The development of a hepatocyte culture system stably expressing a spectrum of differentiated functions is a highly desired objective for pivotal applications in the fields of toxicology and bioartificial liver systems. Various approaches have been explored to prevent or delay phenotypic alterations attributable to changes in liver specific gene expression during hepatocyte isolation and cell culture processes. While a number of advances have been made in engineering substrates and media, a system for hepatocyte cell culture with specific cell functions at levels comparable to those observed in vivo does not yet exist.

MicroRNAs (miRNAs) are endogenously produced 21 to 23 nucleotide non-coding-RNAs that regulate expression of target genes via partial sequence complementarity. Roles for several miRNAs' have been demonstrated that encompass development, apoptosis, growth, fat metabolism and differentiation in diverse organism [1]. For example, Esau, et. al have shown that miR-122 regulates lipid metabolism in the adult liver in vivo [2]. However, regulation of microRNAs as a driving force for loss of differentiated functions in cultured hepatocytes has yet to be investigated.

In order to test the hypothesis that alterations of miRNAs play a significant role in the de-differentiation of hepatocytes during the first days of culture, we extracted the miRNAs from freshly isolated hepatocytes versus those cultured on a single layer of collagen for 4 days and profiled them by microarray analysis. Among the 238 rat miRNA probes, several miRNAs were consistently altered in their expression, including miR-122, which was induced approximately 3.5-fold in cultured vs. freshly isolated hepatocytes. Important targets of miR-122 were predicted computationally, establishing a biochemical network for miRNA regulation of primary rat hepatocytes. These include several liver-specific transcription factor genes. The mRNA levels of miR-122 and several of its targets were monitored and validated using TaqMan RT-PCR. These results demonstrate that microRNAs such as miR-122 are altered in their expression during early hepatocyte culture and suggest that they may play a role in coordinating gene expression programs.

In order to explore a possible causative role for miR-122 on hepatocyte differentiation status, we are currently manipulating its expression using miR-122 mimics and antagomirs. Liver morphology, urea and albumin production are being used in concert with miR-122 target gene expression as markers for hepatocyte differentiation. In addition, we are investigating whether manipulations known to improve performance of hepatocyte cultures – such as cultivation in double layer collagen configuration [3] or the addition of retinoic acid [4] – acts via a miRNA mediated mechanism. These findings set the stage for manipulation of function in hepatocyte culture using microRNAs as master molecular switches of gene expression programs.

References:

[1] Bartel, David P., 2004. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116, 281 - 297

[2] Esau, C., Davis, S., Murray, S, Yu, X., Pandey, S., Pear, M., Watts, L., Booten, S., Graham, M., McKay, R., Subramaniam, A., Propp, S., Lollo, B., Freier, S., Bennett, F., Bhanot, S. and Monia, B., 2006. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metabolism 3, 87-98.

[3] Dunn, J., Tompkins, R., and Yarmush, M. 1991. Long-Term in Vitro Function of Adult Hepatocytes in a Collagen Snadwich Configuration. Biotechnol. Prog. 7, 237-245.

[4] Burley, M., Guzikowski, S. and Roth, C., 2007. Retinoic Acid Induces Morphological and Functional Changes in Primary Rat Hepatocytes. Submitted for publication.