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Ploidy reductions in murine fusion-derived hepatocytes.

Duncan AW, Hickey RD, Paulk NK, Culberson AJ, Olson SB, Finegold MJ, Grompe M - PLoS Genet. (2009)

Bottom Line: Approximately 2-5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction.Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction.Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content.

View Article: PubMed Central - PubMed

Affiliation: Oregon Stem Cell Center, Oregon Health and Science University, Portland, Oregon, United States of America. duncanan@ohsu.edu

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 >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.

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Marker loss in livers repopulated with fusion-derived hepatocytes.(A–D) Marker analysis was performed on sequential liver sections from serially transplanted mice. The transplantation scheme (F>M>F or M>F>F) is indicated. FAH+ nodules (brown) either co-expressed the Y-chromosome (black dots) (A) or were devoid of Y-chromosome staining (B and C). Double asterisks (**) indicate double positive nodules (FAH+ and Y-chromosome+) whereas single asterisks (*) indicate single positive nodules (FAH+ and Y-chromosome−). (D) ß-gal (blue staining) is expressed in a single FAH+ nodule but is absent from peripheral FAH+ tissue. Double diamonds (◊◊) indicate double positive tissue (FAH+ and ß-gal+) and single diamonds (◊) indicate a single positive nodule (FAH+ and ß-gal−). Scale bars are 200 µm. (E) Percentage of transplanted mice containing FAH+ fusion-derived nodules that lack Y-chromosome or ß-gal is shown.
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pgen-1000385-g002: Marker loss in livers repopulated with fusion-derived hepatocytes.(A–D) Marker analysis was performed on sequential liver sections from serially transplanted mice. The transplantation scheme (F>M>F or M>F>F) is indicated. FAH+ nodules (brown) either co-expressed the Y-chromosome (black dots) (A) or were devoid of Y-chromosome staining (B and C). Double asterisks (**) indicate double positive nodules (FAH+ and Y-chromosome+) whereas single asterisks (*) indicate single positive nodules (FAH+ and Y-chromosome−). (D) ß-gal (blue staining) is expressed in a single FAH+ nodule but is absent from peripheral FAH+ tissue. Double diamonds (◊◊) indicate double positive tissue (FAH+ and ß-gal+) and single diamonds (◊) indicate a single positive nodule (FAH+ and ß-gal−). Scale bars are 200 µm. (E) Percentage of transplanted mice containing FAH+ fusion-derived nodules that lack Y-chromosome or ß-gal is shown.

Mentions: After demonstrating that fusion-derived hepatocytes could generate daughter cells with one-half chromosome content, we hypothesized that ploidy reduction events should also lead to marker segregation among daughter cells. If ploidy reduction occurs in a tetraploid fusion-derived hepatocyte, there is a 50% chance of losing a heterozygous or hemizygous marker. Liver sections from mice repopulated by fusion-derived hepatocytes in serial transplantation experiments (Figure 1A) were stained for FAH, Y-chromosome and ß-gal activity (Figures 2A–2D). Typically, FAH is co-expressed with the Y-chromosome (Figure 2A) and ß-gal (Figure 2D). However, while most regenerating nodules expressed all markers of cell fusion, a fraction of FAH positive nodules (2–5%) were Y-chromosome negative (Figures 2B and 2C) or ß-gal negative (Figure 2D). Based on the expected loss-of-heterozygosity frequency of one-half, the observed frequency suggests that 4–10% of the FAH positive nodules were initiated by cells that had undergone ploidy reductions.


Ploidy reductions in murine fusion-derived hepatocytes.

Duncan AW, Hickey RD, Paulk NK, Culberson AJ, Olson SB, Finegold MJ, Grompe M - PLoS Genet. (2009)

Marker loss in livers repopulated with fusion-derived hepatocytes.(A–D) Marker analysis was performed on sequential liver sections from serially transplanted mice. The transplantation scheme (F>M>F or M>F>F) is indicated. FAH+ nodules (brown) either co-expressed the Y-chromosome (black dots) (A) or were devoid of Y-chromosome staining (B and C). Double asterisks (**) indicate double positive nodules (FAH+ and Y-chromosome+) whereas single asterisks (*) indicate single positive nodules (FAH+ and Y-chromosome−). (D) ß-gal (blue staining) is expressed in a single FAH+ nodule but is absent from peripheral FAH+ tissue. Double diamonds (◊◊) indicate double positive tissue (FAH+ and ß-gal+) and single diamonds (◊) indicate a single positive nodule (FAH+ and ß-gal−). Scale bars are 200 µm. (E) Percentage of transplanted mice containing FAH+ fusion-derived nodules that lack Y-chromosome or ß-gal is shown.
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Related In: Results  -  Collection

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pgen-1000385-g002: Marker loss in livers repopulated with fusion-derived hepatocytes.(A–D) Marker analysis was performed on sequential liver sections from serially transplanted mice. The transplantation scheme (F>M>F or M>F>F) is indicated. FAH+ nodules (brown) either co-expressed the Y-chromosome (black dots) (A) or were devoid of Y-chromosome staining (B and C). Double asterisks (**) indicate double positive nodules (FAH+ and Y-chromosome+) whereas single asterisks (*) indicate single positive nodules (FAH+ and Y-chromosome−). (D) ß-gal (blue staining) is expressed in a single FAH+ nodule but is absent from peripheral FAH+ tissue. Double diamonds (◊◊) indicate double positive tissue (FAH+ and ß-gal+) and single diamonds (◊) indicate a single positive nodule (FAH+ and ß-gal−). Scale bars are 200 µm. (E) Percentage of transplanted mice containing FAH+ fusion-derived nodules that lack Y-chromosome or ß-gal is shown.
Mentions: After demonstrating that fusion-derived hepatocytes could generate daughter cells with one-half chromosome content, we hypothesized that ploidy reduction events should also lead to marker segregation among daughter cells. If ploidy reduction occurs in a tetraploid fusion-derived hepatocyte, there is a 50% chance of losing a heterozygous or hemizygous marker. Liver sections from mice repopulated by fusion-derived hepatocytes in serial transplantation experiments (Figure 1A) were stained for FAH, Y-chromosome and ß-gal activity (Figures 2A–2D). Typically, FAH is co-expressed with the Y-chromosome (Figure 2A) and ß-gal (Figure 2D). However, while most regenerating nodules expressed all markers of cell fusion, a fraction of FAH positive nodules (2–5%) were Y-chromosome negative (Figures 2B and 2C) or ß-gal negative (Figure 2D). Based on the expected loss-of-heterozygosity frequency of one-half, the observed frequency suggests that 4–10% of the FAH positive nodules were initiated by cells that had undergone ploidy reductions.

Bottom Line: Approximately 2-5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction.Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction.Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content.

View Article: PubMed Central - PubMed

Affiliation: Oregon Stem Cell Center, Oregon Health and Science University, Portland, Oregon, United States of America. duncanan@ohsu.edu

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 >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.

Show MeSH
Related in: MedlinePlus