RF/6A Chorioretinal Cells do not Display Key Endothelial Phenotypes
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA453564 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7063560.fastq.gz | Fastqsanger.gz | |||
| SRR7063561.fastq.gz | Fastqsanger.gz | |||
| SRR7063562.fastq.gz | Fastqsanger.gz | |||
| SRR7063563.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: The RF/6A chorioretinal cell line originally isolated from a fetal rhesus macaque in 1968 is widely used to model human retinal and choroidal endothelial cells in cell culture. We sought to determine the extent to which this line possesses endothelial cell-specific characteristics. Methods: RF/6A cells obtained from American Type Culture Collection were subjected to next-generation RNA sequencing. Transcriptomic-based cell type profiling was conducted using xCell. Validation of endothelial cell-specific marker expression was conducted by qRT-PCR and western blotting. Functional assays of acetylated low density lipoprotein uptake, TNF-?-induced adhesion molecule expression, shear stress alignment, and endothelial tube formation were assessed. Results: RF/6A transcriptomic profiles obtained de novo or from a publically available repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers (PECAM1, CDH5, KDR, VWF, ICAM2, ENG, or TEK) were very low or undetectable in RF/6A compared to primary human umbilical vein endothelial cells. Compared to primary endothelial cells, RF/6A were unable to uptake acetylated LDL, exhibit induction of E-selectin in response to TNF-? treatment, align in the direction of shear stress, or form regular capillary-like tubes in Matrigel. Conclusions: RF/6A do not exhibit key endothelial cell characteristics or behaviors. Therefore, caution should be employed in designing and interpreting studies using these cells as surrogates for choroidal or retinal endothelial cells.
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Project description:PurposeThe misuse of inauthentic cell lines is widely recognized as a major threat to the integrity of biomedical science. Whereas the majority of efforts to address this have focused on DNA profiling, we sought to anatomically, transcriptionally, and functionally authenticate the RF/6A chorioretinal cell line, which is widely used as an endothelial cell line to model retinal and choroidal angiogenesis.MethodsMultiple vials of RF/6A cells obtained from different commercial distributors were studied to validate their genetic, transcriptomic, anatomic, and functional fidelity to bona fide endothelial cells.ResultsTranscriptomic profiles of RF/6A cells obtained either de novo or from a public data repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers were very low or undetectable in RF/6A compared to primary human endothelial cells. Importantly, RF/6A cells also did not display functional characteristics of endothelial cells such as uptake of acetylated LDL, expression of E-selectin in response to TNF-α exposure, alignment in the direction of shear stress, and AKT and ERK1/2 phosphorylation following VEGFA stimulation.ConclusionsMultiple independent sources of RF/6A do not exhibit key endothelial cell phenotypes. Therefore, these cells appear unsuitable as surrogates for choroidal or retinal endothelial cells. Further, cell line authentication methods should extend beyond genomic profiling to include anatomic, transcriptional, and functional assessments.
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