Limits...
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.

Show MeSH

Related in: MedlinePlus

Potential mechanisms for diploid hepatocyte formation from polyploid fusion-derived hepatocytes.(A) Cytokinesis without mitosis. A binucleated cell undergoes cytokinesis before entering the next cell cycle. This process would not produce marker loss or aneuploidy. (B) Multiple spindles, followed by multipolar (in this case tetrapolar) mitosis. Extreme aneuploidy would result. (C) Mitosis without S-phase with chromosome pairing. This would ensure proper chromosome segregation and would facilitate distribution of hemizygous markers between daughter cells. (D) Horizontal gene transfer. A diploid cell engulfs a neighboring cell undergoing apoptosis. Single chromosomes and/or chromosome fragments would be incorporated into the nucleus while maintaining a nearly diploid karyotype. The parental cell is shown at left, mitotic spindle(s) (when necessary) in the middle and resulting diploid cells on the right. Open circles represent centromeres. Black circles represent centrosomes. Chromosomes are shown in different colors to indicate their lineage. Host hepatocyte chromosomes are red, and donor chromosomes are blue.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2636893&req=5

pgen-1000385-g005: Potential mechanisms for diploid hepatocyte formation from polyploid fusion-derived hepatocytes.(A) Cytokinesis without mitosis. A binucleated cell undergoes cytokinesis before entering the next cell cycle. This process would not produce marker loss or aneuploidy. (B) Multiple spindles, followed by multipolar (in this case tetrapolar) mitosis. Extreme aneuploidy would result. (C) Mitosis without S-phase with chromosome pairing. This would ensure proper chromosome segregation and would facilitate distribution of hemizygous markers between daughter cells. (D) Horizontal gene transfer. A diploid cell engulfs a neighboring cell undergoing apoptosis. Single chromosomes and/or chromosome fragments would be incorporated into the nucleus while maintaining a nearly diploid karyotype. The parental cell is shown at left, mitotic spindle(s) (when necessary) in the middle and resulting diploid cells on the right. Open circles represent centromeres. Black circles represent centrosomes. Chromosomes are shown in different colors to indicate their lineage. Host hepatocyte chromosomes are red, and donor chromosomes are blue.

Mentions: A number of possibilities could explain how diploid hepatocytes are generated from polyploid fusion-derived hepatocytes. First, it is theoretically possible that binucleated fusion-derived hepatocytes could simply complete cytokinesis (Figure 5A). Normal binucleated polyploid hepatocytes are formed through failed cytokinesis [22],[23]. For example, a mononucleated diploid hepatocyte undergoes a regular mitosis, but then separation of the two daughter cells fails, generating a binucleated tetraploid cell with two diploid nuclei [22]. Whether binucleated hepatocytes could resume cytokinesis is unclear, but it remains a possibility. In the context of fusion-derived hepatocytes, the completion of cytokinesis would generate two mononucleated diploid daughter cells, each with the same genotype as the original fusion partners. As seen in Figure 4D, subsets of diploid hepatocytes contained a donor marker (Fah) and a recipient marker (Cre and/or Y-chromosome), proving that these cells were genetically distinct from the original fusion partners. Therefore, a cytokinesis-type mechanism can be excluded.


Ploidy reductions in murine fusion-derived hepatocytes.

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

Potential mechanisms for diploid hepatocyte formation from polyploid fusion-derived hepatocytes.(A) Cytokinesis without mitosis. A binucleated cell undergoes cytokinesis before entering the next cell cycle. This process would not produce marker loss or aneuploidy. (B) Multiple spindles, followed by multipolar (in this case tetrapolar) mitosis. Extreme aneuploidy would result. (C) Mitosis without S-phase with chromosome pairing. This would ensure proper chromosome segregation and would facilitate distribution of hemizygous markers between daughter cells. (D) Horizontal gene transfer. A diploid cell engulfs a neighboring cell undergoing apoptosis. Single chromosomes and/or chromosome fragments would be incorporated into the nucleus while maintaining a nearly diploid karyotype. The parental cell is shown at left, mitotic spindle(s) (when necessary) in the middle and resulting diploid cells on the right. Open circles represent centromeres. Black circles represent centrosomes. Chromosomes are shown in different colors to indicate their lineage. Host hepatocyte chromosomes are red, and donor chromosomes are blue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636893&req=5

pgen-1000385-g005: Potential mechanisms for diploid hepatocyte formation from polyploid fusion-derived hepatocytes.(A) Cytokinesis without mitosis. A binucleated cell undergoes cytokinesis before entering the next cell cycle. This process would not produce marker loss or aneuploidy. (B) Multiple spindles, followed by multipolar (in this case tetrapolar) mitosis. Extreme aneuploidy would result. (C) Mitosis without S-phase with chromosome pairing. This would ensure proper chromosome segregation and would facilitate distribution of hemizygous markers between daughter cells. (D) Horizontal gene transfer. A diploid cell engulfs a neighboring cell undergoing apoptosis. Single chromosomes and/or chromosome fragments would be incorporated into the nucleus while maintaining a nearly diploid karyotype. The parental cell is shown at left, mitotic spindle(s) (when necessary) in the middle and resulting diploid cells on the right. Open circles represent centromeres. Black circles represent centrosomes. Chromosomes are shown in different colors to indicate their lineage. Host hepatocyte chromosomes are red, and donor chromosomes are blue.
Mentions: A number of possibilities could explain how diploid hepatocytes are generated from polyploid fusion-derived hepatocytes. First, it is theoretically possible that binucleated fusion-derived hepatocytes could simply complete cytokinesis (Figure 5A). Normal binucleated polyploid hepatocytes are formed through failed cytokinesis [22],[23]. For example, a mononucleated diploid hepatocyte undergoes a regular mitosis, but then separation of the two daughter cells fails, generating a binucleated tetraploid cell with two diploid nuclei [22]. Whether binucleated hepatocytes could resume cytokinesis is unclear, but it remains a possibility. In the context of fusion-derived hepatocytes, the completion of cytokinesis would generate two mononucleated diploid daughter cells, each with the same genotype as the original fusion partners. As seen in Figure 4D, subsets of diploid hepatocytes contained a donor marker (Fah) and a recipient marker (Cre and/or Y-chromosome), proving that these cells were genetically distinct from the original fusion partners. Therefore, a cytokinesis-type mechanism can be excluded.

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