biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsZhenzhi ChngRNA-seq of coding RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-44064Considerable 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.biostudies-arrayexpressGrowth Protocol - Research corneas were preserved and transported in Optisol-GS at 4M-0C, and were used within 13 days from preservation. Culture of Human Corneal Endothelial Cells: CEC-DM layer was digested enzymatically in collagenase A (2 mg/ml) for at least 2 hours and up to 6 hours. This allowed full detachment of the CECs from the DM, which tended to conglomerate into tightly-packed CEC clusters. The CEC clusters were rinsed once in PBS and further dissociated in TrypLE Express (TE) for 5 minutes. Cell pellets collected after a mild centrifugation (800 g for 5 minutes) were plated in culture-ware coated with FNC coating mixture. Isolated cells were left to adhere overnight in a stabilization medium made up of Human Endothelial-SFM supplemented with 5% FBS and 1x anti-biotic/anti-mycotic. Adhered hCECs were then cultured in F99 medium containing HamM-bM-^@M-^Ys F12 and M199, mixed in a 1:1 ratio, supplemented with 5% FBS, 20 M-NM-<g/ml ascorbic acid, 1x Insulin-Transferrin-Selenium, 1x anti-biotic/anti-mycotic and 10 ng/ml bFGF. When the cultured cells reached 80-90% confluence, they were re-exposed to the stabilization medium for at least one week before passaging. The inclusion of this final step enhanced the general morphology of cultured hCECs (unpublished observation; manuscript in preparation). Confluent hCECs were passaged using TE, and seeded onto FNC-coated culture ware at a plating density of approximately 10,000 cells per cm2 for subsequent passage. All incubation and cultivation of hCECs were carried out in a humidified incubator at 37M-0C containing 5% CO2 unless otherwise stated. Fresh media were replenished every two days.Library Construction - For direct RNA extraction of donor tissues, isolated CEC-DM and corresponding corneal stroma button, punched out from the donor cornea tissue using an 8.0-mm diameter trephine, were rinsed once in PBS and placed directly into 1ml Trizol reagent. It should be noted that although most of the corneal epithelium spontaneously sloughed off the cornea surface during the transport and process of the cornea, remnant cells of the basal corneal epithelium might still be present on the corneal stroma button used for RNA extraction. Tissues were homogenized using a hand-held homogenizer before addition of 200ul of chloroform. After vigorous shaking, samples were spun at 13000 rpm for 15min at 4M-0C. The upper phase, containing total RNA, was transferred to a new tube containing an equal volume of 70% ethanol. The resulting solution was transferred into a QIAGEN RNeasy column and procedures were performed as per manufacturerM-bM-^@M-^Ys protocol with a DNAse digestion step incorporated. Libraries were prepared using an AB Demonstrated Protocol similar to the one reported in Tang et al 2009. Starting amount of RNA was 100pg. SOLiD library preparation kit was used for library generation.Sample Treatment - Research corneas were incubated in three washes of antibiotic/antimycotic solution in PBS, 15 minutes each. Corneoscleral rims were placed endothelial-side-up on a disposable cornea vacuum punch (Ripon, England), and mildly stabilized by the vacuum suction created. A brief 30 seconds treatment with Trypan Blue solution (0.1%) was used to delineate SchwalbeM-bM-^@M-^Ys line. The CEC-DM layer was carefully stripped off, approximately 1 mm anterior to the SchwalbeM-bM-^@M-^Ys line (away from the trabecular meshwork) from the posterior stroma under the dissecting microscope (Nikon, Japan).OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesJodhbir MehtaWishva HerathGary PehPaul RobsonAlan ColmanZhenzhi ChngData Transformation - Reads were alighned with ABI Bioscope version 1.3. Reads were first filtered for rRNA and tRNA samples and then aligned to the human genome (hg19.) The uniquely aligned read counts (wt.merge.score.zone=4) were used for the calculation of RPKM and RPM values based on RefSeq annotation. Genome_build: hg19 Supplementary_files_format_and_content: RPKM values in text format.falseHigh throughput gene expression analysis identifies reliable expression markers of human corneal endothelial cells.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.2013-05-23T00:00:00Z2023-08-30T04:54:20.253Z2022-03-07T00:34:59.337ZE-GEOD-44064SRP018405EFO_0003738