Isolation, Expansion, Differentiation and Growth Kinetics Essay in Mesenchymal Stem Cells Culture from the Bone Marrow of Collared Peccaries (Tayassu tajacu)
Keywords:mesenchymal stem cells, Tayassu tajacu, bone marrow.
Background: There are few studies on stem cell isolation in wild animals that provide isolation and culture protocols of these cells in vitro. Among the wild species studied, we present the collared peccary (Tayassu tajacu) as a model with potential to obtain and use MSC in preclinical studies. These animals are phylogenetically close to the domestic pig, popularly known as peccaries and found naturally in South America, Central America and the South of the United States. The aim of the present study was to establish a protocol for the isolation, in vitro cell expansion, differentiation and assessment of the stromal MSC growth curve before and after thawing.
Materials, Methods & Results: Mesenchymal stem cells (MSC) from collared peccary bone marrow (Tayassu tajacu) were isolated and expanded by centrifuge in Ficoll® solution and cultured in DMEM® High Glucose medium. The culture was assessed by assays of colony forming units CFU-F and growth curve by saturation (GCS). Cultures in the third passage, with 70% confluence, were replicated at 105 cells/mL concentration in the culture media to induce osteogenic cell differentiation and adipogenic cell differentiation, respectively. The MSC were frozen in nitrogen for 40 days, thawed and re-assessed for cell viability and GCS.
Discussion: The bone marrow collected presented high mononuclear cellularity, with a mean variability of 94.5% and 60.83 ± 4.27 UFC were identified in the samples and cells with fibroblast-like-cell morphology were observed. When they were expanded, the mean cell viability was 95%, the mean cell concentration obtained was 233.31 ± 20.04 cells per 25cm2 bottle and the culture reached the growth plateau in GCS between the 13th and 16th day. The osteoblastic cell differentiation assay showed after 18 days, morphology similar to osteoblasts, with irregular cytoplasm limits, cell prolongation formation and flattened appearance. After staining with Alizarin Red, the nucleus presented a wine red coloring and the cytoplasm, more basophilic and well-defined, with calcium deposits inside the cells. The cultures submitted to adipogenic differentiation were large, hexagonal, irregular and presented birrefringent cytoplasm granules after the third week of culture. When stained with Oil Red it was observed that the cytoplasm granules were scattered small fat vacuoles and stained maroon. The viability after thawing was 78% and the mean cell concentration obtained in GCS was 199.71 ± 14.72 cells per 25 cm2 bottle. The curves reached the saturation plateau early, on the eighth day of observation. From then onwards the cultures entered became exhausted and the cell concentration of the samples decreased progressively until minimum values. These results showed the presence of a well-defined MSC population in the collared peccary bone marrow with a high rate of replication in vitro and potential for differentiation confirmed by the adipogenic and osteogenic lines. The cryopreservation technique adopted presented satisfactory results, but indicated a significant cell stress after thawing that justifies investigation of the apoptosis rates induced post thawing in the species. Furthermore, the bone marrow collection did not harm the animals and the facility of stromal MSC isolation and culture qualifies the collared peccary as a viable alternative model to obtain MSC and for studies in the area of cell therapy.
Augello A., Kurth T.B. & Bari C. 2010. Mesenchymal stem cells: a perspective from in vitro to in vivo migration and niches. European Cells & Materials. 20(1): 121-133.
O., Babaei H., Derakhshanfar A., Nematollahi-Mahani S.N., Poursahebi R. & Moshrefi M. 2011. Effects of transplanted mesenchymal stem cells isolated from Wharton’s jelly of caprine umbilical cord on cutaneous wound healing; histopathological evaluation. Veterinary Research Communications. 35(4): 211-222.
Bland M. 2000. An Introduction to Medical Statistics. 3rd edn. NewYork: Oxford University Press, 405p.
Bosnakovski D., Mizuno M., Kim G., Takagi S., Okumura M. & Fujinaga T. 2005. Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells. Cell and Tissue Research. 319(2): 243-253.
Cabrera A., Yepes J. & Wiedner C.C. 1940. Mamiferos Sud-Americanos (Vida, Costumbres y Descripcion). Buenos Aires: Compañia Argentina de Editores, 370p.
Chang Y., Hsieh P.H. & Chao C.C.K. 2009. The efficiency of percoll and ficoll density gradient media in the isolation of marrow derived human mesenchymal stem cells with osteogenic potential. Chang Gung Medical Journal. 32(3): 264275.
Chen K., Baxter T., Muir W.M., Groenen M.A. & Schook L.B. 2007. Genetic resources, genome mapping and evolutionary genomics of the pig (Sus scrofa). International Journal of Biological Sciences. 3(3): 153-165.
Corn D.J., Kim Y., Krebs M.D., Mounts T., Molter J., Gerson S., Alsberg E., Dennis J.E. & Lee Z. 2013. Imaging early stage osteogenic differentiation of mesenchymal stem cells. Journal of Orthopaedic Research. 31(6): 871-879.
Covas D.T., Panepucci R.A., Fontes A.M., Silva Jr. W.A., Orellana M.D., Freitas M.C., Neder L., Santos A.R., Peres L.C., Jamur M.C. & Zago M.A. 2008. Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts. Experimental Hematology. 36(5): 642-654.
Desbiez A.L.J., Santos S.A., Keuroghlian A. & Bodmer R.E. 2009. Niche partitioning among white-lipped peccaries (Tayassu pecari), collared peccaries (Pecari tajacu), and feral pigs (Sus scrofa). Journal of Mammalogy. 90(1): 119128.
Deus G.C., Normanton M., Hamerschlak N., Kondo A.T., Ribeiro A.A.F., Goldberg A.C. & Marti L.C. 2012. Isolation and characterization of mesenchymal stem cells obtained from reusable and disposable bone marrow collection filters. Einstein. 10(3): 296-301.
Dorandeu F., Mikler J.R., Thiermann H., Tenn C., Davidson C., Sawyer T.W., Lallement G. & Worek F. 2007. Swine models in the design of more effective medical countermeasures against organophosphorus poisoning. Toxicology. 233(1-3): 128-144.
Ginis I., Grinblat B. & Shirvan M.H. 2012. Evaluation of bone marrow-derived mesenchymal stem cells after cryopreservation and hypothermic storage in clinically safe medium. Tissue Engineering Part C: Methods. 18(6): 453463.
Goh B.C., Thirumala S., Kilroy G., Devireddy R.V. & Gimble J.M. 2007. Cryopreservation characteristics of adipose-derived stem cells: maintenance of differentiation potential and viability. Journal of Tissue Engineering and Regenerative Medicine. 1(4): 322-324.
Gronthos S, Zannettino A.C., Hay S.J., Shi S., Graves S.E., Kortesidis A. & Simmons P.J. 2003. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. Journal of Cell Science. 116(9): 1827-1835.
Irioda A.C., Zocche L., Souza C.M.C.O., Ferreira R.J., Aliprandini E., Cunha R.C., Francisco J.C., GuaritaSouza L.C., Malvezzi M., Beltrame M.P., Mesquita L.A.F, Kuczera D., Chachques J.C. & Carvalho K.A.T. 2011. Pap test as the first step in screening genetic stability in cell-based therapy. Journal of Stem Cell Research & Therapy. 1(3): 1-6.
Iwata T., Yamato M., Zhang Z., Mukobata S., Washio K., Ando T., Feijen J., Okano T. & Ishikawa I. 2010. Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use. Journal of Clinical Periodontology. 37(12): 1088-1099.
Khatri M., O´Brien T.D. & Sharma J.M. 2009. Isolation and differentiation of chicken mesenchymal stem cells from bone marrow. Stem Cells and Development. 18(10): 1485-1492.
Koerner J., Nesic D., Romero J.D., Brehm W., Varlet P.M. & Grogan S.P. 2006. Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells. Stem Cells. 24(6): 1613-1619.
Linkenhoker J.R., Burkholder T.H., Linton C.G., Walden A., Abusakran-Monday K.A., Rosero A.P. & Foltz C.J. 2010. Effective and safe anesthesia for yorkshire and yucatan swine with and without cardiovascular injury and intervention. Journal of the American Association for Laboratory Animal. 49(3): 344-351.
Liu Y., Liu L., Ma X., Yin Y., Tang B. & Li Z. 2013. Characteristics and neural-like differentiation of mesenchymal stem cells derived from fetal porcine bone marrow. Bioscience Reports. 33(2): 351-360.
Mafi P., Hindocha S., Mafi R., Griffin M. & Khan W.S. 2011. Adult mesenchymal stem cells and cell surface characterization. The Open Orthopaedics Journal. 5(Suppl 2): 253-260.
Mandel, N.S., Henderson JR J.D., Hung L.Y., Wille D.F. & Wiessner J.H. 2004. A porcine model of calcium oxalate kidney stone disease. Journal of Urology. 171(3): 1301-1303.
Meirelles L.S., Chagastelles P.C. & Nardi N.B. 2006. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. Journal Cell Science. 119(11): 2204-2213.
Meirelles L.S. & Nardi N.B. 2003. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. British Journal of Haematology. 123(4): 702-711.
Muscari C., Gamberini C., Basile I., Bonafé F., Valgimigli S., Capitani O., Guarnieri C. & Caldarera C.M. 2010. Comparison between culture conditions improving growth and differentiation of blood and bone marrow cells committed to the endothelial cell lineage. Biological Procedures Online. 12(1): 89-106.
Nakagawa T., Nabeshima Y. & Yoshida S. 2007. Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis. Developmental Cell. 12(2): 195-206.
Nardi N.B. & Meirelles L.S. 2006. Mesenchymal stem cells. Isolation, in vitro expansion and characterization. Handbook of Experimental Pharmacology. 174(1): 249-282.
Orciani M., Mariggio M.A., Morabito C., Di B.G. & Di P.R. 2010. Functional characterization of calcium-signaling pathways of human skin-derived mesenchymal stem cells. Skin Pharmacology and Physiology. 23(3): 124-132.
Penny J., Harris P., Shakesheff K.M. & Mobasheri A. 2012. The biology of equine mesenchymal stem cells: phenotypic characterization, cell surface markers and multilineage differentiation. Frontiers in Bioscience. 17(1): 892-908.
Peterbauer-Scherb A., Griensven M., Meinl A., Gabriel C., Redl H. & Wolbank S. 2010. Isolation of pig bone marrow mesenchymal stem cells suitable for one-step procedures in chondrogenic regeneration. Journal of Tissue Engineering and Regenerative Medicine. 4(6): 485-490.
Purriños M.R., Vazquez F.F., Prado A.P., Altonaga J.R., Ramon C.C., Silverio J.M.A., Orden A. & Orden J.MG. 2011. Ventricular arrhythmias and mortality associated with isoflurane and sevoflurane in a porcine model of myocardial infarction. Journal of the American Association for Laboratory Animal Science. 50(1): 73-78.
Ranera B., Lyahyai J., Romero A., Vázquez F.J., Remacha A.R., Bernal M.L., Zaragoza P., Rodellar C. & Martín-Burriel I. 2011. Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue. Veterinary Immunology and Immunopathology. 144(1-2): 147-154.
Redzić A., Smajilagić A., Aljicević M. & Berberović L. 2010. In vivo osteoinductive effect and in vitro isolation and cultivation bone marrow mesenchymal stem cells. Collegium Antropologicum. 34(4): 1405-1409.
Ribeiro G., Massoco C.O. & Lacerda Neto J.C. 2012. Viabilidade celular da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo de equinos após o processo de congelamento e descongelamento. Pesquisa Veterinária Brasil. 32(Suppl 1): 118-124.
Rocha A.R., Alves F.R., Argôlo Neto N.M., Santos L.F., Almeida, H.M., Carvalho Y.K.P., Bezerra D.O., Ferraz M.S., Pessoa G.T. & Carvalho M.A.M. 2012. Hematopoietic progenitor constituents and adherent cell progenitor morphology isolated from black-rumped agouti (Dasyprocta prymnolopha, Wagler 1831) bone marrow. Microscopy Research Technique. 75(10): 1376-1382.
Shi C., Zhu Y., Su Y. & Cheng T. 2006. Stem cells and their applications in skin cell therapy. Trends in Biotechnology. 24(1): 48-52.
Short B., Brouard N., Occhiodoro-Scott T., Ramakrishnan A. & Simmons P.J. 2003. Mesenchymal stem cells. Archives of Medical Research. 34(6): 565-571.
Souza A.W.S., Neves R.M.S., Oliveira K.R. & Sato E.I. 2006. Tratamento de arterite de Takayasu. Revista Brasileira de Reumatologia. 46(Suppl 1): 2-7.
Tsai M.S., Lee J.L., Chang Y.J. & Hwang S.M. 2004. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Human Reproduction. 19(6): 1450-1456.
Uccelli A. & Prockop D.J. 2010. Why should mesenchymal stem cells (MSCs) cure autoimmune diseases? Current Opinion Immunology. 22(6): 768-774.
Webb T.L., Quimby J.M. & Dow S.W. 2012. In vitro comparison of feline bone marrow-derived and adipose tissuederived mesenchymal stem cells. Journal of Feline Medicine and Surgery. 14(2): 165-168.
Xu J., Qian J., Xie X., Lin L., Zou Y., Fu M., Huang Z., Zhang G., Su Y. & Ge J. 2012. High density lipoprotein protects mesenchymal stem cells from oxidative stress-induced apoptosis via activation of the PI3K/Akt pathway and suppression of reactive oxygen species. International Journal of Molecular Sciences. 13(12): 17104-17120.
Xu S., Becker A., Camp B.V., Vanderkerken K. & Riet I.V. 2010. An improved harvest and in vitro expansion protocol for murine bone marrow-derived mesenchymal stem cells. Journal of Biomedicine and Biotechnology. October: 1-10.
Zhou Y., Yan H., Guo M., Zhu J., Xiau Q. & Zhang L. 2013. Reactive oxygen species in vascular formation and development. Oxidative Medicine and Cellular Longevity. January: 1-14.
How to Cite
This journal provides open access to all of its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Such access is associated with increased readership and increased citation of an author's work. For more information on this approach, see the Public Knowledge Project and Directory of Open Access Journals.
We define open access journals as journals that use a funding model that does not charge readers or their institutions for access. From the BOAI definition of "open access" we take the right of users to "read, download, copy, distribute, print, search, or link to the full texts of these articles" as mandatory for a journal to be included in the directory.
La Red y Portal Iberoamericano de Revistas Científicas de Veterinaria de Libre Acceso reúne a las principales publicaciones científicas editadas en España, Portugal, Latino América y otros países del ámbito latino