Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats

Organs from nonheart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for nonheart-beating donor cells. We studied nonheart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In nonheart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16 or 34 hours after death, respectively, compared with heart-beating donors. These differentially-expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, as well as other discrete pathways. We successfully isolated viable hepatocytes from nonheart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to hepatocytes from heart-beating donors, p<0.05. Similarly, although hepatocytes from nonheart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors, p<0.05. Gene expression profiling in hepatocytes isolated from nonheart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. Conclusion: Liver tissue remained intact over prolonged periods after death in nonheart-beating donors but extensive molecular perturbations following reperfusion/reoxygenation impaired viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from nonheart-beating donors offers opportunities for improving donor cell viability, which will advance utility of nonheart-beating donor organs for cell therapy or other applications.