RNA-Seq reveals shortcomings of experimentation models

Microglia are resident myeloid cells in the CNS that are activated by infection, neuronal injury, and inflammation. Established BV2 microglial cell lines have been the primary in vitro models used to study neuroinflammation for more than a decade because they reduce the requirement of continuously maintaining cell preparations and animal experimentation models. However, doubt has recently been raised regarding the value of BV2 cell lines as a model system.

Researchers at Hanyang University, Korea used triplicate RNA sequencing (RNA-seq) to investigate the molecular signature of primary and BV2 microglial cell lines using two transcriptomic techniques: global transcriptomic biological triplicate RNA-seq and quantitative real-time PCR. They analyzed differentially expressed genes (DEGs) to identify transcription factor (TF) motifs (-950 to +50 bp of the 5′ upstream promoters) and epigenetic mechanisms.

Sequencing assessment and quality evaluation revealed that primary microglia have a distinct transcriptomic signature and express a unique cluster of transcripts in response to lipopolysaccharide. This microglial signature was not observed in BV2 microglial cell lines. Importantly, the researchers observed that previously unidentified TFs (i.e., IRF2, IRF5, IRF8, STAT1, STAT2, and STAT5A) and the epigenetic regulators KDM1A, NSD3, and SETDB2 were significantly and selectively expressed in primary microglia (PM). Although transcriptomic alterations known to occur in BV2 microglial cell lines were identified in PM, they also observed several novel transcriptomic alterations in PM that are not frequently observed in BV2 microglial cell lines.

RNA-seq analyses reveals LPS-induced inflammatory response-related genes
and their downstream effectors in BV2 cell lines and PM.


a A heat map representing the top 150 inflammatory genes that were up-regulated by 2- and 4-h LPS stimulation in BV2 cell lines and PM (P ≤ 0.01, and log2 fold change ≥1.5). Each row shows the relative expression level for a single gene, and each column shows the expression level of a single sample. Biological replicates (n = 3) for each condition were performed. b, c Pie chart displaying the number of up or down-regulated genes at 4-h LPS stimulation in BV2 cell lines and PM. d The area of overlap indicates the number of unique or shared up-regulated genes after 4 h of LPS stimulation in BV2 cell lines and PM. e, f Gene ontology analysis of the functional annotations that were associated with the top 150 up-regulated genes at 4 h after LPS stimulation in the BV2 cell lines and PM.

Collectively, these unprecedented findings demonstrate that established BV2 microglial cell lines are probably a poor representation of PM.

Das A, Kim SH, Arifuzzaman S, Yoon T, Chai JC, Lee YS, Park KS, Jung KH, Chai YG. (2016) Transcriptome sequencing reveals that LPS-triggered transcriptional responses in established microglia BV2 cell lines are poorly representative of primary microglia. J Neuroinflammation 13(1):182. [article]

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