Figure 1.
Differentiation of hiPSCs into retinal pigment epithelial cells.
(A) Schematic diagram of the culture procedure for retinal differentiation. Photomicrograph (B) and N-cadherin staining (C) shows that both primary RPE cells and hiPSC-derived RPE cells form polygonal, cobblestone-like morphology. Scale bars, 100 µm. (D) Flow cytometry analysis of CRALBP and GP-100 expression in hiPSCs (red), hiPSC-derived RPE cells (green) and primary RPE cells (blue). (E) Time-course analysis of expression of RPE cell markers, CRALBP (left) and RPE65 (right), using qRT-PCR. Error bars represent the standard deviation of the measurements (n = 3).
Figure 2.
Soft agar colony formation assay of hiPSCs and teratocarcinoma PA-1 cells.
(A) Phase-contrast images of hiPSCs, primary RPE cells, hiPSC-derived RPE cells and PA-1cells spiked into primary RPE cells (1%) cultured in soft agar medium for 30 days. Arrows indicate the colonies of PA-1 cells. (B) PA-1 cells (1%, 100 cells; 0.5%, 50 cells; 0.25%, 25 cells; 0%, 0 cells) were spiked into 1.0×104 primary RPE cells and grown in soft agar for 10, 20 and 30 days. Cell growth was quantified using a CytoSelect kit. Results were expressed as a relative fold change of the value of blank well. Statistical significance was determined using two-way ANOVA and Bonferroni's post-hoc test (*P<0.05 compared with the 0% control). (C) HiPSC-derived RPE cells, three lots of primary RPE cells and PA-1 cells spiked into primary RPE cells were grown in soft agar for 30 days. Quantification of the results is described in (B). Limit of detection was calculated as the mean plus 3.3 fold the standard deviation of the measurement of the three lots of primary RPE cells. Error bars represent the standard deviation of the measurements (n = 3).
Figure 3.
Detection of undifferentiated hiPSCs by flow cytometry assay.
(A) Flow cytometry analysis of hiPSCs (blue) and primary RPE cells (red). Cells were fixed, permiabilized and stained with anti-TRA-1-60, anti-TRA-1-81, anti-Sox2, anti-Oct3/4 and anti-Nanog antibodies labeled with fluorophore. (B) Five lots of primary RPE cells were analyzed by flow cytometry with anti-TRA-1-60 antibody. (C) HiPSCs (0.1%, 2.5×102 cells; 0.01%, 25 cells) were spiked into primary RPE cells (2.5×105 cells) and analyzed by flow cytometry with anti-TRA-1-60 antibody. (D) Flow cytometry analysis of hiPSC-derived RPE cells was performed with anti-TRA-1-60 antibody. Ten thousand cells (A) and 1×105 cells (B–D) were used for one assay of flow cytometry analysis. The numbers indicate the quantity of cells contained in the gate.
Figure 4.
Detection of undifferentiated hiPSCs by qRT-PCR assay.
(A) The relative mRNA expressions in primary RPE cells of Lin28, Oct-3/4, Sox2, Nanog, Rex1, Klf4, and c-Myc were determined by qRT-PCR analysis. (B–D) qRT-PCR analysis of hiPSCs spiked into primary RPE cells and five lots of primary RPE cells. Single-cell hiPSCs (1%, 2.5×103 cells; 0.1%, 2.5×102 cells; 0.01%, 25 cells) were spiked into 2.5×105 primary RPE cells, and total RNA was isolated from the mixed cells. The mRNA levels of Nanog (B), Oct3/4 (C) and Lin28 (D) are shown as a relative expression. Limit of detection was calculated as the mean plus 3.3 fold the standard deviation of the measurement of the five lots of primary RPE cells. (E) Lin28 expression of hiPSCs differentiating into RPE and purified hiPSC-derived RPE cells (passage 3 and 4). All values are expressed as mRNA levels relative to those in undifferentiated hiPSCs. Results are means ± standard deviation (n = 3).
Table 1.
Comparison of the tumorigenicity-associated assays.