Fig 2: Survival and differentiation of retinal organoid cells as analyzed by immunofluorescence microscopy after transplantation. (a, b) The SC121-positive cells expressing green fluorescent protein (GFP), which were used for construction of the retinal organoids, were clearly visible in the healthy eye (transplanted with a retinal organoid, but receiving no immunosuppressive treatment) and the Oz-eye (eye with ocular hypertension (OHT) transplanted with a retinal organoid and treated with dexamethasone), indicating that cells from the transplanted organoids were alive in the host retina. However, in (c) the RAP-eye (OHT eye transplanted with a retinal organoid and treated with rapamycin) and (d) the OHT-eye (OHT eye transplanted with a retinal organoid, but receiving no immunosuppressive treatment), no SC121-positive or GFP-positive cells were detected. HuD- and RBPMS-positive retinal ganglion cells (RGCs) were also observed in (e, i) the healthy eye and (f, j) the Oz-eye. (m) Tubulin- and (q) NEFL-positive axons were observed within and near the transplants in the healthy eye. (n) Tubulin- and (r) NEFL-positive axons were observed within and near the transplants in the Oz-eye. In (c, g, k, o, s) the RAP-eye and (d, h, l, p, t) the OHT-eye, no cells or axons expressing these markers were detected. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer.

Fig 3: Comparisons of different marker expression profiles among the control, rapamycin- (RAP-) treated, and dexamethasone- (DEX-) treated groups. Two weeks after immunosuppressant treatment, (a) cells in the retinal organoids treated with RAP expressed higher levels of the retinal ganglion cell (RGC) markers Atoh7, Islet1, and Brn3b, the dendrite marker MAP2, the retinal progenitor marker PAX6 and CHX10, and photoreceptor marker CRX, compared to levels in the control group. These cells also expressed lower levels of the axon markers TUBB3 and NEFL than cells in the control group. In DEX-treatment group cells, dendrite, retinal progenitor, and photoreceptor markers (MAP2, CHX10, and CRX) were upregulated compared to levels in the control group. (b) However, 4 weeks after immunosuppressant treatment, both RAP- and DEX-treated cells showed higher expression levels of only PAX6 and CRX as compared to those in the control group. (c) Representative histogram of flow cytometry results. Each wave represented one group; the proportion of each group was calculated by the Blk group. There were 79.87 ± 5.92% of the RGCs in the control group, 60.97 ± 7.36% in the RAP group, and 63.6 ± 10.35% in the DEX group. CTRL: control group; RAP: rapamycin-treatment group; DEX: dexamethasone-treatment group; iPSCs: negative control group in RT-PCR; Blk: blank control group in flow cytometry.