Abstract
Purpose :
Stem cell-derived photoreceptor precursor cells have the potential to restore vision to patients with extensive vision loss from retinal degenerative diseases. Creating these cells from the patients for whom they are intended has the advantage of perfect immunologic matching. The purpose of this study was to develop methods for manufacturing transplantable retinal cells in a low-cost, non-profit environment, under current good manufacturing practices (cGMP).
Methods :
Skin biopsies were obtained from legally blind adult patients (n=35) with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. For seven of the patients with the poorest vision (light perception or worse), patient-specific induced pluripotent stem cells (iPSCs) were generated via transient expression of the OCT4, SOX2, KLF4, and c-MYC. Karyotyping and TaqMan Scorecard analysis were used for clonal validation. Retinal photoreceptor precursor cells were derived using a stepwise cGMP-compliant 3D differentiation protocol. Immunocytochemical analysis and confocal microscopy were used to monitor retinal development. Bolus injections of differentiated cells (1-5x106) were transplanted into immune compromised mice to assess safety.
Results :
We succeeded in establishing iPSC lines from all 7 blind individuals ranging in age from 32-86 years. Thirty days post-differentiation, polarized neural epithelium containing PAX6-, SOX2-, and OTX2-positive retinal progenitor cells were seen. After 60 days, PAX6- and OTX2-positive cells were found to segregate into distinct lamina corresponding to presumptive RPE and photoreceptor precursor cells. After 90 days of differentiation, post-mitotic photoreceptor precursor cells positive for CRX, NRL and NR2E3 were observed. Four weeks after transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation.
Conclusions :
cGMP-compliant protocols were developed for skin biopsy and dermal fibroblast isolation, iPSC generation and retinal specific differentiation. Clinical-grade retinal cells were successfully generated from all 7 patients attempted, including 4 patients over the age of 70 years. Post-mitotic photoreceptor precursor cells retain their retinal phenotype and do not form tumors following transplantation in immune compromised recipients.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.