A Genomic Comparison of in vivo and in vitro Brain Microvascular Endothelial Cells

Abstract

The blood-brain barrier (BBB) is composed of uniquely differentiated brain microvascular endothelial cells (BMEC). Often, it is of interest to replicate these attributes in the form of an in vitro model, and such models are widely used in the research community. However, the BMEC used to create in vitro BBB models de-differentiate in culture and lose many specialized characteristics. These changes are poorly understood at a molecular level, and little is known regarding the consequences of removing BMEC from their local in vivo microenvironment. To address these issues, suppression subtractive hybridization (SSH) was used to identify 25 gene transcripts that were differentially expressed between in vivo and in vitro BMEC. Genes affected included those involved in angiogenesis, transport and neurogenesis, and real-time quantitative polymerase chain reaction (qPCR) verified transcripts were primarily and significantly downregulated. Since this quantitative gene panel represented those BMEC characteristics lost upon culture, we used it to assess how culture manipulation, specifically BMEC purification and barrier induction by hydrocortisone, influenced the quality of in vitro models. Puromycin purification of BMEC elicited minimal differences compared with untreated BMEC, as assessed by qPCR. In contrast, qPCR-based gene panel analysis after induction with hydrocortisone indicated a modest shift of 10 of the 23 genes toward a more 'in vivo-like' gene expression profile, which correlated with improved barrier phenotype. Genomic analysis of BMEC de-differentiation in culture has thus yielded a functionally diverse set of genes useful for comparing the in vitro and in vivo BBB.

Paracellular Tightness and Claudin-5 Expression is Increased in the BCEC/Astrocyte Blood–Brain Barrier Model by Increasing Media Buffer Capacity During Growth

“…This was partly caused by poor recognition of influx transporter substrates in BBB models, which to a wide extent was attributed to paracellular leakage leading to failure to identify active influx. Poor transporter expression has been supported by other reports showing an incomplete transporter expression pattern in in vitro BBB models (42,43). These studies have been performed in model systems different from ours.…”

Single-cell transcriptomics of the human placenta: inferring the cell communication network of the maternal-fetal interface

“…The transcriptome of these cells exhibits most of the known markers of decidualization. A potential limitation to these data is that in vitro cell cultures could lack some gene expression that arises due to the in vivo context or by interaction with adjacent cell types (e.g., Calabria and Shusta 2008;Gellersen and Brosens 2014;LoVerso et al 2015). Future in vivo decidual cell transcriptomes will provide an answer to this question.…”

Culture-Induced Changes in Blood—Brain Barrier Transcriptome: Implications for Amino-Acid Transporters in vivo

“…Given that in vitro the proliferative cellular functions likely take precedence over differentiative ones (e.g., transendothelial transport), examining culture-induced changes may provide clues as to which transporters are central for each function. In this regard, Calabria and Shusta (2008) hypothesized a general decrease in critical BBB transendothelial vesicular transporter expression as a result of culture. We, too, identified decreasing expressions for particular Slc family members in cultured MBMECs (Figures 3 and 4; Supplementary Tables 2 and 3).…”