Eight weeks after transplanting cultured cell sheets prepared from GFP rats.

<p>(A) Slit lamp photograph of a reconstructed nude rat cornea. (B) HE staining showing that the reconstructed cornea was covered with 3–4 epithelial cell layers. (C, D) Immunostaining results showing that a reconstructed cornea still expressed p63 in the basal layers. (E, F) K14 was expressed in all epithelial layers after cell sheet transplantation. (G, H) The expression of CD 31 was observed in the basal epithelial layer and the superficial stroma in the peripheral cornea. (I) Corneal surface completely covered with GFP-positive epithelial cells. (J) Fluorescence microscopy showing that surviving GFP-positive epithelial cells were on the surface of a transplanted cornea. (K) CFAs for peripheral and central epithelial cells prepared from a postoperative eye and limbal and central epithelial cells prepared from a normal cornea. (L) The mean CFE values for the peripheral cells removed from the transplanted cornea was significantly greater than that for the central cell, which was also observed in a normal cornea (N = 4, *p<0.05). Epi, epithelium; St, stroma. Scale bars = 20 µm (C, D, E, F, G, H) and 25 µm (B, I).</p

Successful generation of transplantable epithelial cell sheets from GFP rats.

<p>(A) Phase-contrast image of oral mucosal epithelial cells. (B) Fluorescence microscopy showing GFP-positive cells from a donor GFP rat. (C) Cross-section of a GFP-positive cell sheet. (D) Basal cells of cultured epithelial cell sheets express p63. (E) Both primary oral mucosa and cultured cell sheets contained sufficient numbers of progenitor cells. Scale bars = 200 µm (A, B) and 50 µm (C, D).</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

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

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

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

DNA methylation analysis of corneal epithelium-related genes.

<p>Methylation analysis of individual genes was performed. Methylation frequency in K12, K3, Pax6, p63, and K14 genes were not statistically different among L1B41, 253G1, and 201B7 (significance level; p<0.001 [ = diffscore >33 or <−33]).</p

iPS cells were established from human corneal limbal epithelial cells (HLECs).

<p>(A) Three iPS-like cell colonies (L1B41, L1C51, and L1B34) were cloned after reprogramming of HLEC using Yamanaka 4 factors. (B) Immunofluorescent analysis showed that all 3 isolated iPS cell lines expressed the pluripotent stem-cell markers Nanog, Oct3/4, Sox2, and SSEA4. (C) Karyotype analysis showed no obvious aberration in the 3 iPS cell clones derived from HLEC. (D) Global expression analysis among iPS cells by microarray showed that the 3 HLEC-derived iPS cells exhibited similar expression to HDF-derived iPS cells. (E) HLEC-derived L1B41 was able to form a teratoma that contained the tissue originating from the 3 germ layers in the testis of SCID mice. Scale bar: (A) 200 µm, (B) 100 µm.</p