Wnt and EGF inhibit serum-induced osteogenic differentiation in a murine marrow-derived stromal cell line, 489-2.1

• Main Authors: Hse, Ke Hsun, 許可勳
• Other Authors: Chung-Leung Li
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/29533809848244802523

碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 98 === 489-2.1 is a marrow-derived stromal cell line (MSC) which is derived from murine bone marrow and has the capacity to differentiate into cells of skeletal and adipocyte lineages. Using this cell line we re-evaluate the traditional inducers commonly used in the osteogenic and adipogenic differentiation of the mesenchymal stem cells. We observed that serum alone is sufficient to induce osteogenic differentiation as measured by the early marker, ALP and matrix mineralization requires the presence of β-glycerol phosphate. Dexamethasone or ascorbic acid is not absolutely required during the osteogenic differentiation. In contrast, ascorbic acid is a critical factor for in vitro adipogenic differentiation of the MSC on top of the presence of Dex, Insulin, IMBX and Indomethacin (DIXIn). Using the 489-2.1 cells as a cell model to study in vitro osteogenic differentiation, we confirm and extend the previous observation that most batches of fetal calf serum contained ALP-inducing/BMP-like activity. We demonstrate such an activity in the serum based on the (i) Smad reporter assay, and (ii) the use of BMP antagonist, Noggin, and a small chemical BMP signaling inhibitor, dorsomorphin. We also confirm previous observation that in addition to the Smad pathway, MAPK and PI3K signaling pathways are integral during in vitro osteogenic differentiation We have also established a serum-free culture medium to demonstrate that recombinant BMPs, but not Wnt3a, can induce osteogenic differentiation. In addition we observed that EGF can inhibit both serum- or BMP-mediated osteogenic differentiation. The role of Wnt signaling in the in vitro osteogenic differentiation of MSC is examined. We showed that activated Wnt signaling, either through the use of (i) small chemical, CT99021 and LiCl, or (ii) L-Wnt3aCM or recombinant mWnt3a protein, will inhibit serum-mediated ALP induction and matrix mineralization in the 489-2.1 cells. Real-time quantitative RT-PCR revealed suppression of bone-related transcription factors’ (Dlx5 and OSX) expression, and the down-stream markers (ALP and OCN). To address how valid our observation on the factors affecting the in vitro osteogenic differentiation of a cloned cell line, 489-2.1, we take the advantage that our laboratory routinely isolates the MSC-enriched population (CD45- Ter119- CD31- Sca-1+) from the marrow depleted bone cell fraction. We confirm that serum alone is sufficient to induce (i) the formation of ALP+ colonies in primary cultures and (ii) osteogenic differentiation of these cells expanded in the culture. Furthermore, though individual addition of Wnt3a-CM and EGF in the culture medium did not significantly inhibit the formation of ALP+ colonies, a combined use of both growth factors synergistically inhibits the ALP expression of the primary colonies. Whether these ALP- colony cells expanded in this culture medium will still possess their in vivo and in vitro osteogenic and adipogenic differentiation potential remains yet to be studied in the near future. In conclusion, 489-2.1 is a useful MSC cell line for (i) the study of in vitro osteogenic and adipogenic differentiation and (ii) identification of factor(s) in suppressing osteogenic differentiation and maintaining the differentiation competency of the culture-expanded MSC.