title: Use of induced pluripotent stem-cell technology to understand photoreceptor cytoskeletal dynamics in retinitis pigmentosa.
abstract: BACKGROUND
  Retinitis pigmentosa, which affects one in 3000 people, causes blindness and has no treatment. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause 20% of all cases. Recent work suggests that RPGR, localised to the photoreceptor connecting cilium, regulates rhodopsin transport to the outer segment through its effect on the turnover of actin. We set out to establish a novel model for RPGR disease to test the hypothesis that RPGR mutations lead to retinal degeneration due to a dysregulation of the actin cytoskeleton.
  
  
  METHODS
  Patients with RPGR mutations and their unaffected relatives were recruited and skin biopsy samples taken. Fibroblast lines were established and reprogrammed to generate induced pluripotent stem cell (iPSC) lines. A three-dimensional organogenesis protocol was optimised whereby embryoid bodies were formed and patterned towards an eye field fate in a 100-day retinal differentiation protocol, allowing three-dimensional optic cups to form. RPGR-mutated cultures were compared with their healthy controls.
  
  
  RESULTS
  Mutant and wild-type iPSC lines were generated and characterised. Differentiation of all lines resulted in the generation of optic cups in a self-organising manner after 100 days in culture. These cultures contained mature photoreceptors, as evidenced by morphology and both RNA and protein expression. Photoreceptor cultures from RGPR-mutated iPSCs had increased actin polymerisation compared with controls (mean confocal pixel intensity count 59·02 [SD 16·24] vs 23·70 [8·20], p=0·0081). This finding was confirmed by assessment of F-actin with western blot. Pathways regulating actin turnover were explored; western blot analysis showed a reduction in both Src and ERK phosphorylation in RGPR-mutated photoreceptor cultures. An unbiased protein array confirmed this reduction in ERK and Src activation. Several other pathways were also shown to be dysregulated in the RGPR-mutated photoreceptor cultures.
  
  
  CONCLUSIONS
  This study supports the hypothesis that RPGR mutations lead to actin dysregulation. We have identified several pathways that are interrupted in RPGR-mutant photoreceptor cultures and could be contributing to disease. This study is the first use, to our knowledge, of human iPSCs with retinitis pigmentosa-causing mutations to look at pathophysiology of disease.
  
  
  BACKGROUND
  Wellcome Trust.
authors: Scottish Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK. Electronic address: rolymegaw@ed.ac.uk.
authors: Roly
authors: R
authors: Megaw
authors: Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors: Carla
authors: C
authors: Mellough
authors: Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
authors: Alan
authors: A
authors: Wright
authors: Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors: Majlinda
authors: M
authors: Lako
authors: Scottish Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors: Charles
authors: C
authors: Ffrench-Constant