Considerable research efforts in liver biology are focused on deriving new sources of hepatocytes for liver directed cell therapies, bioartificial liver-assist devices and other applications. Emphasis is being placed on investigating progenitor cells, fetal hepatocytes or human embryonic stem cells (ESC); cells that could first be expanded and then induced to express liver-specific function. Recently, we developed a novel genetically-engineered human fetal hepatocyte cell line containing reconstituted human telomerase reverse transcriptase (hTERT) subunits and capable of more than 300 cell doublings. Traditional cell culture approaches vary biological inducers one-at-a-time, requiring large number of cells and considerable time investment for thorough analysis. These methods are not well suited for defining precise composition of the microenvironment required to induce hepatic phenotype in novel, fetal- or stem cell-derived cell lines. Here we propose to employ microfabrication- and microarraying-based cell culture approaches to allow for small groups of cells to be cultured under precisely-defined microenvironment conditions and to be exposed to multiple biological stimuli in parallel. These novel cell culture tools will be applied to induce liver-specific differentiation in the immortalized fetal hepatocytes by providing an appropriate combination of the cell-cell and cell-extracellular matrix (ECM) interactions. Proposed cell culture platforms will be complemented with a new approach for evaluating hepatic function in the context of the local environment. Laser capture microdissection will be used in tandem with real-time quantitative RT-PCR to retrieve small groups of hepatic cells from locally engineered microenvironment and to analyze expression of liver specific genes. Upregulation in such genes as albumin, transferrin and alpha 1-antitrypsin will indentify a given combination of biological stimuli as having a positive effect on hepatic differentiation. In the future, proposed microfabricated devices will be used to induce differentiation of human ESC toward hepatic lineage. ? ? ?
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