title: 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.
authors.affiliation: Center for Vision Research, School of Ophthalmology and Optometry, Eye Hospital of Wenzhou Medical College, Wenzhou, China. jinzb@mail.eye.ac.cn
authors.forename: Zi-Bing
authors.initials: ZB
authors.lastname: Jin
authors.forename: Satoshi
authors.initials: S
authors.lastname: Okamoto
authors.forename: Ping
authors.initials: P
authors.lastname: Xiang
authors.forename: Masayo
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D016895:Culture Media, Serum-Free; D059865:Endoplasmic Reticulum Stress; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000166:cytology / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000473:pathology*; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D029082:Sendai virus / Q000235:genetics / Q000378:metabolism; D016662:Virus Integration
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D012243:Rhodopsin