Preclinical safety studies of human embryonic stem cell-derived retinal pigment epithelial cells for the treatment of age-related macular degeneration

Abstract

Abstract As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)-derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology. Significance statement This study evaluated the preclinical safety of in vitro differentiated retinal pigment epithelial cells from embryonic stem cells by (a) examining karyotype and performing whole genome sequencing to assess genome stability; (b) performing single-cell RNA sequencing to ensure purity and absence of undifferentiated cells; and (c) examining biodistribution and tumorigenicity of transplanted cells to rule out malignant growth and migratory properties. The derived cells proved to be safe, and this study altogether provided a strategy for preclinical evaluation of PSC-based therapies. Also, the whole genome sequencing analysis illustrates that the preexisting load of germline variants is significantly higher than the introduced variants acquired through vitro culture or differentiation, which enforces the importance to evaluate the genome integrity at a deeper level than just karyotype.