High glucose induces adipogenic differentiation of muscle-derived stem cells

Aguiari et al. 10.1073/pnas.0711402105.

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Fig. 6. Differentiation of Adipose-derived Stem Cells into Osteogenic, Chondrogenic and Adipogenic lineage. Three classical differentiation protocols into osteoblasts, chondrocytes and adipocytes were tested for adipose-derived stem cells (ADSC), and the results verified by means of morphological analyses (histological and electron microscopy analyses) and molecular approaches. In brief, for osteogenic phenotype cells were maintained in culture for 14 days in presence of Dulbecco's modified Eagles Medium (DMEM) supplemented with 10% FBS, 1% L-glutamine, 50 mg/ml L-ascorbic acid [Sigma], 10 ng/ml fibroblast growth factor (FGF) [Calbiochem, CA], dexamethasone 10 nM, b glycerophosphate 10 mM (namely osteogenic medium (OM).

Then, a sample was analyzed by the three approaches (column A). In the first, the cultured cells were fixed and stained for alkaline phosphatase activity (Ai). The histochemical reactions turned out markedly positive (blue cells Fig. Ai), whereas chondrocyte markers (immunocytochemistry for type II collagen, typical of mature collagen) and adipocyte markers (presence of lipid droplets, as revealed by Oil Red O) were completely negative (not shown). Ultrastructural analysis of the sample confirmed the notion of bone differentiation (Aii), by showing a typical osteoblastic phenotype: a large ovoid nucleus and extensive granular endoplasmic reticulum. Aii illustrates a mineralized area with matrix vesicles in the intracellular spaces. These cells, which contained a large amount of granular endoplasmic reticulum, were completely surrounded by fully mineralized bone matrix. These results well matched the molecular analysis by real-time PCR (RT-PCR), carried out in parallel batches of cells, that revealed expression of bone differentiation markers, such as alkaline phosphatase, collagen type I, osteopontin, osteonectin and osteocalcin (Aiii), and negativity for adipocyte markers (fatty acid binding protein 4 (FABP4), peroxisome proliferators activated receptor-gamma (PPARg), lipoprotein lipase (LPL), glucose transporter 4, (GLUT4)) and chondrocyte markers (type II collagen).

For triggering chondrogenic differentiation (chondrogenic medium, CM), DMEM was supplemented with 10% FBS, 1% L-glutamine, 50 mg/ml L-ascorbic acid [Sigma], 1 ng/ml transforming growth factor-b1 (TGF-b1) [Calbiochem, CA], 1 ng/ml of insulin [Sigma], 1 ng/ml epidermal growth factor (EGF), [Sigma] and 10 ng/ml basic fibroblast growth factor (EGF) [Sigma].

14 days after initiation of the differentiation protocol, a sample was analyzed as in the former case. The cells stained positive at the immunocytochemistry for type II collagen (Bi), while completely negative for alkaline phosphatase and lipid staining (data not shown). Transmission electron microscopy (TEM) showed (Bii) that the cells contained a certain number of well preserved mitochondria, an expanded cytoplasm rich in free ribosomes and abundant rough endoplasmic reticulum, a well developed Golgi apparatus and a relevant number of glycogen granules. Cell membranes showed contact points and finger-like protrusions; microfibrils, were present in the intercellular spaces, frequently connected to cell membranes from which they looked extruded. The RT-PCR results confirmed chondrogenic differentiation, by revealing expression of type II collagen and total negativity for osteogenic and adipose marker (Biii).

Finally, differentiation of ADSCs into adipocytes was driven by a classical adipogenic differentiation medium, i.e., supplemented with 10% FBS, 1% L-glutamine, 1 mm dexamethasone (Sigma,), 0.1 mm indomethacin (Sigma), 0.5 mm 3-isobutyl-1-methylzanthine (IBMX) (Sigma), and 10 mm insulin (Sigma). 14 days after the application of the differentiation medium, a sample was analyzed as above. Oil Red O staining of the cells revealed loading with lipid droplets (Ci), and negativity for alkaline phosphatase and type II collagen (data not shown). The EM pictures showed the typical features of adipocytes (Cii; presence of lipid droplets), and RT-PCR revealed the expression of adipocyte markers, such as FABP4, PPARg, LPL and GLUT4 (Ciii). We concluded from these results that, upon application of suitable differentiation protocols, mesenchimal stem cells derived from lipoaspirates (ADSCs) retained the capability of efficiently differentiating into bone, cartilage and adipose cells.

SI Figure 7

Fig. 7. RT-PCR analyses of adipocyte-specific transcripts in cells cultured in the bioreactor. Time course of adipogenic (PPARg, LPL adiponectin, GLUT4 and SREBP1c), chondrogenic (type II collagen) and osteogenic markers (osteopontin, osteonectin, osteocalcin) mRNA expression analyzed by semiquantitative real time PCR in ADSCs (A) and in MDSCs (B) cultured on 3D scaffolds in LG and HG medium after 7 and 14 days. Results for each experiment are from quadruplicate experiments and values are expressed in relative units as the mean ± SD. * P < 0.05.