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Modeling retinal degeneration using patient-specific induced pluripotent stem cells.

Jin ZB, Okamoto S, Osakada F, Homma K, Assawachananont J, Hirami Y, Iwata T, Takahashi M - PLoS ONE (2011)

Bottom Line: 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.Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan.

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.

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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).
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pone-0017084-g004: 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).

Mentions: We next asked how the patient-derived rod photoreceptors degenerate. We evaluated apoptosis and cellular stresses in each cell line at both day 100 and day 120, respectively. Interestingly, in the RP9-iPS (K10 and K11) cells, a subset of Recoverin+ cells co-expressed cytoplasmic 8-hydroxy-2'-deoxyguanosine (8-OHdG), a major oxidative stress marker, indicating the presence of DNA oxidation in RP9 patient-derived photoreceptors by differentiation day 100 (Fig. 4A and Fig. S8). More caspase-3+ cells were presented in the Crx+ photoreceptor cluster of RP9-iPS than in those from other lines (Fig. 4C–D). After maturation of the rod photoreceptors from RP9-iPS cells, Rhodopsin+ cells co-expressed Acrolein, a marker of lipid oxidation (Fig. 4E), while no Rhodopsin+/Acrolein+ cells were observed in iPS cells derived from other patients carrying different mutations or in normal iPS cells (Fig. 4F). This pattern was similar to the cases of 8-OHdG and activated caspase-3. Thus, we conclude that oxidation is involved in the RP9-rod photoreceptor degeneration.


Modeling retinal degeneration using patient-specific induced pluripotent stem cells.

Jin ZB, Okamoto S, Osakada F, Homma K, Assawachananont J, Hirami Y, Iwata T, Takahashi M - PLoS ONE (2011)

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).
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pone-0017084-g004: 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).
Mentions: We next asked how the patient-derived rod photoreceptors degenerate. We evaluated apoptosis and cellular stresses in each cell line at both day 100 and day 120, respectively. Interestingly, in the RP9-iPS (K10 and K11) cells, a subset of Recoverin+ cells co-expressed cytoplasmic 8-hydroxy-2'-deoxyguanosine (8-OHdG), a major oxidative stress marker, indicating the presence of DNA oxidation in RP9 patient-derived photoreceptors by differentiation day 100 (Fig. 4A and Fig. S8). More caspase-3+ cells were presented in the Crx+ photoreceptor cluster of RP9-iPS than in those from other lines (Fig. 4C–D). After maturation of the rod photoreceptors from RP9-iPS cells, Rhodopsin+ cells co-expressed Acrolein, a marker of lipid oxidation (Fig. 4E), while no Rhodopsin+/Acrolein+ cells were observed in iPS cells derived from other patients carrying different mutations or in normal iPS cells (Fig. 4F). This pattern was similar to the cases of 8-OHdG and activated caspase-3. Thus, we conclude that oxidation is involved in the RP9-rod photoreceptor degeneration.

Bottom Line: 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.Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan.

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.

Show MeSH
Related in: MedlinePlus