Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty
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PROVIDER: PRJNA726726 | ENA |
REPOSITORIES: ENA
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Project description:Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in mammals (rabbits) and non-human primates (monkeys). We injected cryopreserved hESC-derived CECs in rabbits and monkeys either immediately after removing 8 mm of the central portion of the CE or a few days later when corneal edema developed. All clinical models developed deturgesced and clear corneas 2-3 weeks following the CEC injection and remained comparable to the CE of the untreated eye. Confocal scanning microscopy confirmed an intact structure of hexagonal/polygonal cells and immunohistochemical analysis illustrated a monolayer expressing barrier and pump function proteins in the regenerated CE. The necropsy examination confirmed no remarkable change in multiple tissues examined for teratoma formation. In conclusion, our data demonstrate the efficacy of cryopreserved hESC-derived CECs to form a functional CE on the denuded Descemet’s membrane.
2021-09-29 | GSE173692 | GEO
Project description:Genome wide DNA methylation profiling of normal human corneal endothelium and human corneal endothelium from FECD cases. The Illumina Infinium MethylationEPIC 850K BeadChip was used to obtain DNA methylation profiles across approximately 850,000 CpGs in genomic DNA from human corneal endothelium samples. Samples included 11 non-FECD donors, 17 FECD cases.
2023-02-15 | GSE198917 | GEO
Project description:The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity, and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelium (evHCEnC) with that of primary HCEnC and HCEnC lines, and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome datasets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level primary HCEnC most closely resemble evHCEC and thus represent a viable therapeutic option for managing corneal endothelial dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of the potential clinical utility of the cultured HCEnC for the management of corneal endothelial cell failure. Transcriptomes from ex vivo corneal endothelium, primary cultures and three cell lines were compared. Three samples of each endothelial cell group were submitted for RNA sequencing for a total of 15 samples. The transcriptome for the ex vivo corneal endothelium was used as the reference (i.e., proxy for in vivo corneal endothelium). Transcript abundances for a subset of genes associated with corneal endothelial cell function or disease were validated with qPCR and western blot. Samples of ex vivo endothelium used for validation were independent replicates not used for RNA-sequencing.
2015-09-04 | E-GEOD-65991 | biostudies-arrayexpress
Project description: Not available
| S-EPMC8452521 | biostudies-literature
Project description:The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity, and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelium (evHCEnC) with that of primary HCEnC and HCEnC lines, and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome datasets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level primary HCEnC most closely resemble evHCEC and thus represent a viable therapeutic option for managing corneal endothelial dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of the potential clinical utility of the cultured HCEnC for the management of corneal endothelial cell failure.
2015-09-04 | GSE65991 | GEO
Project description:Defining the normal and age-dependent HCEnC transcriptome will further refine our understanding of the functional roles that the endothelium plays in the cornea and will provide a basis upon which to compare transcriptomes of normal and dystrophic endothelium for the subsequent development of gene-targeted therapies. We used microarrays to comprehensively characterize human corneal endothelial cell (HCEnC) gene expression, age-dependent differential gene expression and to identify expressed genes mapped to chromosomal loci associated with the corneal endothelial dystrophies PPCD1, FECD4 and XECD
2014-11-12 | GSE58315 | GEO
Project description:PURPOSE: To compare the gene expression profiles of normal human corneal endothelium with Fuchs' corneal endothelium, by using serial analysis of gene expression (SAGE). METHODS: Three pairs of normal human corneas were obtained from eye banks. Thirteen bisected Fuchs' corneal buttons were processed at the time of corneal transplantation. The endothelia of normal and Fuchs'-affected corneas were stripped, and total RNA was isolated. Serial analysis of gene expression (SAGE) was performed to identify and quantify gene transcripts. Genes over- and underexpressed by Fuchs' endothelium were limited to P < 0.01 by the method of Audic and Claverie. RESULTS: A total of 19,136 tags were identified with 9,530 from normal and 9,606 from Fuchs' endothelium. The expression of 18 transcripts was upregulated, and 36 transcripts were downregulated in Fuchs' endothelium compared with normal tissue. Upregulated transcripts included serum amyloid A1 and A2, metallothionein, and apolipoprotein D. Of the downregulated transcripts, 26 matched known genes, 3 matched expressed sequence tags (ESTs), and 7 were unknown to current databases. One downregulated transcript involved a newly reported bicarbonate transporter. Decreased transcripts related to antioxidants and proteins conferring protection against toxic stress were noted in Fuchs' versus normal endothelium including nuclear ferritin, glutathione S-transferase-pi, and heat shock 70-kDa protein. Nine different SAGE tags matching mitochondrial sequences accounted for 25% of the ESTs that were decreased in Fuchs' endothelium. CONCLUSIONS: SAGE analysis comparing normal to Fuchs' endothelium demonstrates diminished expression of mitochondrial, pump function, and antiapoptotic cell defense genes. A complete report of gene expression profiles of normal human corneal endothelium can be found in PMID 12583793. Keywords: other
2003-07-16 | GSE505 | GEO
Project description:The regenerative capacity of corneal endothelial cells (CECs) differs between species; in bigger mammals such as humans and sheep, CECs are arrested in a non-proliferative state. Damage to these cells can compromise its function leading to corneal opacity and impaired vision. Corneal transplantation is the current treatment for the recovery of a clear eyesight, but the donor tissue demand is higher than the offer and there is the need to develop novel treatment modalities. In contrast, rabbit CECs retain a high proliferative profile and have the capacity to repopulate the endothelium upon injury. There is currently a lack of fundamental knowledge to explain the difference in the proliferation capacity of these cells across species. Gaining information on the transcriptomic differences across species could allow the identification of crucial proliferation drivers of CEC proliferation to develop novel regenerative medicine therapies. In this study we have analyzed at the transcriptomic level corneal endothelial samples originating from human, sheep, and rabbit. To understand the differences across the CECs proliferation capacity in each species, we have generated an automated pipeline for the analysis of pathways with different activity. Our results revealed that 52 pathways had commonly different activity when comparing species with non-proliferative endothelium (human and sheep) to species with proliferative endothelium (rabbit).
2023-01-20 | GSE222458 | GEO
Project description:Fuchs’ endothelial corneal dystrophy (FECD) is a progressive vision impairing disease caused by thickening of Descemet’s membrane and gradual degeneration and loss of corneal endothelial cells. The aim of this study was to identify differentially expressed genes between FECD-affected and unaffected corneal endothelium to gain insight into the pathophysiological mechanisms underlying this disease. Microarray gene expression analysis was performed on total RNA from FECD-affected and unaffected corneal endothelium-Descemet’s membrane (CE-DM) specimens using the Illumina HumanHT-12 v3.0 expression array. RNA from pools of FECD-affected (n=3 per pool) and individual unaffected (n=3) specimens was used for comparison. Altered expression of a sub-set of differentially expressed genes was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in independent specimens. Bioinformatics analysis was performed using InnateDB to reveal functional relationships among the differentially expressed genes and molecular pathways involved in the disease. A total of 16,513 genes were found expressed in the corneal endothelium of which 142 genes were differentially expressed between FECD-affected and unaffected endothelium (log2 fold-change ≥1.5, corrected p-value ≤0.05). Most of the genes were up-regulated (126) and a small proportion down-regulated (16) in affected corneal endothelium. Of the twelve genes prioritised for validation, differential expression of 10 genes, including those ranked 57th and 81st by significance validated by qRT-PCR (8 up-regulated and 2 downregulated, corrected p ≤0.05), one gene showed a trend for up-regulation in affected endothelium, consistent with the microarray analysis and another was up-regulated in an independent study indicating robustness of the differential expression dataset. Bioinformatic analysis revealed significant over-representation of differentially expressed genes in extracellular matrix reorganisation, cellular remodelling, immune response, and inflammation. Network analysis showed functional inter-relatedness of the majority of the dysregulated genes and revealed known direct functional relationships between 20 of the genes; many of these genes have roles in macrophage differentiation, phagocytosis and inflammation. This is the second report of microarray gene expression analysis in FECD. This study revealed a set of highly dysregulated genes in the corneal endothelium in FECD. More than a third of the dysregulated genes in the disease have been discovered for the first time and thus are novel. The dysregulated genes strongly suggest the presence of phagocytic cells, most likely immune cells, and inflammation in corneal endothelium in the disease. This study provides a molecular framework for delineating the mechanisms underlying these cellular processes in FECD.
2021-10-15 | GSE171830 | GEO
Project description:Considerable interest has been generated for the development through cell-tissue engineering of suitable corneal endothelial graft alternatives, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as DescemetM-bM-^@M-^Ys Stripping Endothelial Keratoplasty (DSEK) and DescemetM-bM-^@M-^Ys Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types were also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. A total of 20 donor corneas consisting of 10 single donor corneas and 5 paired donor corneas were used in this study. Donor age ranged from 19 - 76. This RNA-seq study included 15 pooled corneas (5 each) used form CEC old, CEC young and stroma samples.
2013-05-23 | E-GEOD-44064 | biostudies-arrayexpress