doi: 10.15389/agrobiology.2019.2.395eng
UDC: 619+616.5]:57.085.23
Acknowledgements:
The work was done according to project No. 0578-2018-0006 “Creation of new mammalian stem cell-based systems, including farm animals, with desired properties based on for veterinary medicine, virology and biotechnology”
CHARACTERIZATION OF MESENCHYMAL STEM CELLS ISOLATED FROM FELINE AND CANINE ADIPOSE TISSUE
I.P. Savchenkova, S.A. Vasilyeva, D.G. Korovina, А.А. Shabeikin, A.M. Gulyukin
Federal Science Center Kovalenko All-Russian Research Institute of Experimental Veterinary RAS, 24/1, Ryazanskii pr., Moscow, 109428 Russia, e-mail s-ip@mail.ru (✉ corresponding author), s.vasileva89@yandex.ru, darya.korovina@gmail.com, viev@mail.ru, plych@mail.ru
ORCID:
Savchenkova I.P. orcid.org/0000-0003-3560-5045
Shabeikin А.А. orcid.org/0000-0003-3413-8131
Vasilyeva S.A. orcid.org/0000-0001-5946-9071
Gulyukin A.M. orcid.org/0000-0003-2160-4770
Korovina D.G. orcid.org/0000-0003-2186-6084
Received December 13, 2018
Multipotent mesenchymal stem cells (MMSCs) are a promising tool of regenerative medicine for treatment of various small animal diseases. MMSCs have a high proliferative activity, multipotent properties, low immunogenicity, as well as the ability to migrate to the damaged tissue and promote its healing and regeneration. Currently, the methods of regenerative medicine are actively developing to solve problems that are difficult to cope with alternative treatments. However, data on the use of these cells in the clinic are ahead of the study of the properties of these cells in culture. This paper is our first report on isolation cells with phenotype similar to mammalian multipotent mesenchymal stem cells from feline and canine adipose tissue. The aim of the presented research was to study the cells properties with a phenotype similar to MMSC isolated from feline and canine adipose tissue (AT) in vitro. Isolation of cells was achieved by mechanical and enzymatic treatments of the AT. For enzymatic dissociation, the tissues were treated with a 0.01 % collagenase type II solution based on DMEM-LG (PanEco, Russia) with low glucose (1 g/l) at 37 °С for 60 min. The comparative analysis of properties of the derived cellular populations is carried out. Cells isolated from feline and canine adipose tissue had similar morphological characteristics and were represented by two cellular types: small round cells and larger narrow spindle like fibroblast. They had a strong adhesion to cultural plastic and high colonies formation ability, 88.3±0.10 % for feline MMSCs and 88.0±0.15 % for canine MMSCs. The generation time of feline MMSCs was 34.6±0.02 h, while in canine MMSCs it was 50.0±0.01 h. Mitotic index of feline and canine MMSCs was 3.4 ‰ and 2.7 ‰, respectively. The ability of the MMSCs to induced osteo-, chondro- and adipogenic differentiation in vitro was demonstrated using StemPro® Osteogenesis Differentiation Kit, StemPro® Chondrogenesis Differentiation Kit и StemPro® Adipogenesis Differentiation Kit (Gibco, USA), respectively. Adipogenic differentiation accompanied by the appearance of rounded cells with lipid vesicles in the cytoplasm that were identified with the specific dye Oil red O. Specific staining of feline and canine MMSCs for endogenous alkaline phosphatase was positive on day 14 of culture in the induction medium. MMSCs stained by von Kossa revealed extracellular matrix formation on day 21 after induction. Alcian blue staining of cells cultured in chondrogenic medium for 21 days visualized formation of round structures with isogenic groups similar to the lacunae of hyaline cartilage. Thus, it was shown that cells isolated from feline and canine adipose tissue exhibit in culture the properties of MMSC. The derived cell cultures were propagated and deposited to Kovalenko VIEV Specialized Collection of somatic cell cultures of farm and commercial animals.
Keywords: multipotent mesenchymal stem cells, adipose tissue, culture, induced differentiationin vitro, feline, canine.
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