2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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O.E. Smith, B.D. Murphy & L.C. Smith. 2011. Derivation and Potential Applications of Pluripotent Stem Cells for Regenerative Medicine in Horses. ssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s273 - s283. cterization tests. On the other hand, this inability to form teratomas may be advantageous for therapeutic use by lowering the risk of uncontrolled differentiation once the ES-like cells are injected in the horse’s injury site. There has yet to be a clinical experiment done on horses to see if these cells would help regenerate cells on a damaged tissue without tumor formation. V. INDUCED REPROGRAMMING OF ADULT CELLS A landmark in stem cell research was the establishment of a protocol to reprogram differentiated cells back to their initial stage of pluripotency. Although it was possible to reprogram differentiated cells to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with ES cells, little was known about the factors that induce reprogramming (Figure 2). Induced pluripotent stem (iPS) cells were generated by the expression of a set of typical stem cell transcription factors (Oct4, Sox2, Klf4, c-Myc, NANOG and Lin28) in adult somatic cells [34]. These iPS cells show great potential for regenerative therapy as they can differentiate into all three embryonic germ layers and can be expanded to large quantities in vitro. Apart from murine rodents, iPS cells have been produced in a few species such as humans [40] and porcine [39] and most recently in equine [24]. The later was achieved by incorporating via piggyBac transposon-based method that, once the transposon electroporated into the equine fetal fibroblast, delivers transient transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc. The ectopic expression of these factors is doxycycline-dependant and the presence of this antibiotic in the culture media initiates the somatic cells’ reprogramming. Equine iPS cells show a stable karyotype after extended culture, express pluripotent properties, and are able to form teratomas composed of all three embryonic germ layers in immunodeficient mice [24]. Once iPS cells are confirmed to be safe for therapeutic application, they could have a major impact in regenerative medicine especially since they can be produced for specific patients without raising the ethical issue that ES cells tend to. This new discovery represents a new way of approaching musculoskeletal injury treatment in horses, providing stem cells with control over the expression of the pluripotent factors. Injecting these cells in the injury site of a horse being treated with doxycyclin allows the cells to proliferate and expand in the damaged tissue. Once the animal’s doxycyclin treatment is withdrawn, the iPS cells will differentiate into the intended tissue that requires repair. Cell specification could either happen naturally, because of the tissue surrounding factors inducing the right differentiation, or by outside influence via incorporation of specific factors for the iPS cells. This control over the expression of the pluripotency markers could also reduce the chances of unwanted teratoma formation. iPS cells could represent a better source for long-term tissue repair because of its higher pluripotent state than MSCs and, for equine, ES cells. Their capacity to expand in large quantities in vitro is an advantage towards ES cells, and their painless and easy accessibility, requires only a skin biopsy, is more appealing than the methods used for adult MSC harvest. To date only fetal fibroblasts have successfully been used to derive equine iPS, which is somewhat disadvantageous for clinical applications. Allogenic iPS cells could have a higher risk of rejection if implanted in an injured horse. Ideally, one would use the injured animal’s own adult fibroblast to produce and proliferate iPS cells that would then be transplanted to the damaged tissue. However, adult cells might be a bigger challenge to derive, their reprogramming efficiency being affected by age, origin and cell type [15], which will most likely also have an impact on the resulting iPS cell quality. VI. CONCLUSION Presently, equine MSCs are more commonly used in regenerative therapies due to their autologous and tissue specific differentiation properties. For musculoskeletal damages, bone marrow MSCs seem to be the most effective cells to improve early healing response, but their low plasticity hinders long-term tissue restoration, which can lead to facilitated re-injuries. Extra-embryonic stem cells could be a valid option of stem cell source because of their wider plasticity and their less advanced cell state, compared to adult-derived stem cells. Studies have recently shown that UCB-MSC transplantation into fractured mouse femurs allowed the accelerated the repair of the tissues and bone substitution [23]. However, no s280
O.E. Smith, B.D. Murphy & L.C. Smith. 2011. Derivation and Potential Applications of Pluripotent Stem Cells for Regenerative Medicine in Horses. ssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s273 - s283. clinical applications have yet been conducted on the horse. It is also possible to derive autologous pluripotent stem cells in horses by using both ES and iPS cell-derived approaches. Although ES cell lines are generally the main source of stem cells used for regenerative medicine in other species, due to its pluripotency and easy proliferation, a real equine ES cell line has yet to be derived. The risk of uncontrollable division once implanted in the animal is also an important factor against ES cells for clinical applications. Alternatively, equine iPS cells have effectively been produced and show excellent stability during prolonged in vitro culture. They additionally have the ability to differentiate into the three germ layers in vivo, suggesting that they could soon be used in pre-clinical trials. Since they are antibiotic dependent, it is possible that their proliferative power could be controlled and unwanted tumor formation avoided. Although further studies need to be performed to assess their regenerative properties, iPS cells will most possibly become one of the most important sources of stem cells for future clinical applications. REFERENCES 1 Arnhold S.J., Goletz I., Klein H., Stumpf G., Beluche L.A., Rohde C., Addicks K. & Litzke L.F. 2007. Isolation and Characterization of Bone Marrow-derived Equine Mesenchymal Stem Cells. American Veterinary Medical Association Journals. 68 (10): 1095-1105. 2 Bourzac C., Smith L.C., Vincent P., Beauchamp G., Lavoie J.P. & Laverty S. 2010. Isolation of equine bone marrowderived mesenchymal stem cells: a comparison between three protocols. Equine Veterinary Journal. 42(6): 519-27. 3 Chrisoula A., Toupadakis A., Wong A., Genetos D.C., Cheung W.K., Borjesson D.L., Ferraro G.L., Galuppo L.D., Leach J., Owens S.D. & Yellowley C.E. 2010. Comparison of the Osteogenic Potential of Equine Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue, Umbilical Cord Blood, and Umbilical Cord Tissue.” American Veterinary Medical Association Journals. 71: 1237-1245. 4 Cowan C. A., Klimanskaya I., McMahon J., Atienza J., Witmyer J., Zucker J.P., Wang S., Morton C.C., McMahon A.P., Powers D. & Melton D.A. 2004. Derivation of Embryonic Stem-Cell Lines from Human Blastocysts. The New England N Journal of Medicine. 350(13): 1353-1356. 5 Dardick I., Poznanski W.J., Waheed I. & Setterfield J. 1976. Ultrastructural observations on differentiating human preadipocytes cultured in vitro. Tissue Cell. 8(3): 561-571. 6 Del Bue M., Ricco S., Ramoni R., Conti V., Gnudi G. & Grolli S. 2008. Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo. Veterinary Research Communications. 32 (Suppl 1): S51-55. 7 Del Bue M., Riccò S., Conti V., Merli E., Ramoni R. & Grolli S. 2007. Platelet lysate promotes in vitro proliferation of equine mesenchymal stem cells and tenocytes. Veterinary Research Communications. 31(Suppl 1): 289-292. 8 Desmarais J.A., Demers S-P., Suzuki J., Laflamme S., Vincent P., Laverty S. & Smith L.C. 2011. Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos. Reproduction. 141: 321- 332. 9 Dominici M., Le Blanc K., Mueller I., Slaper-Cortenbach I., Marini F., Krause D., Deans R., Keating A., Prockop D. & Horwitz E. 2006. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8(4): 315-317. 10 Durando M.M., Zarucco L., Schaer T.P., Ross M. & Reef V.B. 2006. Pneumopericardium in a horse secondary to sternal bone marrow aspiration. Equine Veterinary Education. 18: 75–78. 11 Frisbie D.D. & Smith R.K. 2010. Clinical update on the use of mesenchymal stem cells in equine orthopaedics. Equine Veterinary Journal. 42: 86-89. 12 Fortier L.A. 2005. Stem cells: classifications, controversies, and clinical applications. Veterinary Surgery. 34(5): 415-423. 13 Galli C., Lagutina I., Duchi R., Colleoni S. & Lazzari G. 2008. Somatic cell nuclear transfer in horses. Reproduction in Domestic Animals. 43: 331-337. 14 Gimble J.M. & Guilak F. 2003. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy. 5(5): 362-369. s281
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