Abstract
Purpose :
There is an unmet clinical need to treat especially bilateral LSCD. For this allogeneic source of limbal stem cells (LSCs) differentiated from human induced pluripotent stem cells (hiPSC) could provide a solution. In previous studies, we have developed efficient methods to produce an enriched population of ABCG2-positive hiPSC-LSCs and our hypothesis is that these cells including both quiescent (p27 and GPHA2 positive) and proliferative sub-populations could represent high regenerative potential to form corneal epithelium in vivo.
Methods :
In this study, hiPSC-LSCs were differentiated, characterized by morphology, immunostaining (PAX6, ABCG2, p40/63, p27, GPHA2), flow cytometry (ABCG2) and colony forming assay and finally cryopreserved. For transplantation, hiPSC-LSCs where thawed and cultured on fibrin membrane. Cells were transplanted to cyclophosphamide-induced neutropenia mouse (Swiss Webster) model with mechanically created LSCD and the eyes received TobraDex gel before tarsorrhaphy. After 5-6 days, eyes were harvested for imaging and histological analyses. Wound only eyes were used as control.
Results :
The limbal phenotype of the ABCG2-enriched (around 80%) population of hiPSC-LSCs was confirmed before transplantation assessment. The mechanical scraping with algerbrush followed by thermal cauterization successfully removed both corneal and limbal epithelium for the induction of the LCSD in a mouse model with chemically induced neutropenia. Histological sectioning and staining with human specific antibody (Ku80) confirmed the successful integration and stratification of the hiPSC-LSCs after 5-6 days of transplantation in mice without obvious signs of xeno-rejection. Further staining with markers including PAX6 and p63 confirmed corneal phenotype of the transplanted cells.
Conclusions :
The neutropenia mouse model with total LSCD was successfully used for the short-term efficacy assessment of ABCG2-postive hiPSC-LSCs. For the long-term efficacy and safety assessment, immunodeficient animal model will be needed.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.