SnRNAseq of human limbus tissue
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE225355 | GEO | 2024/08/01
REPOSITORIES: GEO
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Project description:Regulatory T cells (Tregs) play a key role in immunomodulation and tissue regeneration. Limbal stem cells (LSCs) maintain corneal epithelial homeostasis, and limbal stem cell deficiency (LSCD) leads to visual impairment. Current LSCD treatments face donor shortages and graft rejection risks. The present study explored Tregs’ therapeutic potential for LSCD in a mouse model of graded LSCD, and further explore the direct effect of Tregs on LSCs function by in vitro coculturing human-derived cells. Subconjunctival Tregs injection effectively treated mild and moderate LSCD in mouse models. Coculturing human LSCs with human Tregs promotes LSCs migration, proliferation, and stemness maintenance. Furthermore, amphiregulin (AREG), secreted by Tregs, was crucial to their therapeutic effects, as AREG-/- Tregs resulted in diminished efficacy on LSCD mice compared to wild-type Tregs. These findings highlighted Tregs as a promising treatment for LSCD, enhancing LSC function partially via AREG production.
2025-04-16 | GSE291731 | GEO
Project description:snRNAseq of human limbus tissue
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Project description:Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LEC). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos enhanced wound healing in cultured N-LSC and increased proliferation rate in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos treated LSC reduced keratocyte markers ALDH3A1 and lumican, and increased MSC markers CD73, CD90 and CD105 vs. control LSC. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.
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Project description:Limbal stem cells (LSCs), known as corneal epithelial stem cells, are located at the basal epithelial layer of the corneal limbus and serve an important function in maintaining the homeostasis of the corneal epithelium. Several putative molecular markers of LSCs have been previously identified. However, the specificity of these markers remains largely controversial. To address this gap in the current understanding of LSCs, we performed a transcriptome profiling of heterogeneous corneal limbal basal cells using single-cell transcriptomics technology to identify LSCs and their exclusive markers. We isolated limbal basal cells from two young donors, constructed scRNA-seq libraries, generated RNA-sequencing data using the 10x Genomics platform, and then finally obtained the transcriptome of about 18,000 individual single-cells using Cell ranger (https://10xgenomics.com). Next, we performed quality control, filtering and data integration using Seurat package (https://satijalab.org/seurat), about 16,400 cells were retained for further downstream analysis such as dimensional reduction, unsupervised clustering, and Differentially expressed gene selection, trajectory analysis and visualization. We identified 11 unique clusters of cells assigned to a putative cell type based on published known biomarkers for differentiation, proliferation and putative epithelial stem cells. As a results, we found 2 Terminally Differentiated Cell (TDC), 3 Post-Mitotic Cell (PMC), 1 Transient Amplifying Cell (TAC), 2 Limbal Progenitor Cell (LPC), Putative Limbal Stem Cell (LSC) and 2 Melanocyte (MC) sub-cell type clusters. Furthermore, we confirmed the trajectory order of LSC differentiation for 9 clusters using Pseudotemporal and Functional PCA analysis. Lastly, we validated each assigned cell subtypes and determined their location in human corneal limbus tissue with 9 markers using RNAscope We were able to reveal the heterogeneity of corneal limbal basal epithelium by defining novel dynamic trajectories for cell types in a pseudotemporal manner. This approach allowed us to identify the distinct clusters of LSCs and progenitors with exclusively expressed markers, and might apply for translational research on regenerating a normal corneal epithelium and restoring vision.
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Project description:Limbal stem cells including epithelial and stromal/Mesenchymal stem cells that contribute to sustained corneal homeostasis, maintain their ability to act as self-renewal progenitor cells by virtue of their limbal niche and intercellular communication. Extracellular vehicles (EVs), including exosomes (Exos), are important paracrine mediators through their cargo transfer for intercellular communication in various stem cell niches. Previously we have shown the differential cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos, in limbal epithelial cells (LEC) in normal (N) and diabetic (DM) limbal niche. In the present study, to have a comprehensive knowledge of reciprocal LEC-LSC crosstalk, we investigated the proteomics and miRNA profile of exosomes derived from LEC and their regulatory roles in LSC in N and DM limbus. Our study showed wound healing and proliferation rates in primary N-LSC were significantly enhanced upon treatment by normal LEC-derived Exos (N-Exos), but not by diabetic Exos (DM-Exos). Further, N-Exos treated LSC showed downregulation of keratocyte markers, ALDH3A1 and lumican, but not keratocan, and upregulation of MSC markers, CD105, CD90, and CD73 compared to the DM-Exos treated LSC. Using next generation sequencing (NGS) and proteomics analysis, we revealed some miRNAs and proteins in the Exos that affect the cellular crosstalk and the function of the cornea. We also documented differences in DM vs. normal LEC-derived Exo’s cargos. Overall, DM-Exos have less effect on LSC proliferation, wound healing, and stem cell maintenance than N-Exos, likely by transferring their cargo proteins and/or regulatory miRNAs targeting cell cycle, ERK/MAPK, TGF-β, EMT, PI3K-Akt-mTOR signaling molecules. This suggests that the small RNA and protein cargo differences in DM vs. N LEC-derived Exos could contribute to the disease state. Our study revealed a complex contribution of Exos to health and diabetic state of corneal homeostasis and suggests the potential of EV therapeutics for diabetic cornea regenerative medicine
2023-10-23 | PXD040918 | Pride
Project description:Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintaining ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.
2023-12-04 | GSE248934 | GEO
Project description:Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintaining ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.
2023-12-04 | GSE248928 | GEO
Project description:Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintaining ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.
2023-12-04 | GSE248926 | GEO
Project description:Simple limbal epithelial transplantation (SLET) is a novel in vivo limbal stem cells (LSCs) amplification technique used to reconstruct the ocular surface in¬¬¬ limbal stem cell deficiency (LSCD). However, SLET is limited to two-dimensional (2D) culture systems, and the tissue pieces are easily lost. The developments in hydrogel technology have provided the possibility of three-dimensional (3D) amplification of stem cells. In this study, an injectable photocrosslinkable hydrogel was developed based on porous gelatin methacryloyl (GelMA)/silk fibroin glycidyl methacrylate (SilMA), allowing the 3D culture of LSCs with enhanced expansion efficiency and simplified serial cell culture operations to construct an ocular surface with a uniform cell distribution. The porous GelMA/SilMA hydrogels demonstrated excellent transparency and good adhesion. More importantly, this hydrogel maintained the viability and supported the adhesion of the encapsulated rabbit LSCs and promoted their migration in vitro in 3D culture environments. Furthermore, the immunofluorescence staining results showed positive expression of CK3, CK12, P63 and CK14 in differentiated cells. The above results demonstrated that this porous GelMA/SilMA hydrogel 3D culture of LSCs is beneficial for maintaining the phenotype of stem cells and promoting their differentiation into corneal epithelial cells. These findings provide valuable insights into stem cell therapies and regenerative medicine for ocular surface reconstruction.
2023-11-29 | PXD047365 |
Project description:To date, there is no specific marker for limbal epithelial stem cells. The identification of a marker that is expressed in the limbal epithelium but not in the cornea or conjunctiva epithelium has been a growing need. To search for limbal-specific marker(s), we performed preferential gene profiling in the limbus in direct comparison to that in the cornea and conjunctiva using microarray technique.
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