In vitro and in vivo studies of hESCs-derived insulin producing cells functionality.

<p><b>A</b>: Immunohistochemical analyses of pancreas sections from control mice (Pancreas CTR) and STZ-induced diabetic mice (Pancreas STZ). Upper panel shows hematoxylin and eosin stain (H&E stain) of Langerhans Islets, which are completely compromised in diabetic mice. Lower panel shows the complete loss of insulin stain (<i>red</i>) in diabetic pancreas. <b>B</b>: Nonfasting blood glucose measurements of control mice (<i>blue line</i>, n = 4), STZ-induced diabetic mice (<i>red line</i>, n = 3) and STZ-induced diabetic mice transplanted (Tx) with 2000 islet-like clusters derived from hESCs differentiation (<i>green line</i>, n = 3). <b>C</b>: Percentage of body weight variations in control mice (<i>blue line</i>, n = 4), STZ-induced diabetic mice (<i>red line</i>, n = 3) and STZ-induced diabetic mice transplanted with 2000 islet-like clusters derived from hESCs differentiation (<i>green line</i>, n = 3). <b>D</b>: Immunohistochemical analyses of a transplanted kidney showing H&E stain of the engraftment and a dispersed insulin staining (<i>red</i>) throughout the renal parenchyma. Scale bar 75 μm and 25 μm respectively.</p

Recapitulation of pancreas organogenesis for efficient hESCs differentiation.

<p><b>A</b>: Schematic representation of the steps involved in ESCs differentiation toward a β-cell fate and the factors and signaling pathways involved in this process. ESC, embryonic stem cells; ME, mesendoderm; DE, definitive endoderm; PG, primitive gut; PF, posterior foregut; PE, pancreatic endoderm; EP, endocrine precursors; BC, β-cells. <b>B</b>: Schematic representation summary of the step-wise differentiation protocol used to obtain hESC-derived insulin-producing cells. RA, retinoic acid; Fib, fibronectin; ITS, insulin-transferrin-selenium.</p

Characterization of the differentiated islet-like clusters obtained at the end of the differentiation protocol.

<p><b>A</b>: RT-PCR detection of definitive endoderm, endocrine precursors and islet cells specific markers. β-actin was used as the input control. SD: hESCs spontaneously differentiated; -RSV: hESCs subjected to our differentiation protocol without RSV addition; +RSV: hESCs subjected to our differentiation protocol with RSV addition. H Pancreas: human pancreas as positive control. <b>B</b>: Immunofluorescence analysis of PDX1, GCG, INS and C-PEP expression in spontaneous differentiated cells (SD) and in differentiated β-cell-like cells without (-RSV) or with RSV addition (+RSV). Nuclei are stained with Hoechst (<i>blue</i>). Scale bar 50 μm. <b>C</b>: RT-PCR detection of definitive endoderm, endocrine precursors and islet cells specific markers of hiPSCs-derived β-cell-like cells. β-actin was used as the input control. <b>D</b>: q-PCR comparison of the levels of expression of some β-markers in β-cell-like cells derived from hiPSCs (<i>red bars</i>) or hESCs (<i>blue bars</i>) without or with RSV treatment (-RSV, +RSV).</p

Mechanism of action of resveratrol.

<p><b>A</b>: Analysis of <i>PDX1</i> expression by q-PCR in hESCs derived insulin-producing cells after RSV treatment. Values are mean ±SE of 5 independent experiments. (*) <i>p<0</i>.<i>05</i>, (**) <i>p<0</i>.<i>01</i>. <b>B</b>: Schematic representation of the signaling pathways proposed to be the targets of RSV action for the induction of <i>PDX1</i> and other specific β-cell genes transcription. <b>C</b>: Immunoblotting of hESCs-derived β-cell-like cells extracts treated with (+RSV) or without (-RSV) RSV. Levels of phosphorylated AMPK, PI3K and AKT proteins (pAMPK, pPI3K, pAKT) and total kinases were assessed. β-actin was used as loading control. <b>D</b>: Densitometry quantification of phosphorylated proteins normalized to total proteins. Values are mean ±SE of 3–4 independent experiments. (**) <i>p<0</i>.<i>01</i>, (***) <i>p<0</i>.<i>001</i>. <b>E</b>: Analysis of <i>PDX1</i>, <i>GLUT2</i>, <i>GK</i> and <i>INS</i> expression by q-PCR in hESCs derived insulin-producing cells after RSV treatment. Values are mean ±SE of 3–5 independent experiments. (*) <i>p<0</i>.<i>05</i>, (***) <i>p<0</i>.<i>001</i>. Results of experiments shown in the panels <b>F</b> and <b>G</b> are carried out on cells harvested at day 11 of the differentiation protocol; which corresponds with the first peak of <i>PDX1</i> expression. <b>F</b>: Western blot representing the phosphorylated state of AMPK, PI3K, MAPK and AKT kinases with or without the addition of specific inhibitors. β-actin was used as loading control. <b>G</b>: q-PCR analysis of <i>PDX1</i> expression after treatment with specific inhibitors of upstream signaling pathways. SD: spontaneous differentiation; DP: cells subjected to the differentiation protocol (until day 11); cells subjected to the differentiation protocol and treated during last 48 hours with specific inhibitors: LY 294002 50 μM, Wortmannin 2 μM and Dorsomorphin 10 μM. Values are mean ±SE of 2 independent experiments. (*) <i>p<0</i>.<i>05</i>, (**) <i>p<0</i>.<i>01</i>, (***) <i>p<0</i>.<i>001</i>.</p

Temporal dynamics of gene expression during hESCs differentiation.

<p><b>A</b>: HS181 cells were differentiated to β-cell-like cells as described above. Cell samples were collected at days 0, 5, 8, 11, 14 and 22 and were analyzed by q-PCR for <i>SOX17</i>, <i>FOXA2</i>, <i>HNF1B</i>, <i>HNF4A</i>, <i>PDX1</i>, <i>NGN3</i>, <i>NKX2</i>.<i>2</i> and <i>INS</i> gene expression. For each sample, relative expression was normalized to day 0. <b>B</b>: Overview of the results obtained from q-PCR analysis of the genes described above. The schematic representation shows the sequential expression and temporal variation of these genes during our differentiation protocol.</p

Effects of resveratrol on maturation of hESCs-derived insulin-secreting cells.

<p><b>A</b>: Comparative confocal immunofluorescence expression of insulin (<i>red</i>) and C-peptide (<i>green</i>) expression in hESCs spontaneously differentiated cells (SD) and in differentiated β-cell-like cells without (-RSV) or with (+RSV) RSV addition. Nuclei are stained with Hoechst (<i>blue</i>). Scale bar 25 μm. <b>B</b>: Quantification by MetaMorph analysis of insulin positive cells (%). <b>C</b>: Immunofluorescence analysis of insulin content. Nuclei are stained with Hoechst (<i>blue</i>). Scale bar 25 μm. Graphs show the quantification by MetaMorph analysis of Integrated Intensity of insulin-positive cells (upper graph) and Average Intensity of insulin-positive cells (lower graph). Values are mean ±SE of 3 to 4 independent experiments. (*) <i>p<0</i>.<i>05</i>, (**) <i>p<0</i>.<i>01</i>. <b>D</b>: ELISA quantification of insulin secretion. Insulin release was measured using a Mercodia ELISA kit after 1 hour incubation period with glucose 7 mM. Values are mean ±SE of 3 to 4 independent experiments. (*) <i>p<0</i>.<i>05</i>, (**) <i>p<0</i>.<i>01</i>.</p