engleski

Evaluation of Osteogenic Differentiation by Embryonic Stem Cells.

Recent studies reported the successful differentiation of mouse or human ES cells into osteoblasts. To achieve osteogenic differentiation, some procedures utilize generation of embryoid bodies, while others use a direct differentiation protocol. It has been reported that ES cells can be induced to express a mature osteoblastic phenotype by culture in the presence of various osteo-inductive molecules (Vit D3, dexamethasone, ascorbic acid, ß-glycerol phosphate and/or BMP2). It is conceivable that osteogenic differentiation requires that ES cells undergo differentiation through an intermediary step involving a mesenchymal lineage precursor. These cells are known as ESC-derived, mesenchymal stem cells (ES-MSCs) and they can be identified by functional and morphological criteria. To identify ES-MSC we have generated ES cells from previously developed transgenic mice in which an α-SMA promoter directs the expression of red fluorescent protein (RFP) to adult mesenchymal progenitor cells. To track the transition of ES-MSC into mature osteoblast lineage cells we have utilized a Col2.3 promoter driving green fluorescent protein (GFP). Osteogenic differentiation in the ES cells derived from dual transgenic mice was evaluated. Following osteogenic induction we have observed expression of alkaline phosphatase and subsequent mineralization as detected by von Kossa staining. However, gene expression analysis of the time course during the differentiation period showed an absence of markers of the mature osteoblast lineage (osteocalcin, bone sialoprotein). After one week of osteogenic induction, ES cells begin to express αSMA-RFP. This expression was localized to the peripheral area encircling a typical ES cell colony. Nevertheless, these α-SMA-RFP positive cells did not show activation of the Col2.3-GFP promoter even after 4 weeks of osteogenic differentiation. Our results indicate that expression of alkaline phosphatase and mineralization occuring during induction of ES cells are not sufficient critera to detect osteogenic maturation using ES cells. The procedures utilizing protocols for differentiation without the step of embryoid bodies formation require further studies. In addition, we are currently examining the activation of Col2.3GFP using an in vivo assay of teratoma formation. This would provide us with assurance that we can identify two steps during osteogenic differentiation of murine ES cells.

embrionic stem cells; osteoblast differentiation; transgenic mouse

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