The Significance of Polyploid Hepatocytes During Aging Process

Abstract

The mammalian liver contains numerous polyploid hepatocytes, and polyploidization is significantly enhanced during aging.1Kudryavtsev B.N. et al.Virchows Arch B Cell Pathol Incl Mol Pathol. 1993; 64: 387-393Crossref PubMed Scopus (113) Google Scholar In some tissues, such as bone marrow, diploid stem cells constitutively proliferate for tissue renewal and regeneration, whereas polyploidization leads to cell cycle arrest and differentiation enhancing cellular functions.2Ravid K. et al.J Cell Physiol. 2002; 190: 7-20Crossref PubMed Scopus (219) Google Scholar In contrast, notably, polyploid hepatocytes are an important source of liver regeneration under the stress conditions.3Matsumoto T. et al.Cell Stem Cell. 2020; 26: 34-47Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Polyploidy in hepatocytes has also been hypothesized to contribute to enhancing metabolic functions, and could influence functional properties of the liver.4Gentric G. et al.Clin Res Hepatol Gastroenterol. 2012; 36: 29-34Crossref PubMed Scopus (55) Google Scholar However, it has been unknown how polyploid hepatocytes play roles in physiological cellular renewal and impact on liver (dys)functions during the aging process. To examine whether polyploid hepatocytes contribute to cellular renewal during normal aging, livers of aged Rosa-RGBow+/- mice5He M. et al.Neuron. 2016; 91: 1228-1243Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar (27 months old) were compared with their young 3-month-old counterparts (Figure 1A). The Rosa-RGBow allele consists of multiple fluorescent reporter genes, and a subset of polyploid cells are labeled as bicolored cells after Cre recombination in heterozygous Rosa-RGBow mice (Figure 1A).3Matsumoto T. et al.Cell Stem Cell. 2020; 26: 34-47Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar As we previously showed,3Matsumoto T. et al.Cell Stem Cell. 2020; 26: 34-47Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar rAAV8-Ttr-Cre (6 × 1010 vector genomes) efficiently (about 75%) and specifically labeled hepatocytes in adult Rosa-RGBow+/- mice (Figure 1B, Supplementary Figure 1A), and all bicolored hepatocytes were polyploid (Supplementary Figure 1B). Interestingly, aged livers at >700 days after rAAV8-Ttr-Cre administration contained a substantial number of bicolored hepatocytes at a frequency similar to young livers (Figure 1C). Because murine hepatocytes divide every 200–300 days during normal homeostasis, this result suggests that abundant polyploid hepatocytes, which account for >90% of hepatocytes in the liver, repeatedly divide to maintain normal turnover of hepatocytes during aging. Indeed, young hepatocytes underwent further polyploidization during aging by going through additional cell cycles (Figure 1D). Furthermore, microscopic analysis revealed that both young and aged livers exhibited a similar bias of distribution of bicolored hepatocytes among liver zones. More bicolored hepatocytes were consistently located in pericentral zone 3 than in periportal zone 1 in both young and aged mice (Figure 1B and E). This is consistent with a biased distribution of diploid hepatocytes to the periportal area,6Tanami S. et al.Cell Tissue Res. 2017; 368: 405-410Crossref PubMed Scopus (43) Google Scholar and argues against dynamic migration of hepatocytes from zone 1 to zone 3 or vice versa during aging. Importantly, despite the constant frequencies of bicolored hepatocytes during aging, binucleated cells among bicolored hepatocytes were significantly fewer in the aged liver than in the young liver (Figure 1F). Given that hepatocyte polyploidization occurs via cytokinesis failure and generates binucleated cells at first,7Guidotti J.E. et al.J Biol Chem. 2003; 278: 19095-19101Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar these findings indicate that bicolored binucleated polyploidized cells underwent mitoses with complete cytokinesis, and generated mononucleated cells over time. Taken together, these findings suggest that polyploid hepatocytes proliferate and are an important contributor to maintenance of the liver during normal aging. Next, the impact of polyploidization on aging-related functional changes of hepatocytes was analyzed by RNA sequencing using diploid and polyploid hepatocytes sorted from young and aged livers (Supplementary Figure 2C and D). Heatmap and principal component analysis showed that each sample exhibited similar gene expression patterns among the 4 groups analyzed (Figure 2A, Supplementary Figure 1E). The global pattern of gene expression changes between diploid and polyploid hepatocytes was similar in young and aged livers (Figure 2B). Defining differentially expressed genes as those with P < .05 and absolute value of log2 fold change >1 identified hundreds of common differentially expressed genes especially in downregulated genes in polyploids (Figure 2C, Supplementary Table 1). Gene set enrichment analysis revealed that genes related to immune responses were commonly downregulated in polyploids compared with diploids both in young and aged livers, whereas genes associated with mitochondrial functions were commonly upregulated in polyploids (Figure 2D and E, Supplementary Table 2). Interestingly, downregulation of immune-related genes and upregulation of genes activating mitochondrial functions were previously reported in decidual polyploids in the uterus,8Ma X. et al.PLoS One. 2011; 6e26774Crossref PubMed Scopus (43) Google Scholar suggesting that polyploid cells exhibit somewhat similar expressional characteristics regardless of cell types and animal age. Influence of aging on polyploid hepatocytes was further examined by comparing bicolored polyploid hepatocytes in aged livers with polyploid counterparts in young livers. Because aged livers were labeled about 2 years before harvest, bicolored cells in aged livers had been polyploid for a long time. Interestingly, gene set enrichment analysis found that mitochondrial function genes were significantly downregulated in polyploids after aging, whereas there was no significant gene ontology upregulated in aged polyploids (Figure 2F, Supplementary Table 3). Mitochondrial dysfunction is one of the features of aging,9Lopez-Otin C. et al.Cell. 2013; 153: 1194-1217Abstract Full Text Full Text PDF PubMed Scopus (7830) Google Scholar and given that mitochondrial function genes are upregulated in polyploids at baseline, polyploid cells may be predisposed to mitochondrial dysfunction during aging. However, 96 differentially expressed genes that were uniquely upregulated in polyploids after aging were significantly enriched with genes annotated to lipid and fatty acid metabolism by pathway analysis, which was consistent with a previous report that compared unsorted young and aged liver tissues (Figure 2G, Supplementary Table 3).10Bochkis I.M. et al.Cell Rep. 2014; 9: 996-1006Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Aging-related metabolic changes of the liver may be mainly attributed to that of polyploid hepatocytes. In summary, we showed here that the abundantly present polyploid hepatocytes in the mouse liver are the source of physiological hepatocyte turnover during aging. No evidence for a dominant contribution of a diploid liver stem cell during aging was found. Similar processes may occur in the polyploids of other organs with slow cellular turnover, such as acinar cells in the pancreas. In addition, polyploids exhibited characteristic gene expressions compared with diploids, which may be predetermined irrespective of cell types. Moreover, polyploidy-related upregulation of mitochondrial function genes was especially impaired during aging, and polyploid hepatocytes showed higher expressions of lipid and fatty acid metabolic genes than young cells. Although the mechanisms that link polyploidy and age-related liver dysfunction need further investigation, findings here underscore the importance of polyploid hepatocytes in aging-related metabolic liver diseases. The rAAV-Ttr-Cre construct was kindly provided by Dr Holger Willenbring of University of California San Francisco. RNA sequencing FASTQ data were submitted to the NCBI GEO: Accession number GSE161171. Download .xlsx (.21 MB) Help with xlsx files Supplementary Tables 1–3 Download .doc (.12 MB) Help with doc files Supplementary Data

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