Single Cell RNAseq of adult and stem cell derrived iris muscle

We performed single-cell RNA sequencing (scRNAseq) on iris tissue from three adult human donors and human embryonic stem cell (hESC)-derived whole eye organoids, termed “SEAM,” using the 10X Genomics Chromium platform. From the adult human datasets, we identified an iris muscle population based on the co-expression of PAX6 and smooth muscle proteins (ACTA2 and TAGLN). This dataset was further used to characterize the expression profile of the non-neuronal, crest-derived iris muscle. Additionally, we differentiated hESCs into whole eye organoids (SEAM) and performed scRNAseq after 7 weeks of differentiation. In the organoids, we identified cell populations corresponding to most ocular cell types, spanning neurons, distal optic cup, periocular mesenchyme, and ocular surface epithelium. Notably, we identified iris muscle-like cells in the developing eye organoids. We conducted an integrated analysis using the Seurat R package to compare the hESC-derived eye organoids with one of the adult donor irises (IRIS91 and SRAM48). This analysis revealed a shared iris muscle cluster containing cells from both datasets. The hESC-derived iris muscle represents a developing tissue with lower expression of mature muscle genes and higher expression of transcription factors, such as PAX6 and LHX2. Human globes from donors aged 50–74 years were obtained within 24 hours postmortem from the Eye-Bank for Sight Restoration, NY, and Miracle in Sight, NC, with informed consent provided by the donors or their next of kin. This study was reviewed by the Icahn School of Medicine at Mount Sinai IRB oversight and deemed not to be human subject research. We received approval for use of H9 cell lines by WiCell.