[HTML][HTML] Ploidy reductions in murine fusion-derived hepatocytes

AW Duncan, RD Hickey, NK Paulk, AJ Culberson… - PLoS …, 2009 - journals.plos.org
AW Duncan, RD Hickey, NK Paulk, AJ Culberson, SB Olson, MJ Finegold, M Grompe
PLoS genetics, 2009journals.plos.org
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 …
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.
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