Novel method to examine hepatocyte-specific gene expression in a functional coculture system.
To mimic native tissue function, coculture systems are an extremely useful model. In many cases, differentiated functions can be maintained only through the interactions of various cell types. Therefore, methods for examining the interactions between cocultured cells are necessary. The assessment of cell-to-cell cross-talk at the level of gene expression is one such method to examine interactions between different cell types. However, it is generally difficult to determine the gene expression of specific cell types in coculture without first separating cell populations. To overcome these obstacles, we have established a novel method to determine gene expression levels of a targeted cell population in coculture, using species-specific primers. With this approach, we were able to determine hepatocyte-specific gene expression of Fao cells (a rat hepatocyte cell line) in culture with human umbilical vein endothelial cells (HUVECs). Expression of both albumin and apolipoprotein A-I (apoA-I) increased time dependently for 10 days and maintained significantly higher expression in the coculture system as compared with isolated Fao cells. This indicates that hepatocyte function increased gradually in our coculture system and could be maintained long-term, suggesting that the construction of mature cell-to-cell communication between the two cell lines required a considerable amount of time. The expression of HNF-4 and HNF-1alpha, which are liver-enriched transcription factors, did not differ between the monolayer and cocultured Fao cells, suggesting that expression of HNF-4 and HNF-1alpha was not responsible for the increased expression albumin and apoA-I. Our findings suggest that this novel method for the detection of gene expression of targeted cell populations can be a useful tool in determining the molecular mechanisms that regulate communication between different cell types.
Kurosawa
Yasunori
Y
Bionic Materials Technology Group, Biomaterials Center, National Institute for Materials Science, Tsukuba, Ibaraki, Japan.
Taniguchi
Akiyoshi
A
Okano
Teruo
T
D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis; D002450:Cell Communication / Q000502:physiology*; D002460:Cell Line; D018920:Coculture Techniques / Q000379:methods; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D005821:Genetic Techniques; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000096:biosynthesis; D051557:Hepatocyte Nuclear Factor 4 / Q000096:biosynthesis; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D006801:Humans; D051381:Rats; D012709:Serum Albumin / Q000096:biosynthesis; D013045:Species Specificity; D014471:Umbilical Veins / Q000166:cytology / Q000502:physiology*
D016632:Apolipoprotein A-I; D051538:Hepatocyte Nuclear Factor 1-alpha; D051557:Hepatocyte Nuclear Factor 4; C496489:Hnf1a protein, rat; D012709:Serum Albumin