Single-Cell Transcriptomic Profiling of Xenotransplanted Retinal Organoids Derived from the WA09 CRX-tdTomato Human Embryonic Stem Cell Line

Single-cell gene expression profiles were generated for human retinal organoids derived from the WA09 CRX-tdTomato human embryonic stem cell line. Fragmented organoids were transplanted into the subretinal space of normal canine retinas and analyzed three days post-transplantation. In parallel, fragmented organoids from the same batch, maintained in culture for the same duration, were used as controls. Comparative single-cell transcriptomic analysis provided insights into the early molecular changes associated with the transplanted environment versus in vitro culture conditions, shedding light on the cellular responses and adaptation of retinal organoids post-transplantation. Retinal organoids (ROs) derived from the WA09 CRX-tdTomato human embryonic stem cell line were cultured for 113–126 days. These ROs were mechanically triturated into smaller fragments and subsequently injected into the subretinal space of normal canine retinas. Fragments from the same batch of ROs, subjected to identical trituration, were maintained in culture as controls. After three days, both the transplanted RO fragments and their cultured counterparts were collected and processed for single-cell RNA sequencing (scRNAseq). This process was repeated across three independent experiments to ensure reproducibility and robustness of the findings.