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Fig. 2 | Stem Cell Research & Therapy

Fig. 2

From: Inhibition of the RhoGTPase Cdc42 by ML141 enhances hepatocyte differentiation from human adipose-derived mesenchymal stem cells via the Wnt5a/PI3K/miR-122 pathway: impact of the age of the donor

Fig. 2

Pharmacological targeting of Cdc42 activity by ML141 (ML) on the yield, growth, adherence, apoptosis, and immunomodulatory characteristics of hADSCs. Cells derived from young and aged subjects were cultured for 96 h. Cells were treated with or without ML141 (10 μM, 24 h). Proliferative potential as indicated by cell number (a) and time for population doubling (b). Kinetics of hADSC adherence as evaluated by the number of adhered cells and expressed as the percentage of applied cells (c,d). Cell viability (e) and apoptotic index (f) as assessed by labeled cells with Annexin V/propidium iodide (PI)/7AAD. The cell viability and apoptotic index were expressed respectively as the percentage of Annexin V(−)/PI(−)/7AAD(−) or Annexin V(+) cells divided by total cells. g Early from late-apoptotic cells were identified as Annexin V(+)/PI(−)/7AAD(−) vs Annexin V(+)/PI(+)/7AAD(+), respectively. h Real-time quantitative RT-PCR determination of p16INK4a, p53, and p21 mRNA levels. The results are expressed as fold variations ± SEM over the young group (R) after normalization to β-actin. i,j Cell culture supernatants from undifferentiated hADSCs were collected and undiluted samples were analyzed for the detection of cytokines as indicated in the Methods. Three to five measures were realized by group and results are the mean ± SEM presented in fold of variation relative to young. The cytokine limits of detection (pg/ml) were: tumor necrosis factor (TNF)-α (2.17), interferon (IFN)-γ 6.26, IFN-α (10.4), granulocyte/macrophage colony-stimulating factor (GM-CSF) (0.20), interleukin (IL)-2 (1.52), IL-4 (34.2), IL-5 (0.27), IL-6 (0.077), IL-9 (32.7), IL-10 (2.76), IL-12 (3.44), and IL-17A (0.7). The results represent individual expression per subject (young (n = 19), aged (n = 20) and aged+ML141 (n = 20)) and are the means of two independent experiments performed in triplicate. §*P < 0.05, §§**P < 0.01, §§§***P < 0.001; §aged versus young and *aged treated with ML141 versus untreated

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