Modeling retinal degeneration using patient-specific induced pluripotent stem cells

Abstract

Retinitis pigmentosa (RP) is the most common inherited human eye disease resulting in night blindness and visual defects. It is well known that the disease is caused by rod photoreceptor degeneration; however, it remains incurable, due to the unavailability of disease-specific human photoreceptor cells for use in mechanistic studies and drug screening. We obtained fibroblast cells from five RP patients with distinct mutations in the RP1, RP9, PRPH2 or RHO gene, and generated patient-specific induced pluripotent stem (iPS) cells by ectopic expression of four key reprogramming factors. We differentiated the iPS cells into rod photoreceptor cells, which had been lost in the patients, and found that they exhibited suitable immunocytochemical features and electrophysiological properties. Interestingly, the number of the patient-derived rod cells with distinct mutations decreased in vitro; cells derived from patients with a specific mutation expressed markers for oxidation or endoplasmic reticulum stress, and exhibited different responses to vitamin E than had been observed in clinical trials. Overall, patient-derived rod cells recapitulated the disease phenotype and expressed markers of cellular stresses. Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. iPS cells derived from RP patients.

Mutations identified in patients K21 (RP1) (A), K11 and K10 (RP9) (B), P101 (PRPH2) (C), and P59 (RHO) (D). Patient-derived fibroblast cells (E) were reprogrammed into iPS cells (F). The iPS cells expressed SSEA-4 (G) and Nanog (H). A teratoma formation test confirmed iPS cells' ability to generate all three germ layers: endoderm (I), mesoderm (J) and ectoderm (K). Karyotype analysis (I). Scale bars, 50 µm.

Figure 2. Induction of patient-specific retinal photoreceptor cells.

Retinal cells were induced sequentially by in vitro differentiation. (A) Experimental schema. (B) Neural retina progenitor cells (Pax6+Rx+) and RPE progenitor cells (Mitf+) were separated in the culture dish (C). Patient-specific RPE cells exhibited hexagonal morphology and pigmentation (D) and expressed the tight junction marker ZO-1 (E). Photoreceptor cells were positive for immature photoreceptor markers Crx and Recoverin on day 60 (F). Recoverin+ cells did not co-express Ki67, a proliferating cell marker (G). Differentiation of rod photoreceptors (Rhodopsin+) and cone photoreceptors (Opsin+) from patient iPS cells (H). Rhodopsin + rod photoreceptors induced from K21-iPS at day 120 (I). K11-derived rod photoreceptors were observed at day 120 (J). No Rhodopsin+ cells were detected, but Recoverin+ cells were present at day 150(K). Induced rod photoreceptor cells (Crx+) labeled with lentiviral vectors encoding GFP driven by a rod photoreceptor-specific promoter Nrl (L: Nrl-GFP) or Rhodopsin (M: Rho-GFP). Arrows indicate cells co-expressing Crx and GFP. (N) Whole-cell recording of rod photoreceptor cell differentiated human iPS cells. Recorded cells expressed GFP under the control of the Rhodopsin promoter. (O) Relationship between voltage and membrane current (i) produced a non-linear curve, suggesting that voltage-dependent channels exist in iPS cell-derived rod photoreceptors Rec, Recoverin; Rho, Rhodopsin. Scale bars, 50 µm.

Figure 3. RP patient-derived rod photoreceptors undergo degeneration in vitro.

iPS cells were differentiated into Rhodopsin+ rod photoreceptors in serum-free culture of embryoid body-like aggregates (SFEB culture). The percentages of Rhodopsin+ rod photoreceptors were evaluated at both day 120 and day 150, respectively. Data were from three independent iPS cell lines derived from the patients. ANOVA followed by Dunnett's test. * p<0.05; ***p<0.001. Values in the graphs are means and s.e.m.

Figure 4. Cellular stress in patient-derived rod photoreceptor cells.

Oxidative stress and apoptosis in differentiated rod photoreceptor cells derived from RP9-iPS (A,C,E) and RP1-iPS (B,D,F). (A) 8-OHdG, a marker for DNA oxidation, was found in K11- or K10-iPS−derived differentiated cells (day 100), but not in K21-iPS (B). Arrow indicates a cell double-positive for 8-OHdG and Recoverin. (C) The number of activated Caspase-3+ cells was greater in K11-iPS differentiation than in K21-iPS (D). From day 120, rod photoreceptor cells (Rhodopsin+) derived from RP9-iPS co-expressed the oxidative stress marker Acrolein (E); whereas RP1-iPS derivatives did not (F). (G–L) Abnormal cellular localization of Rhodopsin proteins and endoplasmic reticulum stress in RHO-iPS−derived rod photoreceptors. High magnification revealed cytoplasmic localization of Rhodopsin in rod photoreceptor cells carrying a RHO mutation (G) and a normal localization in the cell membrane in K21 cells (H). Rod cells derived from RHO-iPS co-expressed the ER stress markers BiP (I) and CHOP (K). K21-iPS−derived rod cells did not express BiP (J) or CHOP (L). Arrows indicate double-positive cells. Rec, Recoverin; Rho, Rhodopsin. All scale bars are 50 µm except for G and H (20 µm).

Figure 5. Disease modeling of patient-derived rod photoreceptor cells.

α-Tocopherol treatment of patient-specific rod photoreceptors yielded a significant beneficial effect in RP9 mutant cells. Two-way ANOVA Bonferroni post-test showed no significance in other group (n = 3–8). Data represent 1–2 selected iPS cell lines of each patient. ***p<0.001. Values in the graphs are means and s.e.m.