399 ISOLATION AND CHARACTERIZATION OF BOVINE MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE

Abstract

Stem cells have been widely used because of their multi-differentiation ability. Compared with embryonic stem cells, mesenchymal stem cells (MSC) are more easily sourced and cultured, besides being easily obtained from adult individuals. In this regard, bovine MSC is of great interest because of its wide application in basic and applied research, e.g. in somatic cell nuclear transfer (SCNT). Success rates of SCNT are expected to be enhanced with the use of MSC as donor cells because they exhibit a more undifferentiated condition when compared with the most widely used cell type, fibroblast. There are few reports on MSC referring to this species. Therefore, our aim was to isolate and culture adipose MSC from cattle. Mesenchymal stem cells were induced to differentiate into adipocytes and osteocytes to prove their multi-differentiation ability. A small piece of adipose tissue was sourced from the base of the tail of a cow and extensively washed in cold saline solution containing 5% antibiotic. Recovered tissue was minced and digested in 0.001% collagenase 1 and incubated for 3 h. Collagenase was inactivated in alpha minimal essential medium (MEM) supplemented with 15% FCS. The treated tissue was centrifuged and the pellet plated on plastic dishes in alpha MEM supplemented with 15% FCS and 1% antibiotic. Culture medium was replaced every 2 days, and cells were plated on new dishes before reaching 75% confluence. To test their resistance to grow after thawing, cells were frozen after each passage, thawed, and cultured. Cell growth was accompanied through several weeks, and cells were tested for differentiation ability after 3 passages, when adipocyte and osteocyte differentiation was accomplished using a standard protocol. Briefly, adipocyte differentiation was inducted in DMEM containing 2% FCS, isobutyl-methylxanthine, dexamethasone, insulin, and indomethacin. Osteocyte differentiation was carried out in alpha MEM without FCS containing ascorbate-2-phosphate, dexamethasone, and beta-glycerophosphate.To confirm differentiation, a tissue-specific staining was carried out using Oil Red for adipocyte staining and Alizarin Red for osteocyte staining. The cells adhered to plastic dishes shortly after plating, presented fibroblast-like morphology, and showed an exponential growth curve during the first 6 passages. The cells were capable of growing after thawing similarly to unthawed cells. After 1 week under differentiation protocols, standard morphological changes were observed in the cells. Mesenchymal stem cells subjected to differentiation into adipocytes showed an increase in their size, developed lipid-like vesicles, and stained positive for Oil Red. An increase in the cell size was also observed in the cells subjected to differentiation into osteocyte. Moreover, these cells stained positive for Alizarin Red. Altogether these results provide evidence that these cells are multi-potent MSC. Therefore, we conclude that the current methodology was efficient in isolating bovine MSC from adipose tissue, and the cells resemble those isolated from other mammals. Bovine MSC constitute a significant source of nuclear donor cells for future experiments in SCNT.

Financial support was provided by FAPESPA and Cnpq.