Stable production of hESC/iPSC-derived retinal progenitor spheroid for cell-based therapies using label-free ghost cytometry sorting

Abstract

Purpose : Human iPSC-derived retinal organoid sheets were used as a graft in the recent clinical trial. Although they showed functional potency in preclinical animal studies, the size of individual sheets is up to about 1.0mm². The goal of this research is to obtain retinal progenitor cells in the early differential stage, which can be used to prepare a larger sheet of the desired size. To avoid the risk of contamination by unwanted factors, label-free sorting is a desirable solution to enrich target cells. Thus, we constructed the label-free ghost cytometry (LFGC) sorting system and studied LFGC-sorted retinal spheroids in vitro and after transplantation.

Methods : RAX::Venus cell line and CRX::Venus cell line (both hESC-KhES-1 cell lines), and hiPSC-WJs531 cell line were differentiated into retinal organoids. We first trained a classifier based on support vector machine by obtaining GC waveforms of the RAX::Venus cell line on differentiation day (DD) 25. We then conducted LFGC-based sorting of the dissociated single cells from retinal organoids of the above cell lines at DD25. We defined the sorted cells as Sorted, the others as Waste, and unsorted cells preserved on ice as Control. The expressions of the marker genes of retinal progenitor cells and other cell types were tested by reverse transcription real-time quantitative PCR in each group. Spheroids were formed from the cells of each group and evaluated by immunohistochemistry (IHC) at DD60-70. iPSC-derived spheroids were also transplanted at around DD60 into the retinal degeneration model nude rats and evaluated by IHC at 4-5 months after surgery.

Results : With GC, the mean area under the receiver operating characteristic curve (ROC-AUC) for the classification of RAX::Venus++ cells and the others was 0.73 ± 0.01 (range; 0.71-0.76). Retinal progenitor marker genes were increased while non-retinal genes were decreased in Sorted compared to Control, which was reversed in the Waste. Using IHC, DD60-70 Sorted spheroids reproducibly included the retinal region, while Waste and Control spheroids became non-retinal tissue. Grafted spheroids survived and developed mature photoreceptors in the degenerating host retina.

Conclusions : LFGC-based sorting reproducibly enriched retinal progenitor cells of several different cell lines. These enriched cells can be used to prepare a larger graft sheet for cell-based therapies.

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