Generation of Clinical Grade iPSCs for use in Ocular Cell Therapy

Abstract

Purpose : To generate iPSCs that are regulatory compliant and can be used to generate retinal pigment epithelial (RPE) cells for allogeneic cell therapy.

Methods : Dermal fibroblasts were isolated and banked using GMPs collected from 18 eligible female donors between the ages of 23 and 34. The Cytotune 2.1 Sendai virus kit was used to reprogram fibroblasts from 4 of these donors using GMPs. Procedures described in the Cytotune 2.1 user guide were modified to be regulatory compliant and to optimize iPSC clone growth and survival. Resulting iPSC clones were screened in an assay using PEDF secretion as an endpoint to assess their potential to differentiate into a pure population of RPE cells with a high yield that did not require selection of a subpopulation of cells. Successful clones were further expanded and banked.

Results : A total of 20 regulatory compliant clones were isolated from 3 donors (300-BIOTR-0022, 300-BIOTR-0020, and 300-BIOTR-0019). Additional clones isolated from a fourth donor following Cytotune 2.1 kit instructions were determined to have potential compliance issues surrounding materials used. Of the more than 20 clones isolated from the 4 donors, 3 clones (300-BIOTR-0020 Clone 1 and 300-BIOTR-0022 Clones 1 & 6) met yield and purity criteria for use in commercial production of iPSC-derived RPE. RPE from these clones formed pigmented cobblestone monolayers of cells that expressed Bestrophin 1, RPE65, CRALBP, and mer-tk based on Western blot analysis. These RPE secreted PEDF and were phagocytosis competent. Analysis of purity using flow cytometry indicated that purity of >98% RPE could be achieved. Of these, fewer than 1 in 5000 cells were determined to be iPSCs based on qRT-PCR analysis of lin28a and ZSCAN10 expression.

Conclusions : We have produced regulatory compliant iPSCs from 3 adult human donors. Of the more than 20 total clones produced, 3 have the potential to be used for the commercial production of RPE cells for clinical human allogeneic cell therapy pending production and testing of Master Cell Banks.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.