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Single Cell RNAseq of adult and stem cell derrived iris muscle

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE297613 | GEO | 2025/09/01

REPOSITORIES: GEO

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Project description:Ocular pigment epithelium (PE) cells promote the generation of T regulators (PE-induced Treg cells). Moreover, T cells exposed to PE acquire the capacity to suppress the activation of bystander T cells via TGFbeta. Membrane-bound TGFbeta on iris PE cells interacts with TGFbeta receptors on T cells, leading to the conversion of T cells to CD8(+) Treg cells via a cell contact-dependent mechanism. Conversely, soluble forms of TGFbeta produced by retinal PE cells can convert CD4(+) T cells into Treg cells in a manner that is independent of cell contact. In this study, we looked at the expression of immunoregulatory factors (TGFbeta, thrombospondins, CD59, IL-1 receptor antagonist, etc.) in PE cells as identified via an oligonucleotide microarray. Several thrombospondin-binding molecules were detected, and thus we focused subsequent analyses on thrombospondins. Via the conversion of latent TGFbeta to an active form that appears to be mediated by thrombospondin 1 (TSP-1), cultured iris PE and retinal PE cells induce a PE-induced Treg cell fate. After conversion, both ocular PE and PE-induced Treg cells express TSP-1. Regulatory T cell generation was amplified when the T cells also expressed TSP-1. In addition, PE-induced Treg cells significantly suppressed activation of bystander T cells via TSP-1. These results strongly suggest that the ability of ocular PE and PE-induced Treg cells to suppress bystander T cells depends on their capacity to produce TSP-1. Thus, intraocular TSP-1 produced by both ocular parenchymal cells and regulatory T cells is essential for immune regulation in the eye. Experiment Overall Design: Comparison of gene expression pattern in Mouse Primary Cultured Cell Lines (RPE, IPE, CBPE). Experiment Overall Design: Adult (6- to 8-week-old) C57BL/6 purchased from CLEA Japan Inc. (Tokyo, Japan) were used as donors ocular pigment epithelium (PE). To cultivate iris PE (IPE) and ciliary body PE (CBPE), iris and ciliary body tissues in the eyes were dissected out, and then incubated in 1 mg/ml Dispase and 0.05 mg/ml DNase I (both from Boehringer-Mannheim, Mannheim, Germany) for 1 hr. To cultivate retinal PE (RPE) Eyes were cut into two parts along a circumferential line posterior to the ciliary process, creating a ciliary body-deficient posterior eyecup. The eyecup was then incubated in 0.2% trypsin (Biowhitaker) for 1 hr. These tissues were triturated to make a single cell suspension, and then re-suspended in the medium.

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Single Cell RNAseq of adult and stem cell derrived iris muscle

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