エナメル蛋白の分子多様性の意義に関する分子組織化学的･分子生物学的研究

研究期間:平成14-15年度 ; 研究種目:基盤研究C2 ; 課題番号:14571736原著には既発表論文の別刷を含む

The comparison of gene expression analysis of Cp and/or Tp versus pluripotent stem cells.

<p>Schatter plot of Cp and iPSCs (A), Cp and ESCs (B), Tp and iPSCs (C), Tp and ESCs. Pluripotent markers were indicated by block charcters. NCC markers were indicated by violet characters.</p

Quantitative PCR analysis of selected genes based on transcriptome analyses.

<p>EGFP⁺ and EGFP⁻ cells obtained from craniofacial and trunk regions at E9.5, E10.5, E11.5 and E12.5 were examined. (A) NCC markers up-regulated in cNCCs (Pax7, Msx1, Barx1 and snail2). (B) Mesenchymal markers up-regulated in cNCCs (PDGFRα, PDGFRβ, Lhx8 and Akp2. (C) Candidate markers up-regulated in cNCCs (Foxf1a, Rspo2, S100a4 and Frk). (D) NCC markers up-regulated in tNCCs (Sox10 and FoxD3). (mean±SD, n = 5 per group, *p<0.05, **p<0.005).</p

Differential expression profiles of cNCCs and tNCCs in <i>P0-Cre/Floxed-EGFP</i> mouse embryos.

<p>(A) Scatter plot of Craniofacial EGFP⁺cells (Cp) and Trunk EGFP⁺cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP⁺ cells (blue) and Trunk EGFP⁺ cells (red), compared with those in the EGFP⁺ cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP⁻ cells, and Tn; trunk EGFP⁻ cells.</p

Sphere-forming capacity of EGFP⁺ and EGFP⁻ cells from craniofacial and trunk regions.

<p>(A) Schematic illustration of the experimental design for isolation and differentiation of P0-EGFP⁺ cells from <i>P0-Cre/Floxed-EGFP</i> mouse embryos at E12.5. (B) Phase-contrast and direct EGFP fluorescence images showing spheres formed by EGFP⁺ and EGFP⁻ cells derived from craniofacial and trunk regions, respectively, after 5 DIV. Scale bar, 50 µm. (C, D) The percentage of sphere-forming cells assessed by culturing EGFP⁺ and EGFP⁻ cells from each region at a cell density of 5×10³ cells/ml and counting the number of formed spheres. (mean ± SD; n = 5 per group, *p<0.05,**p<0.005). A significantly higher frequency of primary spheres (C) and secondary spheres (D) were formed by craniofacial EGFP⁺ cells, compared with those formed by trunk EGFP⁺ cells.</p

Isolation of NCCs from <i>P0-Cre/Floxed-EGFP</i> mouse embryos by fluorescence-activated cell sorting.

<p>(A) Craniofacial and trunk regions were indicated in the whole body by observation of direct EGFP fluorescence in E12.5 mice. (B) Representative EGFP-gated flow cytometric analysis charts clearly showed two populations, EGFP positive and negative at all examined embryonic ages. (C) The ratio of collected EGFP⁺ cells showed a significantly higher frequency in the craniofacial region than that in the trunk region of all examined embryonic ages. Results were evaluated using the Student's <i>t</i>-test. (mean ± SD, n = 5 per group, **p<0.005).</p

Differentiation potential of spheres derived from EGFP⁺ and EGFP⁻ cells from craniofacial and trunk regions.

<p>(A) Broad range of differentiation potential in spheres derived from craniofacial EGFP⁺ cells. All groups of spheres differentiated into neurons, glial cells, myofibroblasts and adipocytes. Chondrocytes were differentiated from only spheres derived from craniofacial EGFP⁺ cells and trunk EGFP⁻ cells. Osteocytes were differentiated from all groups of spheres except for spheres derived from trunk EGFP⁺ cells. (B–F) Spheres derived from trunk EGFP⁺ cells showed peripheral neuronal lineages. Quantitative analyses of the potential for differentiation into neurons (B), oligodendrocytes (C), glial cells including Schwann cells (D), and myofibroblasts (E) by counting the number of cells positive for specific markers. (F) Quantification of adipocyte differentiation by relative fluorescence units (RFU). Excitation and emission of Adipo Red-stained cells was measured at 485 and 535 nm, respectively. Trunk EGFP⁻ cells showed the highest differentiation potential among the four groups.</p

Additional file 1: Figure S1. of A novel form of necrosis, TRIAD, occurs in human Huntington’s disease

Ultrastructural analysis of additional human HD patients. ER indicates extremely expanded ER that is very homologous to the previously described ballooning of ER in TRIAD [8]. (TIFF 1777 kb