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SRX879494: GSM1612316: pHCEnC_60; Homo sapiens; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2500) run: 19M spots, 948.3M bases, 421.6Mb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Transcriptomic analysis of cultured corneal endothelial cells as a validation for their use in cell-replacement therapy
show Abstracthide Abstract
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. Overall design: 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.
Sample: pHCEnC_60
SAMN03352057 • SRS846899 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: TriReagent extraction of total RNA was performed according to the manufacturer's instructions. Total RNA was further purified using the Qiagen RNeasy Clean-Up Kit. Enrichment for poly(A) RNAs was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module, followed by library preparation using the PrepX Complete ILM DNA Library Kit. Libraries were prepared as per the manufacturer's instructions.
Experiment attributes:
GEO Accession: GSM1612316
Links:
External link:
Runs: 1 run, 19M spots, 948.3M bases, 421.6Mb
Run# of Spots# of BasesSizePublished
SRR180659018,966,759948.3M421.6Mb2015-09-07

ID:
1261458

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