Co-cultivation of hepatocytes with stromal cells (i.e. fibroblasts) provides for a robust in vitro hepatic tissue; however, the mechanisms underlying the ‘co-culture effect' have yet to be fully elucidated. We have developed and characterized a functional genomic approach utilizing DNA microarrays to identify stromal genes that may modulate hepatocyte functions in vitro. We co-cultivated rat hepatocytes with closely related murine fibroblast cell types (3T3-J2, NIH-3T3, mouse embryonic fibroblasts) to allow their classification as high, medium, or low inducers of hepatic functions. These functional responses were then correlated with fibroblast gene expression profiles obtained using Affymetrix GeneChips. Further data analysis allowed us to narrow down the search space of potential mediators of the co-culture effect to a small list of candidate genes (17) that may mediate hepatic functions. To validate our approach, we confirmed the localization of N-cadherin (cell surface protein) and beta-catenin at heterotypic (fibroblast/hepatocyte) junctions using immunofluorescence, providing our first evidence of functional communication between the cell types. Experiments to elucidate these candidates' functional role in hepatic cocultures are underway in our laboratory. In the future, identification of a critical molecule(s) that mediate stabilization of the hepatic phenotype may have implications in hepatic tissue engineering, stem cell biology, and pathophysiology of liver disease.   Khetani et al, 2004.

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Micropatterned Co-Cultures control cell-cell interaction (left). Functional genomic approach elucidates candidate, stromal-derived genes involved in co-culture (right).