Ploidy Reductions in Murine Fusion-Derived Hepatocytes

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Abstract
We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH), and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ß-galactosidase and the Y-chromosome. Approximately 2–5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ß-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ß-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by ≥50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis. The liver comprises many different types of cells, including hepatocytes. Hepatocytes perform numerous physiological functions, such as detoxification, metabolism, and protein synthesis. Hepatocytes have the ability to fuse with blood cells, generating hybrid hepatocytes that contain nuclei from both fusion partners. In cases of genetic liver disease, fusion between diseased hepatocytes and normal blood cells can result in the formation of hybrid hepatocytes that function normally. In this series of experiments, we show that fusion hepatocytes produce daughter cells with one-half the amount of DNA found in the parental fusion hepatocyte. Furthermore, we show that the daughter cells are genetically distinct from each other. The increase in genetic diversity within the liver could give rise to hepatocytes lacking proper growth control, potentially resulting in tumor formation and cancer.

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