第948回千葉医学会例会・第14回千葉精神科集談会

Expression of NGN3 and hairy and enhancer of split-1 (HES1) mRNA following treatment with tyrosine kinase inhibitor CEP-701. Quantitative RTPCR results of four biological replicate samples (A–D) treated with API-1 for four days. RQ, relative quantification of NGN3 and HES1 normalized to the level of cyclophillin A. ΔCt, change in threshold cycle (Ct). Mean and standard error of the mean (SEM), results of Student’s t-test (TTEST) and percentage of control are shown. (DOCX 47 kb

Additional file 5: Figure S5. of Neurogenin 3 is regulated by neurotrophic tyrosine kinase receptor type 2 (TRKB) signaling in the adult human exocrine pancreas

Expression of NGN3 and hairy and enhancer of split-1 (HES1) mRNA following treatment with TRKB agonist 7,8 dihydroxyflavone (78D). Quantitative RTPCR results of four biological replicate samples (A–D) treated with 78D for four days. RQ, relative quantification of NGN3 and HES1 normalized to the level of cyclophillin A. ΔCt, change in threshold cycle (Ct). Mean and standard error of the mean (SEM), results of Student’s t-test (TTEST) and percentage of control are shown. (DOCX 50 kb

Additional file 7: Figure S7. of Neurogenin 3 is regulated by neurotrophic tyrosine kinase receptor type 2 (TRKB) signaling in the adult human exocrine pancreas

Expression of NGN3 and hairy and enhancer of split-1 (HES1) mRNA following treatment with tyrosine kinase inhibitor CEP-701. Quantitative RTPCR results of four biological replicate samples (A–D) treated with API-1 for four days. RQ, relative quantification of NGN3 and HES1 normalized to the level of cyclophillin A. ΔCt, change in threshold cycle (Ct). Mean and standard error of the mean (SEM), results of Student’s t-test (TTEST) and percentage of control are shown. (DOCX 47 kb

Coexpression of neurogenin 3 (NGN3) and CD133 in cultured human exocrine tissue.

<p><b>A-D</b>, Expression of NGN3 and CD133 in exocrine tissue. <b>A</b>, CD133 expression. <b>B</b>, NGN3 expression. <b>C</b>, Nuclei costained with 4',6-diamidino-2-phenylindole (DAPI). <b>D</b>, Overlay of 3 channels. 1-μm confocal sections. Scale bar is 50 μm. <b>E-H</b>, FACS analysis of exocrine cells after 4 days in culture. Gates indicated by red lines. % cells in each gate shown in red. <b>E</b>, CD133+ gate defined by isotype negative control. <b>F</b>, CD133+ cells following anti-CD133 stain. <b>G</b>, Cells within the CD133+ gate following staining with NGN3 isotype negative control. <b>H</b>, Cells within the CD133+ gate following staining with anti-NGN3. <b>I-P</b>, Parallel fluorescence microscopy imaging of cell populations in E-H. <b>I</b>, Cells stained with NGN3 isotype negative control. <b>J</b>, Cells stained with anti-CD133. <b>K</b>, Cells stained with Hoechst 33352. <b>L</b>, Overlay of images in I-K. <b>M</b>, Cells stained with anti-NGN3. <b>N</b>, Cells stained with anti-CD133. <b>O</b>, Cells stained with Hoechst 33352. <b>P</b>, Overlay of images in M-O. Scale bars are 20 μm. <b>Q</b>, Change in the percentage and total number of CD133+ cells over time in culture. Mean ± SEM percentage of CD133+ cells (black bar) and total number of CD133+ cells (white bar) indicated along Y-axis as a percentage of initial level on day 0 of culture. Significance determined by ANOVA with Bonferroni-Holm post hoc analysis, ***, P<0.001, **, P<0.01, (n = 6 exocrine cultures).</p

Expression of Notch pathways genes.

<p><b>A</b>, Western blot analyses of Notch intercellular domain (NICD), hairy enhancer of split 1 (HES1) and endogenous control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells isolated from human exocrine tissue. Whole cell lysates from CD133+ (+) and CD133-depleted (D) cells. Nuclear (N) and cytoplasmic (C) extracts from CD133+ cells. <b>B</b>, Volcano plot of Notch pathway gene mean ± SEM mRNA level (n = 3 exocrine cultures) differences in expression level from CD133+ cells compared to CD133D shown on X-axis as Log2 of fold difference. Significance determined by Student’s t-test shown on Y-axis as p value. Magenta vertical lines mark a 2-fold difference in expression. Blue horizontal line marks the significance cutoff (p<0.05). Selected gene names shown. Genes are: receptor tyrosine-protein kinase erbB-2 (ERBB2), frizzled class receptor 7 (FZD7), E1A binding protein p300 (EP300), MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (MFNG), H19, imprinted maternally expressed transcript (H19), LIM domain only 2 (rhombotin-like 1) (LMO2), inhibitor of DNA binding 1 (ID1), hairy enhancer of split 4 (HES4), cyclin D1 (CCND1), matrix metallopeptidase 7 (MMP7), mastermind-like 2 (MAML2), jagged 1 (JAG1), Notch 2 (NOTCH2), hes-related family bHLH transcription factor with YRPW motif-like (HEYL), snail family zinc finger 2 (SNAI2), recombination signal binding protein for immunoglobulin kappa J region-like (RBPJL). <b>C</b>, Normalized mRNA expression level of neurogenin 3 (NGN3), HES1 and pancreas transcription factor 1 subunit alpha (PTF1A) in exocrine tissue after 4 days of culture in the presence of 20 μM Notch inhibitor DAPT. Results reported as mean ± SEM percent of levels in DMSO carrier control. mRNA levels normalized to the level of cyclophillin A. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). <b>D</b>, Expression of NGN3 protein following treatment with 20 ∞M DAPT and 47 ∞M Notch agonist JAG-1 peptide (JAG-1). Mean ± SEM percent of DMSO carrier only control or 47 ∞M scrambled JAG-1 peptide, respectively indicated on Y-Axis. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). <b>E-H</b>, Orthogonal analysis of colocalized HES1 and NGN3 in nuclei of exocrine tissue after 4 days of culture. Nuclei counterstained with Hoechst 33342 (H). <b>E</b>, Overlay of 3 channels. 0.5 ∞m confocal section. Scale bar is 50 ∞m. <b>F-H</b>, Higher magnification of crosshair region in all three channels shown at right. Scale bars are 20 ∞m. <b>I</b>, Coprecipitation of ID proteins with HES1. Whole cell lysate from exocrine tissue after 4 days of culture immunoprecipitated with antibody to HES1. ID1, 2 and 4 detected following SDS PAGE and western blotting. Predicted molecular weights of ID proteins (ID) and immunoglobulin heavy chain used for precipitation (HC) shown at right. Molecular weight marker positions shown at left in kDa.</p

Relative mRNA expression of pancreas development transcription factors by CD133+ cells isolated after 4 days of exocrine tissue culture.

<p>Mean ± SEM relative expression level of genes in CD133+ cells compared to the CD133-depleted (CD133D) population shown on Y-axis as fold difference. Significance determined by Student’s t-test from 2^-Δct values. ***, p<0.001, **, p<0.01, *, p<0.05 (n = 4 exocrine cultures). Genes ranked in order of overexpression in the CD133+ population. Inset shows gene expression using an enlarged scale. Genes are: Neurogenin 3 (NGN3), One cut homeobox 2 (ONECUT2), NK6 homeobox 1 (NKX6.1), GLIS family zinc finger 3 (GLIS3), motor neuron and pancreas homeobox 1 (MNX1), HNF1 homeobox B (HNF1B), pancreatic and duodenal homeobox 1 (PDX1), SRY (sex determining region Y)-box 9 (SOX9), Hairy enhancer of split 1 (HES1), One cut homeobox 1 (ONECUT1), Forkhead box A2 (FOXA2), Forkhead box O1 (FOXO1), v-maf avian musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB), GATA binding protein 4 (GATA4), Pancreas specific transcription factor 1A (PTF1A), Neuronal differentiation 1 (NEUROD1), ISL LIM homeobox 1 (ISL1), paired box 6 (PAX6). No expression of NK2 homeobox 2 or paired box 4 was detected.</p

Relative mRNA expression level of endocrine development transcription factors by differentiating pancospheres (PS) over time.

<p>Mean ± SEM (n = 3 exocrine cultures) relative expression level reported as fold difference from CD133+ starting population shown on Y-axis. Days after initiating PS formation shown along X-axis. Proliferative (Phase I) and maturation (Phase II) phases shown at bottom. <b>A</b>, Upregulated genes are: marker of proliferation Ki-67 (KI67), Neuronal differentiation 1 (NEUROD1), Neurogenin 3 (NGN3), v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), v-maf avian musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB), paired box 4 (PAX4), paired box 6 (PAX6). <b>B</b>, Down regulated genes are: SRY (sex determining region Y)-box 9 (SOX9), pancreatic and duodenal homeobox 1 (PDX1), ISL LIM homeobox 1 (ISL1), NK6 homeobox 1 (NKX6.1), GLIS family zinc finger 3 (GLIS3), motor neuron and pancreas homeobox 1 (MNX1), Hairy enhancer of split 1 (HES1). <b>C</b>, Isolation and endocrine differentiation of NGN3+ cells. After death, pancreas is removed and transported to an islet isolation facility where a biopsy is taken for histology. The pancreas is then digested and separated into islets and exocrine tissue. Exocrine culture is initiated on day 2 post mortem. On day 6 post mortem, tissue is digested to single cells, which are labeled and sorted for expression of CD133. CD133+ cells are plated on pancosphere day 0. Samples are collected on pancosphere days 4, 6, 9, 13, 15, 19 and 21. Cell proliferation marker KI67 expression peaks at pancosphere day 9. On pancosphere day 19 IGF II is withdrawn and cells begin final maturation. Days between each step are indicated below arrows. Exocrine tissue culture and phases of pancosphere differentiation are shown above the timeline.</p

Expression of hormones, chromogranin A and PDX1 by CD133+ cells following <i>in vitro</i> differentiation.

<p><b>A</b>, Phase microscopic image of pancospheres on day 6 of formation. Scale bar is 100 μm. <b>B</b>, Pancreatic and duodenal homeobox 1 (PDX1) expression in a day 6 pancosphere. Scale bar is 50 μm. <b>C,D</b>, Orthogonal analyses of PDX1 / glucagon (GCG) and PDX1 / insulin C-peptide (CPEP) coexpression in day 21 pancospheres. 1 μm optical sections, scale bar is 50 μm. Inset box in D is magnified and rotated confocal reconstruction of cells indicated by lines. <b>E</b>, Coexpression of GCG and chromogranin A (CHGA) by cells within a day 21 pancosphere. Scale bar is 20 μm. <b>F</b>, Coexpression of CPEP, CHGA and PDX1 by cells within a day 21 pancosphere. Scale bar is 10 μm. B-F, Nuclei stained with Hoechst 33342 (H).</p

Expression of neurogenin 3 (NGN3) in the adult human exocrine pancreas.

<p><b>A-H</b>, Immunohistochemical staining of histologically normal tissue from living subjects undergoing medically indicated pancreas biopsy using anti-NGN3 antibody F25A1B3. <b>A-D</b>, Expression of NGN3 and cytokeratin 19 (CK19) in by duct cells. B-D, Higher magnification of tissue shown in A. <b>B</b>, CK19 expression, <b>C</b>, NGN3 expression, <b>D</b>, Hoechst 33342 stained nuclei (H). <b>E-H</b>, Expression of NGN3 and amylase (AMY) by acinar cells. F-H, Higher magnification of tissue shown in E. <b>F</b>, Amylase expression, <b>G</b>, NGN3 expression, <b>H</b>, Hoechst 33342 stained nuclei. NGN3+ cells indicated by white arrowheads. Scale bars are 20 μm. <b>I</b>, Immunoprecipitation (IP) of NGN3 and E12/47 from human exocrine tissue after 4 days of culture. Presence of IP antibody F25A1B3 shown on top. F25A1B3 and anti-E12/47 detection antibodies shown on left. Presence of human or E14.5 mouse pancreatic epithelia lysate shown on bottom. Detection with anti-NGN3 (top panel) reveals bands in human and mouse lysates corresponding to the predicted molecular mass of NGN3 (~23KDa, arrow) and capture antibody heavy chain (HC). Detection with anti-E12/47 (bottom panel) identifies coimmunopreciptated proteins corresponding in size to E12 and E47. Molecular weight markers shown at left of blots in kDa. <b>J</b>, HEK293T cell lysate expressing human NGN3 tagged with V5 and 6xHIS epitopes (pHS.NGN3V5_HIS) (+) or negative control vector (-) detected with NGN3 antibody F25A1B3 and anti-V5 as indicated. Molecular weight markers shown at left of blots in KDa.</p