Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa

• DOI: 10.7554/elife.00824
• Publication Date: 2013-08-27T15:53:44Z
• Authors: Budd A Tucker; Robert F Mullins; Luan M Streb; Kristin Anfinson; Mari E Eyestone; Emily Kaalberg; Megan J Riker; Arlene V Drack; Terry A Braun; Edwin M Stone
• Keywords: 0301 basic medicine; induced pluripotent stem cell; QH301-705.5; Science; Blotting, Western; Induced Pluripotent Stem Cells; Nerve Tissue Proteins; retinal transplantation; retinal cell differentiation; Mice; 03 medical and health sciences; retinitis pigmentosa; Animals; Humans; Biology (General); Endoplasmic Reticulum Chaperone BiP; Mice, Knockout; Q; R; Cell Differentiation; 3. Good health; Developmental Biology and Stem Cells; Mutation; retinal degeneration; Codon, Terminator; Medicine; next-generation sequencing; Retinitis Pigmentosa; Photoreceptor Cells, Vertebrate

Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration.