Cellular modeling of AIPL1-Leber Congenital Amaurosis using patient-derived induced pluripotent stem cells

Abstract

Purpose : Patient-derived cellular models can provide a specific tool to test new therapeutic approaches for retinal degenerative diseases. Mutations in AIPL1 are associated with Leber Congenital Amaurosis (LCA). Considering that AIPL1 protein is only expressed in photoreceptors we decided to establish a cellular model of AIPL1-LCA by differentiation of induced pluripotent stem cells (iPS) towards photoreceptor lineage.

Methods : Skin biopsy was obtained from a female LCA patient with a confirmed homozygous Cys 89 Arg mutation and from a healthy control after informed consent and approval from the Hospital Ethics Committee. Fibroblast cells were cultured and reprogrammed to iPS using a non-integrative sendai virus vector for the ectopic expression of hKOS, hcMyc and hKfl4. The iPS clones were checked for the expression of pluripotency markers both by rtPCR and immunofluorescence. Differentiation of iPS cell lines towards photoreceptors was performed using a well established protocol from Boucherie et al Stem Cells, 2013, 3, 408-14. Photoreceptor lineage specific gene expression was evaluated by qPCR and immunofluorescence. Western blot was used to compare protein levels of PDE, RHO and RCVRN at the end of the differentiation protocol.

Results : Fibroblasts from the patient and the healthy control have been successfully reprogrammed to iPS cell lines that stably expressed the specific markers of pluripotency SSEA-4, TRA-1-81, OCT4 and NANOG after loosing the expression of the ectopic reprogramming factors. Both normal and AIPL1-iPS cell lines were directed to generate photoreceptor precursors (PPR) and to further differentiate towards rod lineage. At days 5 and 10 of the differentiation protocol, immunofluorescence and rtPCR showed an increase in the expression of specific PPR markers: PAX6, RAX, SOX9, CHX10, RCVRN and NRL. At the end of the differentiation protocol, on day 30, qPCR detected an increase in gene expression of 3.2±1.7 fold for PDE and 7.4±2.1 fold for RHO. Immunofluorescence labeling in photoreceptor cells was positive for NRL, RHO, CRX and TUJ20. As expected from published studies in AIPL1 animal models, AIPL1-iPS cellular model shows low PDE protein levels normalized relative to RHO and RCVRN.

Conclusions : We have been able to generate and characterize a rod photoreceptor-lineage cellular model for AIPL1-LCA by diferentiation of iPS derived from a patient.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.