Fig. 1

Retinal organoid (RO) specifications and mixed lymphocyte reactions: a Specifications of lot release: a1 lot viability (trypan blue test); a2 No OCT4 expression in organoids (qPCR). Stem cells (d0, n = 3) compared with released ROs (n = 12, age 49–60 d) and human fetal retina (HFE) at d137 (n = 2); a3 RO cells express > 75% OTX2 (flow cytometry); a4 paraffin section of RO stained with anti-Retina and Anterior Neural Fold Homeobox (RAX) antibody. RAX is a transcription factor essential to retinal development and its expression is ubiquitous in ROs. Every nucleus in a differentiated organoid expresses RAX. b Mixed lymphocyte reactions. Retina organoids (ROs, n = 9) of three different lots were evaluated for in vitro immunogenicity using a two-way mixed lymphocyte reaction (MLR). After 7 days of co-culture, live T-cells (CD45+ CD3 +) were stained with Ki67. Protein expression was analyzed using flow cytometry. High expression of the proliferation marker Ki67 is indicative of a T-cell response. Control conditions included irradiated human fibroblasts (n = 3) and anti-CD3 microbeads (n = 3), both of which induced a strong response. The retina organoids (n = 9) did produce some proliferation, but it was significantly reduced as compared to controls (p < 0.0001). c Mixed lymphocyte reactions. Cells were gated as live and CD45 positive. The MHC-II expression levels of the responders were measured after 7 days of co-culture. The retina organoids did produce some proliferation, but significantly less compared to αCD3 microbead controls (p < 0.0012). d Graph of flow data shown in (c), showing the low response of lymphocytes to ROs. e Significantly reduced expression of MHC-I in retina organoids in comparison to hESC and irradiated fibroblasts