274 POTENTIAL OF BOVINE MARROW STROMAL CELLS CULTURED AT DIFFERENT PASSAGES TO DIFFERENTIATE INTO ADIPOCYTE-LIKE CELLS

Bovine marrow stromal cells (MSC) would serve as a preclinical large-animal model for investigating the use of the adult pluripotent cells MSC for human cell therapies, but the information is limited. In this study, adipocytic differentiation was examined in bovine MSC at different passages (psg) after various adipogenic treatments, as the first step in characterizing the multilineage differentiation potential during an extensive culture. Bovine MSC were cultured from the femurs of 3-month-old Holstein calves using a previously described method with slight modifications (Xiang et al. 2001 Chin. J. Pathophysiol. 17, 598–601). The MSC at psg 2, 5, 10, 15, and 20 (total cell culture periods of 10, 19, 34, 49, and 64 days, respectively, 4 × 103 cells cm–2) were cultured for 2 days post-psg. Previously, we found that most of mouse MSC became adipocyte-like cells after they were cultured with a base medium (DMEM, 4.5 g L–1 glucose, plus 10% FBS) containing horse serum (HS) for 5 weeks. Therefore, the bovine MSC at each psg (70% confluence) were then cultured in the base medium containing 0.1 μm dexamethasone, 0.1 mm 3-isobutyl-1-methylxanthine, 0.2 mm indomethacin, and 10 μg mL–1 insulin [hormonal-mixture (HM) group], the base medium containing 15% HS (HS group), or the base medium (control group) for adipogenic induction. The cells were stained with oil red O after being cultured in the respective medium for 1, 3, or 5 weeks. In this study, all cells containing oil red O-stained lipid droplets (LD) were counted as ‘oil red O-positive (OP) cells’, regardless of the size and number contained. The rate of OP cells in each group was determined as follows. In the dishes, the total number of cells (60 to 100 cells) and OP cell number were counted in a randomly selected microscopic field (200×), and the rate of OP cells was calculated. A total of 10 random fields of view were examined, and the mean value was obtained for each group. Data were analyzed by ANOVA. In the HM group, OP cells were detectable about Day 7 and were clearly visible by 2 weeks. After 5 weeks, OP cell rates were significantly greater (P < 0.05) in the cells at psg 2 and 5 than in those at psg 10, 15, and 20 (71 and 70% v. 66, 63, and 64%). In the HS group, cytoplasmic LD was detectable about Day 10 and the number increased gradually by 3 weeks. After 5 weeks, OP cell rate was significantly greater (P < 0.05) in the cell at psg 2, 5, and 10 than in those at psg 15 and 20 (53, 57, and 51% v. 47 and 39%). Also, the size and number of LD in each cell tended to be lower in the HS group than in the corresponding HM group. In the control group, 4–5% of the cells were OP after 5 weeks. These results suggest that the ability of bovine MSC to differentiate into adipocyte-like cells decreased after long-term culture, and that horse serum was capable of inducing adipocytic differentiation of bovine MSC in vitro, but its efficacy was less than that of the hormonal mixture. Further studies to detect adipocyte-specific markers, such as G3PDH activity and aP2 expression, from these cells are needed for the quantitative analysis of the differentiation.