Hepatocyte Ploidy Research Articles

Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells.

Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how cell size influences physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be compositionally different. In the present study, we utilize the natural heterogeneity of hepatocyte ploidy and yeast genetics to establish that the ploidy-to-cell size ratio is a highly conserved determinant of proteome composition. In both mammalian and yeast cells, genome dilution by cell growth elicits a starvation-like phenotype, suggesting that growth in large cells is restricted by genome concentration in a manner that mimics a limiting nutrient. Moreover, genome dilution explains some proteomic changes ascribed to yeast aging. Overall, our data indicate that genome concentration drives changes in cell composition independently of external environmental cues.

Mcl-1 deficiency in murine livers leads to nuclear polyploidisation and mitotic errors: Implications for hepatocellular carcinoma

Background & AimsMcl-1, an antiapoptotic protein overexpressed in many tumours, including hepatocellular carcinoma (HCC), represents a promising target for cancer treatment. Although Mcl-1 non-apoptotic roles might critically influence the therapeutic potential of Mcl-1 inhibitors, these functions remain poorly understood. We aimed to investigate the effects of hepatic Mcl-1 deficiency (Mcl-1Δhep) on hepatocyte ploidy and cell cycle in murine liver in vivo and the possible implications on HCC. MethodsLivers of young Mcl-1Δhep and wild-type (WT) mice were analysed for ploidy profile, mitotic figures, in situ chromosome segregation, gene set enrichment analysis and were subjected to two-thirds partial hepatectomy to assess Mcl-1 deficiency effect on cell cycle progression in vivo. Mcl-1Δhep tumours in older mice were analysed for ploidy profile, chromosomal instability, and mutational signatures via whole exome sequencing. ResultsIn young mice, Mcl-1 deficiency leads to nuclear polyploidy and to high rates of mitotic errors with abnormal spindle figures and chromosome mis-segregation along with a prolonged spindle assembly checkpoint activation signature. Chromosomal instability and altered ploidy profile are observed in Mcl-1Δhep tumours of old mice as well as a characteristic mutational signature of currently unknown aetiology. ConclusionsOur study suggests novel non-apoptotic effects of Mcl-1 deficiency on nuclear ploidy, mitotic regulation, and chromosomal segregation in hepatocytes in vivo. In addition, the Mcl-1 deficiency characteristic mutational signature might reflect mitotic issues. These results are of importance to consider when developing anti-Mcl-1 therapies to treat cancer. Impact and implicationsAlthough Mcl-1 inhibitors represent promising hepatocellular carcinoma treatment, the still poorly understood non-apoptotic roles of Mcl-1 might compromise their successful clinical application. Our study shows that Mcl-1 deficiency leads to nuclear polyploidy, mitotic errors, and aberrant chromosomal segregation in hepatocytes in vivo, whereas hepatocellular tumours spontaneously induced by Mcl-1 deficiency exhibit chromosomal instability and a mutational signature potentially reflecting mitotic issues. These results have potential implications for the development of anti-Mcl-1 therapies to treat hepatocellular carcinoma, especially as hyperproliferative liver is a clinically relevant situation.

Implications of Polyploidy and Ploidy Alterations in Hepatocytes in Liver Injuries and Cancers.

Polyploidy, a condition in which more than two sets of chromosomes are present in a cell, is a characteristic feature of hepatocytes. A significant number of hepatocytes physiologically undergo polyploidization at a young age. Polyploidization of hepatocytes is enhanced with age and in a diseased liver. It is worth noting that polyploid hepatocytes can proliferate, in marked contrast to other types of polyploid cells, such as megakaryocytes and cardiac myocytes. Polyploid hepatocytes divide to maintain normal liver homeostasis and play a role in the regeneration of the damaged liver. Furthermore, polyploid hepatocytes have been shown to dynamically reduce ploidy during liver regeneration. Although it is still unclear why hepatocytes undergo polyploidization, accumulating evidence has revealed that alterations in the ploidy in hepatocytes are involved in the pathophysiology of liver cirrhosis and carcinogenesis. This review discusses the significance of hepatocyte ploidy in physiological liver function, liver injury, and liver cancer.

Centriole signaling restricts hepatocyte ploidy to maintain liver integrity.

Hepatocyte polyploidization is a tightly controlled process that is initiated at weaning and increases with age. The proliferation of polyploid hepatocytes in vivo is restricted by the PIDDosome-P53 axis, but how this pathway is triggered remains unclear. Given that increased hepatocyte ploidy protects against malignant transformation, the evolutionary driver that sets the upper limit for hepatocyte ploidy remains unknown. Here we show that hepatocytes accumulate centrioles during cycles of polyploidization in vivo. The presence of excess mature centrioles containing ANKRD26 was required to activate the PIDDosome in polyploid cells. As a result, mice lacking centrioles in the liver or ANKRD26 exhibited increased hepatocyte ploidy. Under normal homeostatic conditions, this increase in liver ploidy did not impact organ function. However, in response to chronic liver injury, blocking centriole-mediated ploidy control leads to a massive increase in hepatocyte polyploidization, severe liver damage, and impaired liver function. These results show that hyperpolyploidization sensitizes the liver to injury, posing a trade-off for the cancer-protective effect of increased hepatocyte ploidy. Our results may have important implications for unscheduled polyploidization that frequently occurs in human patients with chronic liver disease.

Assessment of Hepatocyte Ploidy by Flow Cytometry.

Polyploidy is a common and dynamic feature of mature rodent and human hepatocytes. While polyploidization occurs naturally during growth, alterations in the distribution of diploid and polyploid cells in the liver can be indicative of tissue stress or a pathologic state. Here, we describe a method for flow cytometric quantification of ploidy distribution by staining with propidium iodide. We first outline a hepatocyte isolation procedure from mouse liver through a two-step perfusion system for analysis of cellular ploidy. In an alternative approach, we employ a nuclei isolation protocol to assess nuclear ploidy. Finally, we describe how the use of fluorescent cell markers is compatible with these methods and helps retain information on cellular position within the tissue.

Malaria parasite liver stages

Malaria parasite liver stages

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm–Controlled Gene Expression in Mice

N6-methyladenosine (m6A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m6A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m6A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m6A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m6A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m6A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m6A modifications are critical regulators of liver homeostasis and function.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Hepatocyte ploidy in cats with and without hepatocellular carcinoma

BackgroundDomestic cats rarely develop hepatocellular carcinoma. The reason for the low prevalence is unknown. Reductions in hepatocellular ploidy have been associated with hepatic carcinogenesis. Recent work in mice has shown that livers with more polyploid hepatocytes are protected against the development of hepatocellular carcinoma. Hepatocyte ploidy in the domestic cat has not been evaluated. We hypothesized that ploidy would be reduced in peri-tumoral and neoplastic hepatocytes compared to normal feline hepatocytes. Using integrated fluorescence microscopy, we quantified the spectra of ploidy in hepatocellular carcinoma and healthy control tissue from paraffin embedded tissue sections.ResultsFeline hepatocytes are predominantly mononuclear and the number of nuclei per hepatocyte did not differ significantly between groups. Normal cats have a greater number of tetraploid hepatocytes than cats with hepatocellular carcinoma.ConclusionsTotal hepatocellular polyploidy in normal cat liver is consistent with values reported in humans, yet cellular ploidy (nuclei per cell) is greater in humans than in cats. Tetraploid cat hepatocytes are predominantly mononuclear.

Влияние хронической алкогольной интоксикации и постоянного освещения на морфофункциональное состояние печени

Introduction. Alcohol abuse is one of major health and social problems of modern society, the essence of which is an increase in morbidity and mortality not only from alcohol misuse and its complications, but also from associated comorbid pathologies. The liver is the organ that is affected most by ethanol and its metabolites. A significant factor leading to melatonin-associated morphological manifestations in the liver is the so-called light pollution, a violation of the light–dark regime. At the same time, heavy consumption of alcohol over a short period of time (alcoholic excesses) and other forms of alcoholic illness are often associated with various sleep disorders. The aim of the study was to investigate the morphological and functional state of the liver of Wistar rats under experimental alcohol intoxication in conditions of a fixed light regime and constant illumination. Materials and methods. Male rats (n=160) were kept under a fixed light regime, constant illumination, and at a combination of both light regimes with simultaneous experimental alcohol intoxication. We performed a pathological examination of the liver and determined morphometric parameters, hepatocyte ploidy, and the proportion of binuclear hepatocytes. Results. The consumption of 15% ethanol solution as a drink ad libitum for 3 weeks at a fixed light regime causes the development of fatty degeneration in the liver of laboratory animals. Secondary to it, changing lighting from fixed to constant is accompanied by the development of alcoholic hepatitis in about half of the animals. The results of micromorphometry indicate significant changes in the studied parameters of hepatocytes, which are expressed least of all in the case of ethanol consumption under fixed lighting condi-tions and affect most the cells of the liver parenchyma of rats with chronic alcohol intoxication secondary to constant lighting. Conclusion. Alcohol intoxication that occurs secondary to a melatonin deficiency caused by constant lighting is accompanied by a much greater severity, prevalence, and intensity of inflammatory tissue reactions and a variety of manifestations of liver pathology. Keywords: hepatocyte, alcohol, lighting regime, stress, fatty degeneration

Aged-related Function Disorder of Liver is Reversed after Exposing to Young Milieu via Conversion of Hepatocyte Ploidy.

Previous study showed that senescent hepatocytes from aged liver could be rejuvenated after repopulated in the young recipient liver. The proliferative capacity of hepatocytes was restored with the senescence reversal. However, it is unknown whether metabolic and homeostatic function of aged liver, as well as age-dependent liver steatosis could be rejuvenated or alleviated. Here, we found that senescent hepatocytes from aged liver were rejuvenated after exposing to young blood. An autonomous proliferation of senescent hepatocytes which resulting in ploidy reversal might be the underlying mechanism of senescent reversal. After performing 2/3 partial hepatectomy (2/3PHx) in young blood exposed old liver, delayed DNA synthesis of senescent hepatocytes was rescued and the number of BrdU positive hepatocytes was restored from 4.39±2.30% to 17.85±3.21%, similarly to that in the young mice at 36 hours post 2/3PHx. Moreover, Cyclin A2 and Cyclin E1 overexpression of hepatocytes in aged liver facilitating the G1/S phase transition was contributed to enhance liver regeneration. Furthermore, lipid droplet spread widely in the elderly human liver and old mouse liver, but this aged-associated liver steatosis was alleviated as senescence reversal. Collectively, our study provides new thoughts for effectively preventing age-related liver diseases.

In Search of Zonation Markers to Identify Liver Functional Disorders.

A substantial amount of research is being conducted on zonation markers to identify hepatic injuries and disorders based on the structural and functional zonation of the liver. In contrast to metabolic zonation, hepatocyte ploidy reflects the capability of liver regenerative turnover. Nonetheless, many knowledge gaps remain in the understanding of the links between liver disorders and altered zonation and ploidy, partially owing to the lack of sufficient zonation markers. Under this setting, we recapitulated the currently known and prospective markers used to identify normal and altered liver zonation in different disorders. Furthermore, we discussed new findings from studies that have used advanced methodologies to identify potential markers with greater accuracy. We also elaborated on the perspectives and future applications of zonation research in the early detection of various liver diseases.

PIDDosome‐induced p53‐dependent ploidy restriction facilitates hepatocarcinogenesis

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi‐protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome‐induced p53‐activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro‐tumorigenic effect of PIDDosome‐mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence‐free survival in HCC patients.

Hepatocyte ploidy and pathological mutations in hepatocellular carcinoma: impact on oncogenesis and therapeutics.

Hepatocellular carcinoma (HCC) occurs in the chronic liver inflammation such as viral hepatitis, alcoholic and non-alcoholic steatohepatitis. While anti-viral treatment has been significantly improved, the prevalence of HCC remains high and treatment is still challenging. The continuation of hepatocyte death, inflammation, and fibrosis leads to the accumulation of gene alterations, which may trigger carcinogenesis. Hepatocytes are a unique cell type having more than one complete set of 23 chromosomes, termed polyploidy. Due to gene redundancy, hepatocytes may tolerate lethal mutations. Next generation sequencing technology has revealed gene alterations in HCC related to telomere maintenance, Wnt/β-catenin pathway, p53 cell-cycle pathway, epigenetic modifiers, oxidative stress pathway, PI3K/AKT/mTOR, and RAS/RAF/MAPK pathway with or without a chromosomal instability. Some type of driver gene mutations accumulates in hepatocytes and breaks the orchestration of excessive copies of chromosomes, which may lead to unfavorable gene expressions and fuel tumorigenesis. Recently, molecular targeted drugs, developed with the aim of interfering with these signaling pathways, are being used for HCC patients in the clinics. Therefore, a deeper understanding of hepatocyte ploidy and genetic or epigenetic alterations is indispensable for the establishment of novel therapeutic strategies against HCC.

The Role of Immunomodulator Betaleukin in Recovery of Hepatocytic Ploidy Profile in Delayed Terms after Irradiation.

We studied the radioprotective effect of betaleukin administered to (CBA×C57Bl/6)F1 mice in single doses of 50 and 3 μg/kg 2 and 22 h, respectively, prior to long-term (21 h) whole-body low-intensity (10 mGy/min) γ-radiation (137Сs; total dose 12.65 Gy). Hepatocyte ploidy, a biomarker of metabolic disorders of the liver, was evaluated, and nuclearity and ploidy indices were calculated. In 10 months after irradiation, a significant decrease in the ploidy index was revealed in the group of irradiated mice, while in animal receiving 3 or 50 μg/kg betaleukin, this parameter did not differ and even surpassed the control level, respectively. Thus, in vivo assessment of hepatocytic ploidy profile in mice revealed negative delayed effects of γ-irradiation in a dose of 12.65 Gy and a protective effect a single injection of immunomodulator betaleukin.

Hepatocyte Ploidy in Cats with and without Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer related death in humans, yet it is an uncommon neoplasm in the domestic cat. The difference in prevalence and etiology between these two species is unexplained. Recent work in mice has shown that hepatocyte polyploidy (> 2n) is protective against the development of hepatocellular carcinoma. Further, human hepatocarcinogenesis is associated with a reduction in hepatocyte ploidy. Hepatocyte ploidy in the domestic cat has not been previously evaluated. Our research objective is to establish a baseline for hepatocellular chromosome number in normal cats and cats with HCC. We hypothesize that hepatocyte ploidy is significantly different in cats with or without HCC. Samples were selected from archival cases at both Cummings School of Veterinary Medicine, Tufts University (Tufts SVM) and Louisiana State University, Louisiana Animal Disease and Diagnostic Laboratory (LSU LADDL). Seven confirmed feline HCC cases and seven age and sex matched normal control cases were selected following review by a veterinary anatomical pathologists (IL). Using Hoechst H33342, a nuclear stain that stoichiometrically binds to the minor groove of DNA, fluorescence intensity was measured to determine hepatocellular ploidy. Neoplastic, peri‐tumoral, and normal hepatocytes were analyzed. There was statistical differences between the distribution of intensities between normal to peri‐tumoral, normal to neoplastic, and neoplastic to peri‐tumoral hepatocytes (p<0.0001). In addition, there is a significant difference between the number of 2N vs. 4N cells in normal and peri‐tumoral (p<0.0001) and neoplastic (p<0.0001) cells, and peri‐tumoral to neoplastic (p=0.002). There is no statistical difference in the number of multinucleated cells between groups. Normal feline liver has a greater number of 4N cells than neoplastic samples. This difference may contribute to development or be a cellular response to HCC development.Support or Funding InformationLouisiana State University, School of Veterinary Medicine Summer Scholars Program; Kenneth F. Burns Trust

Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing.

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

E2F-Family Members Engage the PIDDosome to Limit Hepatocyte Ploidy in Liver Development and Regeneration.

E2F transcription factors control the cytokinesis machinery and thereby ploidy in hepatocytes. If or how these proteins limit proliferation of polyploid cells with extra centrosomes remains unknown. Here, weshow that the PIDDosome, a signaling platform essential for caspase-2-activation, limits hepatocyte ploidy and is instructed by the E2F network to control p53 in the developing as well as regenerating liver. Casp2 and Pidd1 act as direct transcriptional targets of E2F1 and its antagonists, E2F7 and E2F8, that together co-regulate PIDDosome expression during juvenile liver growth and regeneration. Of note, whereas hepatocyte aneuploidy correlates with the basal ploidy state, the degree of aneuploidy itself is not limited by PIDDosome-dependent p53 activation. Finally, we provide evidence that the same signaling network is engaged to control ploidy in the human liver after resection. Our study defines the PIDDosome as a primary target to manipulate hepatocyte ploidy and proliferation rates in the regenerating liver.

Mice With Increased Numbers of Polyploid Hepatocytes Maintain Regenerative Capacity But Develop Fewer Hepatocellular Carcinomas Following Chronic Liver Injury

Thirty to 90% of hepatocytes contain whole-genome duplications, but little is known about the fates or functions of these polyploid cells or how they affect development of liver disease. We investigated the effects of continuous proliferative pressure, observed in chronically damaged liver tissues, on polyploid cells. We studied Rosa-rtTa mice (controls) and Rosa-rtTa;TRE-short hairpin RNA mice, which have reversible knockdown of anillin, actin binding protein (ANLN). Transient administration of doxycycline increases the frequency and degree of hepatocyte polyploidy without permanently altering levels of ANLN. Mice were then given diethylnitrosamine and carbon tetrachloride (CCl4) to induce mutations, chronic liver damage, and carcinogenesis. We performed partial hepatectomies to test liver regeneration and then RNA-sequencing to identify changes in gene expression. Lineage tracing was used to rule out repopulation from non-hepatocyte sources. We imaged dividing hepatocytes to estimate the frequency of mitotic errors during regeneration. We also performed whole-exome sequencing of 54 liver nodules from patients with cirrhosis to quantify aneuploidy, a possible outcome of polyploid cell divisions. Liver tissues from control mice given CCl4 had significant increases in ploidy compared with livers from uninjured mice. Mice with knockdown of ANLN had hepatocyte ploidy above physiologic levels and developed significantly fewer liver tumors after administration of diethylnitrosamine and CCl4 compared with control mice. Increased hepatocyte polyploidy was not associated with altered regenerative capacity or tissue fitness, changes in gene expression, or more mitotic errors. Based on lineage-tracing experiments, non-hepatocytes did not contribute to liver regeneration in mice with increased polyploidy. Despite an equivalent rate of mitosis in hepatocytes of differing ploidies, we found no lagging chromosomes or micronuclei in mitotic polyploid cells. In nodules of human cirrhotic liver tissue, there was no evidence of chromosome-level copy number variations. Mice with increased polyploid hepatocytes develop fewer liver tumors following chronic liver damage. Remarkably, polyploid hepatocytes maintain the ability to regenerate liver tissues during chronic damage without generating mitotic errors, and aneuploidy is not commonly observed in cirrhotic livers. Strategies to increase numbers of polypoid hepatocytes might be effective in preventing liver cancer.

InVivo Lineage Tracing of Polyploid Hepatocytes Reveals Extensive Proliferation during Liver Regeneration.

The identity of cellular populations that drive liver regeneration after injury is the subject of intense study, and the contributions of polyploid hepatocytes to organ regeneration and homeostasis havenot been systematically assessed. Here, we developed a multicolor reporter allele system to genetically label and trace polyploid cells in situ. Multicolored polyploid hepatocytes undergo ploidy reduction and subsequent re-polyploidization after transplantation, providing direct evidence of the hepatocyte ploidy conveyor model. Marker segregation revealed that ploidy reduction rarely involves chromosome missegregation invivo. We also traced polyploid hepatocytes in several different liver injury models and found robust proliferation in all settings. Importantly, ploidy reduction was seen in all injury models studied. We therefore conclude that polyploid hepatocytes have extensive regenerative capacity in situ and routinely undergo reductive mitoses during regenerative responses.