Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. Here, we show that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. We also show that enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, we show that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport.epilepsy | planar cell polarity | spiny-legs | EB1-GFP | neurodegeneration E pilepsy, which affects ∼1% of the population, is a disabling and debilitating disease characterized by recurrent seizures. Coupling genetic analysis of human epilepsy cases with functional validation in animal models, we previously showed that mutations in human PRICKLE1 and PRICKLE2, as well as mutations in mouse, zebrafish and fly prickle (pk) orthologs, increase susceptibility to seizures (1-3). We recently showed that both mouse and fly Prickle can associate with Synapsin (4). However, little is known regarding what cellular process connects Prickle with epilepsy. Products of the prickle gene work in concert with a group of cytoplasmic and membrane-associated proteins including Frizzled (Fz) and Dishevelled (Dsh) to establish polarity of structures such as hairs and bristles in the fly epidermis (5-8). The fly pk locus expresses three alternately spliced isoforms, two of which, pk sple (prickle-spiny-legs) and pk pk (prickle-prickle), are expressed in postembryonic animals (5, 7). Homozygous pk pk and pk sple fly mutants show strong planar cell polarity (PCP) phenotypes in the wing and in the legs, respectively, in addition to other regions of the adult body (6, 7). Prickle (Pk) protein isoforms are localized to the proximal end of fly wing cells, whereas Fz and Dsh are localized distally (8).Here, we show that vesicle transport dynamics are altered in neurons of Drosophila mutant for either adult isoform of prickle. Seizure-prone pk sple heterozygotes show enhanced vesicle transport (along with electrophysiological defects similar to other seizure-prone mutant flies), and the seizure phenotypes can be rescued by lowering the dose of vesicle transport motor proteins. However, pk pk heterozygotes show severely reduced vesicle transport, with loss of both copies of pk pk showing a marked decrease i...

Stem cell strategies focused on replacement of RPE cells for the treatment of geographic atrophy are under intense investigation. Although the eye has long been considered immune privileged, there is limited information about the immune response to transplanted cells in the subretinal space of large animals. The purpose of this study was to evaluate the survival of allogenic induced pluripotent stem cell-derived RPE cells (iPSC-RPE) delivered to the subretinal space of the pig as well as determine whether these cells induce an immune response in non-diseased eyes. GFP positive iPSC-RPE, generated from outbred domestic swine, were injected into the subretinal space of vitrectomized miniature swine. Control eyes received vehicle only. GFP positive iPSC-RPE cells were identified in the subretinal space 3 weeks after injection in 5 of 6 eyes. Accompanying GFP-negative cells positive for IgG, CD45 and macrophage markers were also identified in close proximity to the injected iPSC-RPE cells. All subretinal cells were negative for GFAP as well as cell cycle markers. We found that subretinal injection of allogenic iPSC-RPE cells into wild-type mini-pigs can induce the innate immune response. These findings suggest that immunologically matched or autologous donor cells should be considered for clinical RPE cell replacement.

Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.