Differentiating human embryonic stem cells into corneal epithelial-like cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE70152 | GEO | 2015/06/24
SECONDARY ACCESSION(S): PRJNA287750
REPOSITORIES: GEO
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Project description:Transplantation of ex vivo expanded limbal stem cells (LSC) is the main treatment for limbal stem cell deficiency though the clinical problem of donor tissues shortage. Recently, as the development of tissue engineering, embryonic stem cells (ESC) derived corneal epithelial-like cells (ESC-CEC) has become a new direction to this issue.Our group successfully induced ESC into corneal epithelial-like cells, and in the present study we explored various aspects of physiological properties of ESC-CEC. The experiment included three samples: hES, the human embryonic stem cell line H1, RA_SB, the corneal epithelial-like cells derived from hES by differentiation with RA and SB, epithelial_cell, the primary human limbal stem cells from cadaver eyes. hES, the human embryonic stem cell line H1, RA_SB, the corneal epithelial-like cells derived from hES by differentiation with RA and SB, epithelial_cell, the primary human limbal stem cells from cadaver eyes.
2015-06-24 | E-GEOD-70152 | biostudies-arrayexpress
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: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:PURPOSE. Limbal stem/progenitor cells (LSC) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. Previous study revealed that Paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CEC). However, the molecular events downstream of PAX6 remain largely unknown. In this study, we aimed to clarify the regulation network of PAX6 in driving CEC differentiation. METHODS. An air-liquid culture system was used to differentiate LSC into mature CEC. Specific targeting PAX6 short-hairpin shRNAs were used to knock down PAX6 in LSC. RNA-seq was used to analyze shPAX6-transfected CEC and CEC differentiation-associated genes to identify the potential downstream targets of PAX6. RNA-seq analysis, quantitative real-time PCR, and immunofluorescence staining were performed to clarify the function of WNK lysine deficient protein kinase 2 (WNK2), a downstream target of PAX6, and its relationship with corneal diseases. RESULTS. WNK2 expression increased during CEC differentiation and decreased upon PAX6 depletion. The distribution of WNK2 was specifically limited to the central corneal epithelium and suprabasal layer of the limbus. Knockdown of WNK2 impaired the expression of CEC-specific markers (KRT12, ALDH3A1, and CLU), disrupted the corneal differentiation process, and activated the terms of keratinization, inflammation, and cell proliferation, consistent with PAX6-depleted CEC and published microbial keratitis. Thus, aberrant expression of WNK2 was linked to corneal ulcers. CONCLUSIONS. As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.
2024-10-30 | GSE272034 | 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 | GSE248934 | GEO
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.
2021-01-14 | GSE153515 | GEO
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:Transplantation of amniotic membrane-expanded limbal epithelium (AMLE) in place of donor tissue grafts results in significantly improved outcomes for patients suffering from severe limbal stem cell deficiency; however the reasons for such superior results are unclear. The purpose of this study was to identify transcriptional gene profiles specific to AMLE and donor central corneal epithelium (CE), which may contribute to the divergent clinical outcomes observed following transplant. Limbal fibroblasts which underlie the epithelium and secrete extracellular matrix proteins following injury/surgery were also profiled. Using cell culture, immunofluorescence, microarray gene expression profiling and qRT-PCR validation; this study aims to identify enriched biological processes and pathways which characterise AMLE and CE tissues. We hope the study outcomes will shed light onto the factors which contribute to provide the improved clinical outcomes associated with AMLE transplantation. Gene expression profiling of three central corneal buttons, three amniotic membrane-expanded limbal epithelial cultures and three limbal fibroblast cultures
2014-10-02 | E-GEOD-56421 | biostudies-arrayexpress
Project description:Limbal vs. corneal epithelial basal cell gene expression patterns were identified and compared. Experiment Overall Design: 8 limbal and 8 corneal epithelial basal cells samples from 8 mice were dissected and mRNAs were isolated and amplified for microarray analysis
2007-11-22 | E-GEOD-4098 | biostudies-arrayexpress
Project description:Stem cells (SCs) are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. It was several decades ago, when limbal epithelial SCs (LSCs) that regenerate the corneal epithelium were among the first sporadic, quiescent SCs ever discovered. However, LSC dynamics, heterogeneity and genetic signature are largely unknown. Moreover, recent accumulating evidence strongly suggested that epithelial SCs are actually abundant, frequently dividing cells that display stochastic behavior. In this work, we combined single-cell RNA sequencing and advanced quantitative lineage tracing for in-depth analysis of the murine limbal epithelium. The generated data provides an atlas of cell states of the entire corneal epithelial lineage and reveales the co-existence of two novel LSC populations that reside in separate and well-defined sub-compartments. In the “outer” limbus, we discovered a primitive widespread population of quiescent LSCs (qLSCs) that uniformly express Krt15/Gpha2/Ifitm3/Cd63 proteins that serve as SC reservoire and in boundary formation. In the “inner” peri-corneal limbus, we identified prevalent active LSCs (aLSCs) that express Krt15-GFP/Atf3/Mt1-2/Socs3 and maintain homeostasis. We propose that these SC populations are abundant, follow stochastic rules and neutral drift dynamics. Notably, we provide evidence that T cells serve as niche cells for qLSCs, regulating quiescence and wound response. Taken together, we propose that divergent regenerative strategies are tailored to properly support tissue specific physiological constraints.
2021-03-02 | GSE167992 | GEO