Characterization Of The Adipocyte Differentiation From Human Mesenchymal Stem Cells

The increase of adipose tissue mass associated with obesity is due to both hypertrophy and hyperplasia of adipocytes. This hyperplasia results from the differentiation of in situ multipotent stem cells present in the vascular stroma of adipose tissue. The recruitment and then differentiation of mesenchymal stem cells in bone marrow to adipose tissue contributes in part to the hyperplasia. The adipocyte differentiation from mesenchyma stem cells is usually described as two phase. The first phase is the commitment of stem cells to preadipocyte, which cannot be distinguished morphologically from its precursor cell but has lost the potential to differentiate into other cell types. In the second phase known as terminal differentiation, the pre-adipocyte will obtain the characteristics of the mature adipocyte. The terminal differentiation has been extensively characterized using preadipocyte cell lines (for example, the mouse cell lines 3T3-L1,3T3-F442A). But what happens to the earlier stage cells-mesenchymal stem cells during adipocyte differentiation is rarely known, especially for the human derived mesenchymal stem cells. The objective of our research is to charaterize the adipocyte differentiation of human mesenchymal stem cells(hMSCs) from bone marrow, including the relationship between proliferation and differentiation and the role of three key transcription factors C/EBPβ, C/EBPα, and PPARγ.In our study, we first isolated mesenchymal stem cells from human bone marrow with Ficoll density gradient centrifugation and in vitro adherent culture method. The cultured hMSC showed typical spindle-shape phenotype. hMSC proliferated slowly, contact inhibition was not obvious, since the dense cells grows into multilayers in the central of clones. More than 90% cells were at G1/G0 phase indicated by PI staining and flowcytometry. hMSC had a limited lifespan in culture, and serial subcultivation resulted in senescence and grandually die. The cells could be subcultured to passage 12.In order to induce hMSCs to differentiate into adipocytes, PPARy agonist (indomethacin) was added to classic adipocyte differentiation protocol (DMEM containing 10%FBS,0.5mM isobutyl methylxanthine, 1μM dexamethasone and 10μg/ml insulin:MDI), and treated repeatedly. Most cells differentiated after repeated treatment for three times. The expression of adipose-specific gene aP2 by real-time PCR increased along hormone induction. Multiple treatments resulted in increasing numbers of adipocytes. Significant increase of human C/EBP beta expression was not observed during differentiation. C/EBP alpha and PPAR gamma increased upon induction, and decreased in the terminal stage.To examine whether MCE occures during adipocytes differentiation of hMSCs. We compared the cell number of hMSCs pre- and after differentiation. The more cells differentiate into adipocyte, the less cells would be there eventually. BrdU incorporation upon induction and detected when fully differentiation showed that the differentated hMSC were BrdU negative, while differentiated 3T3-L1 were BrdU positive. Together, hMSC did not undergo mitosis clone expansion upon adipocyte differentiation in culture condition, which was different from 3T3-L1 model.To define the role of the three transcription factors in hMSCs. We performed gain-or loss-of function experiments. C/EBPβknockdown in hMSCs with RNAi impared the adipogenic potential, while overexpression of C/EBP(3 stimulated cells to differentiate in response to indomethacin treatment. Overexpression of C/EBPa also potentiated the adipocyte differentiation along with PPARy angonist. Overexpression of PPARy alone was enough to initiate adipocytes differentiation of hMSCs, but not stimulate fully differentiated adipocytes.