Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling

Abstract

We investigated retinitis pigmentosa (RP) caused by a mutation in the gene rhodopsin (RHO) with a patient-specific rod cell model generated from induced pluripotent stem cells (iPSCs) derived from an RP patient. To generate the iPSCs and to avoid the unpredictable side effects associated with retrovirus integration at random loci in the host genome, a nonintegrating Sendai-virus vector was installed with four key reprogramming gene factors (POU5F1, SOX2, KLF4, and c-MYC) in skin cells from an RP patient. Subsequent selection of the iPSC lines was on the basis of karyotype analysis as well as in vitro and in vivo pluripotency tests. Using a serum-free, chemically defined, and stepwise differentiation method, the expressions of specific markers were sequentially induced in a neural retinal progenitor, a retinal pigment epithelial (RPE) progenitor, a photoreceptor precursor, RPE cells, and photoreceptor cells. In the differentiated rod cells, diffused distribution of RHO protein in cytoplasm and expressions of endoplasmic reticulum (ER) stress markers strongly indicated the involvement of ER stress. Furthermore, the rod cell numbers decreased significantly after successive culture, suggesting an in vitro model of rod degeneration. Thus, from integration-free patient-specific iPSCs, RP patient-specific rod cells were generated in vitro that recapitulated the disease feature and revealed evidence of ER stress in this patient, demonstrating its utility for disease modeling in vitro.

Figure 1.

Generation of patient-specific iPSCs. (A): Schematic diagram of this study. Compared with patient-iPSCs generated by the retroviral method, nonintegrative Sendai virus (SeV)-iPSCs from the same retinitis pigmentosa patient with a rhodopsin (RHO) mutation are more appropriate for disease modeling. (B): Sequence chromatogram of the RHO gene G188R mutation identified in patient-derived fibroblasts. (C): Representative result of SeV infection efficiency. The SeV infection efficiency was tested using SeV18+ green fluorescent protein (GFP)/TSΔF. Six days after infection of fibroblast cells, 10.6% of cells were positive for GFP (red histogram). Black histogram indicates a negative control. (D): The colony morphology of the patient-specific iPSCs. (E): Specific expression of the pluripotency marker Oct3/4 in the patient-derived iPSCs. Scale bar = 20 μm. (F): The results of karyotype analysis. (G–I): Histology of three germ layer derivatives in a teratoma formed by patient-derived iPSCs. (J): Ectopic expression of the Sendai viral Oct3/4 gene in the early passage iPSCs. Lane 1, positive control; lanes 2 and 3, 2nd passage iPSC lines of Sev3 and Sev9; lanes 4 and 5, 3rd passage Sev3- and Sev9-iPSC lines; lane 6, 10th passage Sev9-iPSC; lane 7, differentiated (day 40) Sev9-iPSCs. Abbreviations: ACTB, housekeeping β-actin gene; FITC, fluorescein isothiocyanate; iPSCs, induced pluripotent stem cells; Sv, Sendai virus; Sv-Oct3/4, Sendai viral Oct3/4 gene.

Figure 2.

Directed retinal differentiation of the patient-specific induced pluripotent stem cells. On day 40, induced Pax6+Mitf+ RPE progenitor cells (A) and Pax6+Rx+ neuroretinal progenitor cells (B) were observed. (C): On day 60, cells positive for Crx and recoverin appeared. (D): Recoverin+ cells significantly increased by day 90; immature RPE (RPE-like) cells displayed a net-like shape (E) and did not express pigments on day 35 (F). Typical RPE cells displayed hexagonal shape (G) and pigmentation (H) on day 60. (I): Apoptotic cells (caspase-3+) were found in the recoverin+ cell cluster. (J): Induced rod (rhodopsin+) cells on day 120. (K): Induced rod cells were usually observed on the top of ground cells on day 120. (L): Rhodopsin+ cells displayed neural processes and stick-like shape. Scale bars = 20 μm. Abbreviations: Crx, cone-rod homeobox-containing gene; d, day; DAPI, 4′,6-diamidino-2-phenylindole; Rec or Rcvrn, recoverin; RPE, retinal pigment epithelial.

Figure 3.

Patient-specific retinal cells showed endoplasmic reticulum (ER) stress. ER stress in the patient-specific retinal cells. (A–C): CHOP+ cells in recoverin+ cell cluster (day 120). (D–F): Rhodopsin+ cells coexpressed Bip, a conventional marker for ER stress. Scale bars = 20 μm. Abbreviations: Bip, binding immunoglobulin protein (a molecular chaperone of binding immunoglobulin protein/Grp78 involved in ER stress); CHOP, a proapoptotic protein responsive to ER stress; Rec, recoverin.

Figure 4.

Patient-derived rod cells underwent degeneration in vitro. Representative figure of induced rod cells (RHO+) on day 120 (A–C) and day 150 (D–F). (G): Cells positive for Crx and recoverin remained on day 150. (H): No significant differences in differentiated RHO+ cell number among cells derived from a wild-type induced pluripotent stem cell (iPSC) line (201B7) and patient-specific iPSCs obtained through the retroviral (retro-P59) and Sendai viral (Sev-P59) methods. (I): Changes in the number of cells indicated significant rod degeneration under in vitro culture conditions by day 150. Scale bars = 20 μm. ***, p < .001. Abbreviations: Crx, cone-rod homeobox-containing gene; d, day; DAPI, 4′,6-diamidino-2-phenylindole; NS, no statistical significance; Rec, recoverin; Sv, Sendai virus; Wt, wild-type.

Figure 5.

Localization of differentiated neuroretinal cells. (A): RPE cells (black area) and optic cup-like organizations (*) on day 60 (phase). (B): Recoverin+ cell clusters. (C): Overlapped. (D): Schema of RPE and neuroretina differentiation in vitro. Scale bars = 200 μm. Abbreviations: NR, neural retina; RPE, retinal pigment epithelial.