Ploidy reductions in murine fusion-derived hepatocytes.
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 >or=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.
Duncan
Andrew W
AW
Oregon Stem Cell Center, Oregon Health and Science University, Portland, Oregon, United States of America. duncanan@ohsu.edu
Hickey
Raymond D
RD
Paulk
Nicole K
NK
Culberson
Andrew J
AJ
Olson
Susan B
SB
Finegold
Milton J
MJ
Grompe
Markus
M
D000818:Animals; D002459:Cell Fusion; D002478:Cells, Cultured; D033481:Chromosomes, Mammalian / Q000235:genetics; D005260:Female; D022781:Hepatocytes / Q000166:cytology* / Q000201:enzymology; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D007621:Karyotyping; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011003:Ploidies; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D006867:Hydrolases; D001616:beta-Galactosidase; C020156:fumarylacetoacetase