In Vitro Mouse Bone Marrow Stromal Cell Differentiation into Adipocytes
Transcrição
Adipocytes, or fat-storing cells, are the predominant cells of adipose tissue — that secrete adipokines — immune signaling molecules that regulate the immune response.
To generate adipocytes in vitro, begin with a multi-well plate containing an adherent culture of mouse bone marrow stromal cells, BMSCs, — undifferentiated multipotent cells.
Treat the cells with an adipocyte induction medium containing phosphodiesterase enzyme inhibitors and dexamethasone — a synthetic glucocorticoid — to trigger the cells' differentiation.
The inhibitors enter the cells and deactivate the cyclic nucleotide phosphodiesterases, enzymes essential for degrading cyclic adenosine monophosphate, or cAMP. This inhibition results in increased intracellular cAMP levels, critical for cell differentiation.
Dexamethasone binds to intracellular receptors in the BMSCs and enters the nucleus to activate genes involved in lipid synthesis and accumulation. This induces the differentiation of BMSCs into preadipocytes — the precursor cells for mature adipocytes.
Replace the spent medium with a fresh induction medium to maintain the preadipocytes. After a specific duration, treat the cells with an adipocyte base medium containing high glucose and insulin.
Insulin binds to its specific cell surface receptors and activates the translocation of glucose transporters on the cell surface, facilitating glucose uptake into the preadipocytes. The cells utilize excess glucose to synthesize the lipid droplets, leading to adipogenesis — the process of lipid accumulation — which promotes differentiation into mature adipocytes.
Treat the cells with a lipid-staining dye and observe them under a light microscope. Mature adipocytes appear as round cells with multiple red lipid droplets.
