Abstract
Purpose :
To generate and characterize clinical-grade, human induced pluripotent stem cells (hiPSCs) from healthy donor and differentiate them into retinal pigmented epithelium (RPE) for their applications in pre-clinical and clinical trial evaluations.
Methods :
The dermal fibroblast cells derived from skin biopsy of healthy volunteers were reprogrammed into hiPSCs using CTS™ CytoTune 2.1 Sendai reprogramming kit. Three clones derived from a single donor were stably expanded beyond passage 10. One of these hiPSC lines (LVIP11-SV-CTS-NC-1) was stringently characterized for stemness, pluripotency, loss of vector genome, genetic identity, genomic stability by RT-PCR, immunocytochemistry (ICC), FACS, embryoid body formation, karyotyping, STR profiling, HLA typing analysis. This line was further differentiated into retinal lineages using established protocols to prepare neuro-retinal organoids and RPE cells. The RPE progenitors are enriched by antibody-based negative MACS sorting and characterized by ICC, RT-PCR, FACS analysis.
Results :
The clinical grade hiPSC line remained stable at passage 12 and expressed the stemness mRNAs and proteins such as OCT3/4, NANOG, SSEA4, hTERT. Upon random differentiation, it expressed all three lineage-specific markers such as PAX6, OTX2, αSMA, MSX2, GATA6, MIXL1. Karyotype analysis confirmed the normal chromosomal integrity. STR analysis and HLA typing has confirmed its genotype, which matched the healthy female donor. Bioburden testing confirmed the absence of any cryptic infectious agents such as HIV1/2, HBV, HCV, syphilis, mycoplasma and endotoxin levels was within permissible limits (<0.125 EU/mL). Upon retinal lineage differentiation, the iPSCs formed distinct eye field primordia, containing OTX2, SIX6, RX expressing retinal stem cells at 3-4 weeks. The neuro-retinal organoids expressed, PAX6, SOX10, CHX10, CRX, Recoverin. The RPE progenitors expressed MITF and PAX6 and the enriched RPE cultures with pigmentation showed typical cobblestone morphology and expressed RPE65, TYR, BEST1, CRALBP and secreted significant amounts of PEDF.
Conclusions :
The clinical-grade hiPSC line reported here meets the minimal criterion for the stemness, pluripotency, safety and applicability for pre-clinical and clinical trial evaluations and for their ability to differentiate into neuro-retinal organoids and mature RPE cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.