Adipose Tissue-Derived Stem Cells from Type 2 Diabetics Reveal Conservative Alterations in Multidimensional Characteristics

Fig. 1. T2DM-ASCs show similar cytomorphology and immunophenotype. (A) Morphology of ND-ASCs and T2DM-ASCs under same conditions (left, 40× magnification; right, 100× magnification,). (B, C) Immunophenotype of ND-ASCs (n=5) and T2DM-ASCs (n=3) at P6, analysed by flow cytometry. The markers including CD44, CD73, CD90, CD105, CD34 and CD45. The data were shown as the mean±SEM, each experiment was repeated at least twice, one representative example shown. NS: not significant.

Fig. 2. T2DM-ASCs exhibit indiscriminate signatures in cell viability. (A) Cell count of ND-ASCs (n=4) and T2DM-ASCs (n=3) at P5. (B) CCK-8 assay of ND-ASCs (n=3) and T2DM-ASCs for 4, 24, 48 and 72 h. (C) PI staining profiles of ND-ASCs (n=4) and T2DM-ASCs (n=3) by flow cytometry. (D) Histograms statistics of cell cycle status populations. (E) 7-AAD and Annexin V staining profiles of ND-ASCs (n=4) and T2DM-ASCs (n=3) by flow cytometry. (F) Histograms statistics of cell apoptosis, including early apoptosis (Annexin Ⅴ+7-AAD−) and total apoptosis (Annexin Ⅴ+) populations. The data were shown as the mean± SEM, each experiment was repeated at least twice, one representative example shown. NS: not significant. ***p<0.001.

Fig. 3. T2DM-ASCs possess impaired adipogenic and osteogenic differentiation potential. (A) The morphology of adipogenic differentiation of ND-ASCs (n=3) and T2DM-ASCs (n=3) by Oil Red O staining (200× magnification). (B) qRT-PCR assessment of adipogenic marker PPAR-γ. (C) The morphology of osteogenic differentiation of ND-ASCs (n=3) and T2DM-ASCs (n=3) by von Kossa staining (200× magnification). (D) qRT-PCR assessment of osteogenic marker BGLAP. (E) The morphology of chondrogenic differentiation of ND-ASCs (n=3) and T2DM-ASCs (n=3) by Alcian Blue staining (200× magnification). (F) qRT-PCR assessment of chondrogenic marker ACAN. All values are normalized to the negative control (NC) group. The data were shown as the mean±SEM, each experiment was repeated at least twice, one representative example shown. *p<0.05, **p<0.01.

Fig. 4. T2DM-ASCs show comparable migration and immunosuppressive capacity. (A) Cell migration ability of ND-ASCs (n=3) and T2DM-ASCs (n=3) at 0, 12 and 24 hours (40×magnification). (B) Statistical analysis of cell migration area by the ImageJ. (C) ND-ASCs (n=3) and T2DM-ASCs (n=3) cocultured with PBMCs for 3 days, flow cytometry analysis of PBMCs with CD3 staining. (D) Percentages of CD3+ lymphocyte proliferation. The data were shown as the mean±SEM, each experiment was repeated at least twice, one representative example shown. NS: not significant. **p<0.01, ***p<0.001.

Fig. 5. The characteristics of cytokine expression and stemness of T2DM-ASCs and ND-ASCs. (A, B) The comparation of immunomodulation-associated factors (A) and islet function-related factors (B) between T2DM-ASCs (n=3) and ND-ASCs (n=3). (C) The comparation of stemness-associated gene expression (NANOG, POU5F1) between T2DM-ASCs (n=3) and ND-ASCs (n=3). The data were shown as the mean± SEM, each experiment was repeated at least twice. NS: not significant. **p<0.01, ***p<0.001.