A stem cell-based tool for small molecule screening in adipogenesis

Abstract

Techniques for small molecule screening are widely used in biological mechanism study and drug discovery. Here, we reported a novel adipocyte differentiation assay for small molecule selection, based on human mesenchymal stem cells (hMSCs) transduced with fluorescence reporter gene driven by adipogenic specific promoter--adipocyte Protein 2 (aP2; also namely Fatty Acid Binding Protein 4, FABP4). During normal adipogenic induction as well as adipogenic inhibition by Ly294002, we confirmed that the intensity of green fluorescence protein corresponded well to the expression level of aP2 gene. Furthermore, this variation of green fluorescence protein intensity can be read simply through fluorescence spectrophotometer. By testing another two small molecules in adipogenesis--Troglitazone and CHIR99021, we proved that this is a simple and sensitive method, which could be applied in adipocyte biology, drug discovery and toxicological study in the future.

Figure 1. Vector construction and lentivirus transduction, characterization, and differentiation of hMSCs.

a, Construction of pLV/Final-puro-aP2-hrGFP; b, Phase contrast microscopy of hMSCs at Passage 9, one day before transduction; c, Phase contrast microscopy of hMSCs at Passage 11 after puromycin selection; d, Flow-cytometric analysis of cell surface antigens of untransduced hMSCs; e, Flow-cytometric analysis of cell surface antigens of transduced hMSCs; f, Oil red O staining of adipogenic differentiated transduced hMSCs; g, Alizarin red S staining of osteogenic differentiated transduced hMSCs; h, Collagen II staining of chondrogenic differentiated transduced hMSCs. Bar = 50 µm.

Figure 2. Normal adipogenic induction of aP2-hrGFP hMSCs.

a, hrGFP expression of aP2-hrGFP hMSCs in different days of adipogenesis; b, Oil red O staining results of aP2-hrGFP hMSCs in different days of adipogenic differentiation; c, Normalized mRNA expression of aP2 gene determined by real-time PCR analysis during adipogenesis; d, Fluorescence intensity detection of aP2-hrGFP hMSCs by fluorescence spectrophotometer during normal adipogenic induction; Data are presented as mean ± SD of three independent experiments, and the p-values in the graph show the statistical significance of the difference between each test day and Day 0. (^★ p<0.05; ^★★ p<0.01). e, Immunofluorescence staining of aP2 protein on Day 15 of adipogenic induction (e). Bar = 50 µm.

Figure 3. Adipogenic differentiation assays of aP2-hrGFP hMSCs with LY294002 (a–c), Troglitazone (d–f) and CHIR99021 (g–i).

a, Fluorescence observation of LY294002 assay with different concentrations, after 15 days of adipogenic induction; b, Normalized mRNA expression of PPAR gamma, aP2 and GPDH genes determined by real-time PCR in LY294002 assay; c, Fluorescence intensity detection of LY294002 assay on Day 15 of differentiation; d, Fluorescence observation of aP2-hrGFP hMSCs with different concentrations of Troglitazone, on Day 15 of differentiation; e, Normalized mRNA expression of aP2 gene determined by real-time PCR in Troglitazone assay; f, Fluorescence intensity detection of Troglitazone with relative concentrations, after 15 days of adipogenic induction; g, Fluorescence observation of aP2-hrGFP hMSCs with different concentrations of CHIR99021, on Day 15 of differentiation; h, Normalized mRNA expression of aP2 gene determined by real-time PCR in CHIR99021 assay; i, Fluorescence intensity detection of CHIR99021 with relative concentrations, after 15 days of adipogenic induction. Data are presented as mean ± SD of three independent experiments, and the p-values in the graph show the statistical significance of the difference between each test group and the DMSO control group. (^★ p<0.05; ^★★ p<0.01). Bar = 50 µm.