Biology subjects

Refaian, N.

Publications and source records attributed to Refaian, N..

3 recordsLinked to original sources

bioRxiv2025

Human retinal organoid single-cell atlas allows to reconstruct retinal development at high resolution and identify nature restricted transcriptional states in vitro

The vertebrate retina is a highly specialized neural structure crucial for the initial capture and processing of visual information. Perturbations in retinal development contribute significantly to congenital and acquired forms of blindness, underscoring the necessity of elucidating human retinal ontogeny. Human pluripotent stem cell-derived retinal organoids have emerged as a powerful model capable of self-organizing and recapitulating key aspects of retinal morphogenesis, offering valuable insights into retinal development and disease. Despite their utility, the extensive cellular heterogeneity, dynamic state transitions, and existence of unique, the development of this system has not yet been fully resolved, posing significant challenges for interpreting their developmental fidelity and therapeutic applicability. Here, we present the Human Retinal Organoid Cell Atlas (HROCA), a comprehensive single-cell transcriptomic reference spanning retinal organoid development from Day 10 to Day 365, integrating data from 15 studies, 71 batches, and encompassing 458,309 cells. HROCA provides a detailed benchmark for organoid differentiation, identifying known retinal cell classes and novel cellular states at single-cell resolution. By associating HROCA with the human fetal retina atlas (HFR), we offer an in-depth comparison between in vitro and in vivo retinal development, revealing protocol-specific differences, limitations in neuronal maturation, and developmental timing discrepancies. HROCA expands our understanding of retinal developmental plasticity, highlighting unique cell populations such as chimeric, non-canonical, and apatride cells, which do not conform to classical retinal cell types but exist consistently across developmental timelines in vitro. This atlas serves as a crucial resource for understanding retinal development, evaluating organoid models, and guiding future improvements in regenerative medicine strategies.

developmental biology↗

bioRxiv2025

Single-cell transcriptome of retinal myeloid cells in response to transplantation of human neurons reveals reversibility of microglial activation

The host retinal microglia and macrophage activation remains a major challenge for the integration of donor neurons following transplantation. Previously, we and others have shown that it is possible to increase donor retinal ganglion cell (RGC) survival by inhibiting the microglia-RGC interaction with Annexin V or through reprogramming microglia with the soluble Fas ligand. However, the exact mechanisms of the microglia/macrophage activation and their heterogeneity following transplantation remain unknown. To address this question, the donor RGC were differentiated from Brn3b-Tdtomato-Thy1.2 human embryonic stem cells using a 3D protocol, followed by dissociation and RGC purification. RGC were delivered subretinally (1.5x104 viable cells/eye) into 3-6-month-old CX3CR1GFPknock-in mice. Three days after transplantation retinas were dissociated into single-cell suspension and GFP-positive myeloid cells isolated using FACS. Of the sorted cells, up to 10,000 viable cells per sample were used for single-cell RNA library preparation and sequenced using the 10X Genomics Chromium platform. In addition, several retinas were fixed and stained for donor RGC (mCherry) and host microglia/macrophages (Iba1). RNA Velocity was used to reconstruct the myeloid cell population and activation trajectory from scRNAseq data. We observed continuous bi-directional transition of microglia/macrophages from a homeostatic to an activated state. We also observed that the response to the transplant falls into the classic disease-associated-microglia (DAM) activation paradigm with a decrease in expression of the homeostatic gene Tmem119 and an increase in expression of disease-associated genes including Apoe, Lgals3, and Spp1. Our findings show that the host retinal myeloid cell population undergoes activation upon transplantation of stem-cell derived donor RGC, with a molecular profile of the activated cells similar to that of activated myeloid cells associated with neurodegenerative diseases of the brain and the eye. Advanced integrated transcriptomic analysis shows separate activated-to-homeostatic and homeostatic-to-activated trajectories suggesting the reversibility of this process.

neuroscience↗

bioRxiv2024

Retinal microglia exacerbate uveitis by functioning as local antigen-presenting cells

Autoimmune uveitis is a major cause of blindness in the working-age population of developed countries. Experimental autoimmune uveitis (EAU) depends on activation of interphotoreceptor retinoid-binding protein (IRBP) specific CD4+ effector T cells that migrate systemically and infiltrate into the retina. Following systemic induction of retinal antigen-specific T cells, the development of EAU can be broken down into three phases: early phase when inflammatory cells begin to infiltrate the retina, amplification phase, and peak phase. Although studied extensively, the function of local antigen-presenting cells (APCs) within the retina remains unclear. Two potential types of APCs are present during uveitis, resident microglia and infiltrating CD11c+ dendritic cells (DCs). MHC class II (MHC II) is expressed within the retina on both CD11c+ DCs and microglia during the amplification phase of EAU. Therefore, we used microglia specific (P2RY12 and TMEM119) and CD11c+ DC specific MHC II knockout mice to study the function of APCs within the retina using the conventional and adoptive transfer methods of inducing EAU. Microglia were essential during all phases of EAU development: the early phase when microglia were MHC Il negative, and amplification and peak phases when microglia were MHC II positive. Unexpectedly, retinal infiltrating MHC Il+ CD11c+ DCs were present within the retina but their antigen-presenting function was not required for all phases of uveitis. Our data indicate microglia are the critical APCs within the retina and an important therapeutic target that can prevent and/or diminish uveitis even in the presence of circulating IRBP-specific CD4+ effector T cells.

immunology↗