML, Zaragoza P, Rodellar C, Martin-Burriel I: Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue. Vet Immunol Immunopathol 2011, 144(1-2):147-154.
• [44]Nekanti U, Dastidar S, Venugopal P, Totey S, Ta M: Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia. Int J Biol Sci 2010, 6(5):499-512.
• [45]Na KH, Lee HJ, Choi JH, Eun JW, Nam SW, Yoon TK, Kim GJ: Dynamic alterations in integrin alpha4 expression by hypoxia are involved in trophoblast invasion during early implantation. J Cell Biochem 2011, 113(2):685-694.
• [46]Deguchi JO, Yamazaki H, Aikawa E, Aikawa M: Chronic hypoxia activates the Akt and beta-catenin pathways in human macrophages. Arterioscler Thromb Vasc Biol 2009, 29(10):1664-1670.
• [47]Violini S, Ramelli P, Pisani LF, Gorni C, Mariani P: Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12. BMC Cell Biol 2009, 10:29. BioMed Central Full Text
• [48]Ezashi T, Das P, Roberts RM: Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci U S A 2005, 102(13):4783-4788.
• [49]Fehrer C, Brunauer R, Laschober G, Unterluggauer H, Reitinger S, Kloss F, Gully C, Gassner R, Lepperdinger G: Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell 2007, 6(6):745-757.
• [50]Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T: Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol 2006, 207(2):331-339.
• [51]Mathieu J, Zhang Z, Zhou W, Wang AJ, Heddleston JM, Pinna CM, Hubaud A, Stadler B, Choi M, Bar M, et al.: HIF induces human embryonic stem cell markers in cancer cells. Cancer Res 2011, 71(13):4640-4652.