Figure 1.
Characterization of Atoh7-Cre/ROSA-YFP iPS cells.
(A) Phase contrast micrograph of Atoh7-Cre/ROSA-YFP iPS cell colonies cultured on feeder cells. (B) Alkaline phosphatase histochemical staining of Atoh7-Cre/ROSA-YFP iPS cells. (C) RT-PCR comparisons of pluripotent gene expression between mouse J1 ES cells and Atoh7-Cre/ROSA-YFP iPS cells. (D–F) Co-immunolabeling of Atoh7-Cre/ROSA-YFP iPS cells for Oct4 and Nanog proteins. A merged image of a single colony is shown in (F). (G–K) Histological staining of teratomas formed from Atoh7-Cre/ROSA-YFP iPS cells injected into SCID mice. Note the presence of multiple tissue types, including the neural epithelium (G), blood islands (H), tubular structures (I), mesenchymal tissues (J), and striated muscles (K). Scale bars, 50 µm.
Figure 2.
Expression of neural progenitor markers in Atoh7-Cre/ROSA-YFP iPS cultures.
(A) RT-PCRs detect transcripts of genes expressed by retinal progenitor cells. Positive controls were 1 µg cDNA of P0 mouse retina as PC1, and HEK cells infected with a lentivirus expressing GFP as PC2 (*). Negative controls were total RNAs from iPS cells without reverse transcriptase as NC1, and non-infected HEK cells as NC2 (*). Arrowheads indicate the position of the 500 bp size marker. (B, C) Confocal images of immunolabeled Atoh7-Cre/ROSA-YFP iPS cell-derive EB cultures at day 7 are shown. (B) YFP-positive neurite extension, and YFP and Pax6 co-expression in a subset of cells were detected. (C) Pax6 was detected in some but not all Sox2 positive cells. The merged images also include DAPI labeling (blue) of cell nuclei. Scale bar, 50 µm for all panels.
Figure 3.
Effects of γ-secretase inhibitor DAPT on production of the Atoh7 lineage cells.
(A) Representative FACS profiles for Atoh7-Cre/ROSA-YFP iPS cell cultures with or without DAPT at day 14. DAPT was added to the cultures at day 7. Red lines indicate gating for YFP-positive cells. (B) FACS quantification of YFP-positive cells in the Atoh7-Cre/ROSA-YFP iPS cell cultures at day 14. N = 4, the significant p value is shown.
Figure 4.
Expression of YFP and retinal ganglion cell marker Pou4f1 in Atoh7-Cre/ROSA-YFP iPS cell cultures.
Confocal images show Atoh7-Cre/ROSA-YFP iPS cell cultures at day 12. (A) A representative EB co-labeled for YFP (green) and Pou4f1 (red), and also stained by DAPI (blue). (B) Expanded views showing merged images of YFP (green), Pou4f1 (red), and DAPI (blue) from different EBs at day 12. Scale bars, 50 µm.
Figure 5.
Differentiation of retinal ganglion cells from Atoh7-Cre/ROSA-YFP iPS cells.
Confocal images show Atoh7-Cre/ROSA-YFP iPS cell cultures at day 12 co-immunolabeled for YFP and/or various neuronal markers, including (A) YFP and NF68; (B) Pou4f1 and NF145; (C) β-Tubulin (Tubb3) and Islet1; (D) Pax6 and NF68. The bottom panels show merged images including DAPI (blue) staining of nuclei. Scale bar, 50 µm for all.
Figure 6.
Quantitative analyses of retinal ganglion cell differentiation from Atoh7-Cre/ROSA-YFP iPS cells.
(A) Typical FACS profiles show effects of DAPT on YFP and Pou4f1 markers at day 10. (B) Bar graphs show FACS quantification at day 10 of total Pou4f1+ (Q1 and Q2), Pou4f1+YFP+ double (Q2), and Pou4f1+ YFP− (Q1) cells. N = 3, Significant p values are indicated. (C, D) Representative FACS profiles show effects of DAPT on Pou4f1 and YFP at day 14. Different populations of cells shown in (D) are designated as Pou4f1high YFPhigh (group 1), Pou4f1low YFPlow (group 2), and Pou4f1high YFPlow (group 3). (E) Bar graphs show quantification of group 1–3 cells shown in (D) under the influence of DAPI. N = 4, significant p values are indicated.
Figure 7.
Differentiation of photoreceptor precursor cells from Atoh7-Cre/ROSA-YFP iPS cells.
(A, B) Confocal images show Atoh7-Cre/ROSA-YFP iPS cell cultures at day 12 co-immunolabeled for Crx and Recoverin (A), or Crx and YFP (B). The merged image for (A) also shows DAPI labeling of cell nuclei. Scale bars, 50 µm for all. (C–F) Quantitative analyses of photoreceptor precursor differentiation in the iPS cell EB cultures. (C) Typical FACS profiles show effects of DAPT on Otx2 and Crx at day 12. (D) Bar graphs show quantification of various populations of marker positive cells with or without DAPT at day 12. (E) Typical FACS profiles show effects of DAPT on Crx and YFP at day 14. (F) Bar graphs show quantification of Crx+ and Crx+ YFP+ cells among total cells at day 14. N = 4, significant p values are indicated.