Figure 1.
All-Trans Retinoic Acid (ATRA) induces neuronal differentiation of NT2-D1 cells.
(A) Phase contrast images of NT2-D1 cells exposed to ATRA taken during the 1st, 3rd, 4th and 6th week of differentiation. (B) Histograms showing the relative expression of transcripts of markers of specific neuronal subtypes in differentiated NT2-D1 cells (VI week) versus undifferentiated cells. (C) Immunofluorescence images of undifferentiated (upper panels) and NT2-D1 cells exposed to the differentiation protocol for 6 weeks (VI week). GFAP and MAP2 were tested as markers of neural and glial lineages. Histograms represent the fraction (%) of cells positive to the tested markers during the differentiation period. Nuclei are stained in blue by 4′,6-diamidino-2-phenylindole (DAPI). Arrows indicate when, during the differentiation protocol, immunoreactive filaments were first observed. *, **, ***, **** p<0.05 vs I, II, III, and IV column, respectively.
Figure 2.
Characterization and neuronal differentiation of human adipose-tissue derived Multipotent Adult Stem Cells (hAT-MASC).
(A) Representative flow cytometry histograms of a hAT-derived multipotent stem cell population. Plots show isotype control IgG-staining profile (green histogram) versus specific antibody staining profile (red histogram). hAT-MASCs express high levels of mesenchymal stem cell markers (CD90, CD73, CD44, while they are negative for hematopoietic markers CD45 and CD34). (B) Immunofluorescence images of undifferentiated and differentiated hAT-MASC. Undifferentiated cells express the pluripotent state specific transcription factors Oct4 (green), Nanog (purple) and Sox2 (yellow). When exposed to neural differentiation medium these cells express the neural markers ß3 tubulin (TUBB3, green) and acetylcholinesterase (ACHE, brown). Once induced to differentiate into hepatocytes, hAT-MASC uptake acetylated LDL (AcLDL, red). Once exposed to myogenic or endothelial media, these cells express either alpha-sarcomeric actin (ASA, red), smooth muscle actin (SMA, purple) or PE-CAM (CD31, green). Nuclei are stained by DAPI (blue). (C) Phase contrast images of undifferentiated and differentiated hAT-MASC. (D) Histograms showing the relative expression of transcripts of neuronal and glial markers in differentiated hAT-MASC cells (N3, red histogram) versus undifferentiated cells (blue histogram).
Figure 3.
Differentiation towards a neuronal fate increases both intracellular ROS levels and the nuclear localization of APE1.
(A–D) Epifluorescence images of undifferentiated (undiff, A), and differentiated hAT-MASC cultured either in the absence (N3, B) or in the presence (N3+NAC, C) of NAC. The ROS sensitive, green fluorescent dye CM-H2DCFDA was used to quantitate intracellular ROS levels. Histograms (D) show the quantification of the Integrated Fluorescence Intensity (IFI) of the above-described groups in 3 (I–III) independent hAT-MASC lines. (E–G) Immunofluorescence images of APE1 (red) in undifferentiated, N3, and N3+NAC groups. (H–J) Nuclear localization of APE1 is shown superimposing to the above images, the blue fluorescence of DAPI staining. APE1 expression was quantified in the three groups by analyzing the IFI of APE1 nuclear fluorescence. Histograms (K) summarizing data for APE1 nuclear quantitative fluorescence. (L) Representative Western blot of APE1. 10 µg of total protein extracts were loaded onto a 10% SDS-PAGE, blotted and incubated with the APE1 primary antibody and horseradish peroxidase-conjugated secondary antibody. Numbers at the bottom were obtained from densitometric analysis of three independent experiments, normalized versus actin. (M) Cells in the different stages of differentiation were separated into fractions S1 (soluble proteins) and P1 (proteins bound to DNA). Equivalent amounts were analyzed by western blot with antibody against APE1. Numbers at the bottom were obtained from densitometric analysis of three independent experiments, normalized versus a gel run in parallel and stained with Comassie. (N, O) Immunofluorescence images showing colocalization of APE1 (red) with the p65 subunit of NFkB (green) obtained both in undifferentiated and differentiated (N3) hAT-MASC. Cells comprised in the square are shown at higher magnification in the panels localized at the right side of each picture, where the contribution of APE1 and NFkB are shown as separate images. *, ** p<0.05 vs Undifferentiated or N3 cells, respectively.
Figure 4.
Impact exerted by ROS on the differentiation of adult stem cell towards a neural fate.
(A, B) Contrast phase images of hAT-MASC differentiated in the absence (A) or in the presence (B) of NAC. Fluorescence images showing MBP (C, green) and MAP2 (G, red) expression in undifferentiated hAT-MASC and in hAT-MASC differentiated either in the absence (D, H) or presence of NAC (E, I). Histograms (F, J) represent the fraction of cells positive for MBP or MAP2. n = 3 distinct hAT-MASC lines; *, ** p<0.05 vs Undifferentiated or N3 cells, respectively.
Figure 5.
Effects of E3330 on cell viability, proliferation and APE1 expression.
(A) Histograms summarizing the results of a viability assay (MTT) performed following the exposure of undifferentiated hAT-MASC to either positive control (DMSO), negative control (untreated), or 10 µM, 25 µM, 50 µM, 75 µM, and 100 µM of E3330. (B) Histograms summarizing the effects of the exposure of hAT-MASC to E3330 -during either the last step (N3) or the last two steps (N2N3) of differentiation- on the yield of cells at the end of the differentiation protocol. (C) Immunofluorescence images showing the effects the exposure of hAT-MASC to E3330–during either N3 or N2N3- on APE1 expression (green). Nuclei are blue labeled by DAPI. (D) Quantification of Ape1 nuclear expression (IFI) is summarized in histograms. *, **, ***, ***** p<0.05 vs I, II, III, and V column, respectively.
Figure 6.
E3330 increases the neuronal differentiation of hAT-MASC.
(A) Histograms showing the relative expression of transcripts of neuronal and glial markers in undifferentiated and differentiated hAT-MASC, exposed or not to E3330. (B) Immunofluorescence images of differentiated hAT-MASC that were either not exposed (upper 2 panels) or exposed (lower 4 panels) to E3330 during the differentiation protocol. Cells were stained for the glial marker MBP (green) and for the neuronal marker MAP2 (red). * p<0.05 vs Undifferentiated cells.
Figure 7.
E3330 increases the neuronal differentiation of hAT-MASC.
(A) Histograms showing the relative expression of transcripts of markers of specific neuronal subtypes in undifferentiated and differentiated NT2-D1, exposed or not to E3330, at the IV and VI week of differentiation. (B) Immunofluorescence images of differentiated NT2-D1 that were either not exposed (upper panels) or exposed (lower panels) to E3330 during the differentiation protocol. Cells were stained for the glial marker GFAP (green) and for the neuronal marker MAP2 (red). (C) Histograms summarizing the fraction of cells positive to MAP2 and GFAP at the end of the VI week of differentiation in the absence (−E3330) or presence (+E3330) of Ape1 inhibition. (D) Diagram summarizing the main findings of this work on the involvement of Ape1 in neuronal differentiation. Arrows with blunt ends indicate inhibition. * p<0.05 vs I column.