A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7-10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated.

No abstract

Differences in monolayer and three-dimensional (3D) culture systems have been recognized for several years. Despite the recognized importance of 3D systems, low cost and convenience of monolayer culture are still readily used for metabolic and nutritional studies. Here, we present part 1 of a 2-part series that will highlight (1) a novel and cost-effective model for culturing 3T3-L1 preadipocytes in 3D agarose as well as (2) an initial study showing the successful use of this 3D model for experimental analysis of these cells treated with cinnamon extract while suspended in agarose. In part 1, we provide a full characterization of the model system for the 3T3-L1 cells that demonstrate the functionality and convenience of this system. Importantly, we note spontaneous differentiation to adipocytes while cultured under these methods, independent of chemical induction. We present a 2.5-week time course with rounded cells forming vacuoles as early as 24 hours and accumulation of lipid detectable by Oil Red O stain at 0.5 weeks. Serum selection, lipid volume determination, and cell size are characterized. We conclusively demonstrate adipogenesis based on a peroxisome proliferator-activated receptor γ (PPARγ) detection using immunohistochemistry (IHC) of sections from these 3D cultures. Methods, materials and recommendations are described as well as proposed benefits to the use of this culture system for 3T3-L1 cells.

Lysosomal storage diseases such as GM1-gangliosidosis are associated with skeletal abnormalities. Radiological and histological studies, both in human and corresponding animal models, indicate retarded bone formation. Since cartilage maturation leads to bone formation, we developed an in vitro system to study and compare the biological features of cartilage from dogs affected with GM1-gangliosidosis with age-matched controls. Costochondral chondrocytes were grown in monolayer and in agarose culture. Both affected and control cells dedifferentiated in monolayer; however, in agarose culture they re-expressed the chondrocytic phenotype. Cells from affected dogs were enlarged and contained numerous large vacuoles when compared with control cells. This morphology was similar to that seen in vivo. In addition, the affected cells appeared to have a reduction in mitosis and alcian blue staining proteoglycans. Cultures from affected animals contained fewer cells positive for alkaline phosphatase activity. Both affected and control cells expressed collagen types I and II and were positive for the lectin Ricinus communis agglutinin-I. However, the staining of the control culture for type II collagen was more prominent than in the affected cells. These findings suggest that culture of chondrocytes in agarose may be a useful method for studying the biology of cartilage which leads to skeletal abnormalities in lysosomal storage diseases.

Colon cancer is normally refractive to the chemotherapy drug cis‐platinum, (Cis‐Pt). Selenium (Se) containing glutathione peroxidase has been studied because of its role in reduction of reactive oxygen species which would improve the efficacy of Cis‐pt. The influence of Se on the effect of Cis‐PT on the HT‐29 colon cancer cell line was tested using a three dimensional agarose culture model. This method allowed independent analysis of mitosis and cell viability. Single cells were suspended in low temperature agarose and grown for 1 week. Se at low doses did not affect cell viability or mitosis when compared to vehicle controls. To simulate the in vivo scenario, colonies were allowed to form prior to Cis‐Pt exposure. Experiments were conducted to determine the time course of exposure to Se and Cis‐Pt. Some cultures were untreated or pretreated with Se (0.33 μg/ml) at day 0 followed by or in conjunction with Cis‐Pt (3 and 6 μg/ml) on day 4. On day 7 cultures were evaluated for cell viability, using trypan blue exclusion, and mitotic activity by counting single cells and colonies alive and dead. Differences between treatment groups were analyzed using ANOVA at 95% confidence level. Cell growth conditions allowed evaluation of glutathione peroxidase acitivity under each treatment condition following analysis of mitotic and cell viability. Glutathione peroxidase activity was compared to total protein in each cell culture. Initial results indicate that cultures treated with Se in conjunction with Cis‐Pt exhibited higher glutathione peroxidase activity coinciding with increased Cis‐Pt efficacy. These data suggest that supplementation with Se will increase efficacy of Cis‐Pt in colon cancer, specifically in the HT‐29 cell line, thus providing a more effective use of existing treatments. Support for this project was provided by The Raabe College of Pharmacy and the Department of Biological and Allied Health Sciences, Ohio Northern University.