Pdx1 effects on cell viability and Pdx1 internalization.

<p>(A) hBTSCs and HepG2 were exposed to different concentrations of Pdx1 (0.1 μM or 0.5 μM). Incubation with 0.1 μM Pdx1 determined the preservation of cell viability. When cells were treated with 0.5 μM Pdx1, cell viability significantly decreased after 48 hours in both hBTSCs and HepG2 as observed by Trypan blue exclusion assay (data are means ± SD of 6 experiments; **p< 0.01; ***p< 0.001). (B) After 24 hours of treatment with 0.1 μM Pdx1 recombinant protein, Western Blot analyses showed the presence of Pdx1 recombinant protein (His-Tagged protein) in treated hBTSCs and HepG2. The densitometry histograms showed an equal amount of His-Tagged protein in hBTSCs and HepG2 cells treated with 0.1 μM Pdx1 (data are means ± SD of 3 experiments). (C) Immunofluorescence analysis showed the internalization and nuclear translocation of His-Tagged recombinant protein (red nuclei) in treated cells. Nuclei are displayed in blue (DAPI). Original Magnification = 20X left image or 40X right images.</p

Human biliary tree stem cell (hBTSC) differentiation towards pancreatic fate induced <i>in vitro</i> by Pdx1 peptide.

<p>(A) In HepG2 cells the insulin mRNA level was not affected by 14 days of 0.1 μM Pdx1 administration, while it increased significantly (average 6 folds) in hBTSCs cultured for 14 days in Kubota’s Medium (KM) containing 0.1 μM Pdx1in comparison with control medium (KM) (data are means ± SD of 6 experiments; ***p< 0.001). The effect of Pdx1 administration on hBTSCs was similar to treatment with a Hormonally Defined Medium for Pancreatic islet cell differentiation (HDM-P). PANC-1 cells and pancreatic islets (islets) were used as positive controls. (B, C) In hBTSCs cultured for 14 days in KM containing 0.1 μM Pdx1, the glucagon and somatostatin mRNA levels increased 6- (data are means ± SD of 6 experiments; ***p< 0.001) and 2-folds (data are means ± SD of 6 experiments; **p< 0.01), respectively, in comparison with KM. (D) In hBTSCs cultured for 14 days in KM containing 0.1 μM Pdx1, MafA (4.5-folds) (data are means ± SD of 6 experiments; **p< 0.01) or Pdx1 (2-folds) (data are means ± SD of 6 experiments; **p< 0.01) gene expression increased in comparison with KM, while EpCAM mRNA decreased (average 4-folds) (data are means ± SD of 6 experiments; *p< 0.05).</p

Pdx1 effects on cell viability and Pdx1 internalization.

<p>(A) hBTSCs and HepG2 were exposed to different concentrations of Pdx1 (0.1 μM or 0.5 μM). Incubation with 0.1 μM Pdx1 determined the preservation of cell viability. When cells were treated with 0.5 μM Pdx1, cell viability significantly decreased after 48 hours in both hBTSCs and HepG2 as observed by Trypan blue exclusion assay (data are means ± SD of 6 experiments; **p< 0.01; ***p< 0.001). (B) After 24 hours of treatment with 0.1 μM Pdx1 recombinant protein, Western Blot analyses showed the presence of Pdx1 recombinant protein (His-Tagged protein) in treated hBTSCs and HepG2. The densitometry histograms showed an equal amount of His-Tagged protein in hBTSCs and HepG2 cells treated with 0.1 μM Pdx1 (data are means ± SD of 3 experiments). (C) Immunofluorescence analysis showed the internalization and nuclear translocation of His-Tagged recombinant protein (red nuclei) in treated cells. Nuclei are displayed in blue (DAPI). Original Magnification = 20X left image or 40X right images.</p

Pdx1 induced hBTSC-derived β-pancreatic islets are functioning active.

<p>(A) Morphologically (Phase Contrast: Ph-C; Original Magnification = 10X), after 14 days in basal condition, hBTSCs formed large colonies composed of small, densely packed, and uniform EpCAM-positive cells (Immunofluorescence for EpCAM; Original Magnification = 20X). After 14 days in cultures containing 0.1 μM Pdx1, several islet-like structures were present (Phase Contrast: Ph-C; Original Magnification = 10X); these structures were mostly composed of insulin-positive cells (Immunofluorescence for Insulin; Original Magnification = 10X); and some glucagon-positive cells were located at the periphery of these islet-like structures (double immunofluorescence for Insulin and Glucagon; Original Magnification = 20X). (B) Functionally, hBTSCs in Kubota’s Medium (KM) or in KM plus 0.1 μM Pdx1 were exposed to low (5.5 mM) or high (28 mM) glucose concentrations to stimulate C-peptide secretion and the response was compared with human pancreatic islets (islets). Human C-peptide secretion in hBTSCs in KM was not affected by high glucose stimulation (data are means ± SD of 6 experiments). In hBTSCs cultured for 14 days in KM containing 0.1 μM Pdx1, C-peptide secretion was detected at low glucose levels and further increased after exposure to high glucose concentration (data are means ± SD of 6 experiments; *p< 0.05). Human pancreatic islets (islets) were used as a positive control of the high glucose challenge (data are means ± SD of 6 experiments; *p< 0.05).</p