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Z.-B. Jin, S. Okamoto, M. Takahashi; In vitro Induction of Retinitis Pigmentosa-Specific Photoreceptor Cells From Patient-Derived Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1234.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is a group of inherited retinal degeneration characterized by night blindness and visual field defects which are caused by rod photoreceptor death. Most causative genes are involved in phototransduction cascade or signaling specifically in rod photoreceptor, and still are there some widely expressed genes but their mutations only cause rod degeneration. The genetic heterogeneity together with the inter-familiar and intra-familiar phenotypic variances in RP make the disease research complex. It is thus attempted to investigate the phenotype of individual photoreceptor cell with distinct genotype and its response to candidate drugs. In this study, we aimed to generate patient-specific photoreceptor cells by using an induced pluripotent stem (iPS) cells technology and an in vitro differentiation strategy.
This study was approved by local ethical committee with informed consent from patients. RP patients with different mutations/genes were studied. Fibroblast cells were cultured from skin sample and were re-confirmed the genotype. The fibroblasts were infected with retrovirus and/or non-integrating virus harboring four reprogramming factors (OCT3/4, KLF4, c-MYC and SOX2). Established iPS cell lines were amplified for in vitro differentiation. iPS cells were cultured under a serum-free suspension conditions followed by an adherent culture. Immunocytochemistry and gene expression profiling were performed to monitor the differentiation.
Morphologically embryonic stem (ES) cell-like colonies were appeared after infection of each type of virus. These cells were positive for pluripotent markers (Nanog, Oct3/4, Tra-1-60, SSEA3). Furthermore, teratoma formation was confirmed by all three-derm derivatives. Through in vitro differentiation, neural retinal progenitor cells, retinal pigment epithelia (RPE) progenitor cells, RPE, and photoreceptor precursor cells were induced sequentially. And rod photoreceptor cells emerged with specific markers again rhodopsin and recoverin. Additionally, other types of retinal cell, including cone, bipolar cells and ganglion cells, were also confirmed.
We successfully generated iPS cells from RP patients. These RP-derived iPS cells do have differentiation potential into most retinal cells including the rod photoreceptor cells which were lost in the patients. These induced patient-specific rod photoreceptor cells may be useful for drug discovery, disease modeling, and regenerative medicine.
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