Cellular polyploidy in organ homeostasis and regeneration

Abstract

Polyploid cells, which contain more than one set of chromosome pairs, are very common in nature. Polyploidy can provide cells with several potential benefits over their diploid counterparts, including an increase in cell size, contributing to organ growth and tissue homeostasis, and improving cellular robustness via increased tolerance to genomic stress and apoptotic signals. Here, we focus on why polyploidy in the cell occurs and which stress responses and molecular signals trigger cells to become polyploid. Moreover, we discuss its crucial roles in cell growth and tissue regeneration in the heart, liver, and other tissues.

Graphical abstract

Keywords

cellular polyploidy / tissue regeneration / cardiac regeneration / liver regeneration

Introduction

Establishing cellular polyploidy

Polyploidy is a widespread cellular feature in nature and plays an essential role in organism growth. Eukaryotic organisms usually have two complete sets of homologous chromosomes, a eukaryotic cell typically duplicates its chromosomes to produce two equal diploid sets of the genome during S phase, which will evenly divide and generate two identical diploid daughter cells. In contrast, cells that harbor more than two complete sets of homologous chromosomes are considered to be polyploid. This can be observed either in cells with a single polyploid nucleus or in multinucleated cells with diploid or even polyploid nuclei. In plants, polyploidy is likely to affect their fitness, which helps them adapt to the changing environment and provides new genetic material to induce genetic variations, resulting in higher diversity or novel gene functions (Leitch and Leitch, 2008). Polyploidy is also common in insects and vertebrates (Bogart, 1979). In certain types of mammalian cells, such as mammary gland cells (Rios et al., 2016), subperineurial glia (SPG) cells (Unhavaithaya and Orr-Weaver, 2012), cardiomyocytes and hepatocytes (Anatskaya and Vinogradov, 2007), skin keratinocytes (Zanet et al., 2010), and placenta trophoblast giant cells (Sher et al., 2013), the transition from diploid to polyploid appears to be essential for cellular function during their postnatal development (Senyo et al., 2013; Wilkinson et al., 2019a). In specific tissues, the generation of polyploid cells is associated with different cellular stress, like mechanical or metabolic stress, which can be observed in the myocardium and vascular smooth muscle cells when exposed to pressure overload (Vliegen et al., 1995; Hixon et al., 2000). Interestingly, in humans, extra chromosomes, such as triploid and tetraploid fetuses, are usually aborted or die early after birth due to malformations (Jacobs et al., 1982; Guc-Scekic et al., 2002). Therefore, polyploidy only exists in some specific tissues rather than in the whole organism.

Many scientists tried to understand why cells become polyploid and proposed some hypotheses to explain this phenomenon. First, polyploid cells generally are larger than diploid cells, thereby contributing to organ growth and tissue homeostasis (Unhavaithaya and Orr-Weaver, 2012). Second, more genome copies can prevent the unfavorable consequence of mutations and DNA damage, especially in some key genes, such as tumor suppressor genes (Zhang et al., 2018a), thereby improving cellular responsiveness and cell survival in response to stress (Anatskaya and Vinogradov, 2007; Mehrotra et al., 2008). Third, cell division might be a potential threat under certain circumstances; for instance, skin keratinocytes and placenta trophoblast giant cells are essential in protecting tissue integrity, and cell division may temporarily impair this function (Zanet et al., 2010; Sher et al., 2013). Fourth, cells may preserve extra energy via cytokinesis failure (Anatskaya and Vinogradov, 2007). As every condition has pros and cons, there are also some potential drawbacks for being polyploid. For example, if polyploid cells try to complete mitotic division, gene copies and multiple centrosomes may lead to asymmetric distribution and genetic variation in daughter cells and trigger aneuploidy (Duncan et al., 2012a). There is strong evidence for a high frequency of aneuploidy in cancer, such as acute myeloid leukemia (Barnard et al., 2002) and Ewing’s sarcoma (Maurici et al., 1998). Unexpected tetraploidy is also observed in human cancers (Ganem et al., 2007). Tetraploidy is viewed as a metastable intermediate between healthy diploidy and neoplastic aneuploidy, and it has been observed in certain types of diseases like breast cancer (Jonsdottir et al., 2012) and Barrett’s esophagus (Reid et al., 1987). In short, polyploid can either contribute to important tissue function or, on the other hand, is associated with the development of cancer.

This review summarizes the formation of polyploidy and its essential roles in different organs and reveals a potential crosslink between polyploidy and tissue regeneration.

Mechanisms for the genesis of polyploid cells

Different mechanisms can convert diploid cells turn into polyploid cells. Different diploid cells can obtain a polyploidy state through various mechanisms, some even have more than one way to accomplish this state. Here we list four potential routes of polyploid cell formation: endoreplication, cytokinesis failure, cell fusion, and mitotic slippage, and all these mechanisms will be discussed in the following section (Fig. 1).

Endoreplication

Endoreplication, also known as endoreduplication or endocycles, occurs when cells successively copy their genomes without chromosome segregation, thereby leading to the genesis of mononucleated polyploid cells. The key event that triggers endoreplication is inhibition of entry into mitosis, usually mediated by mitotic cyclins under the control of the anaphase-promoting complex/cyclosome (APC/C) (Zielke et al., 2008). Endoreplication can also be induced by cyclin kinase inhibitors P57 and P21 via CDK1 activity suppression (Ullah et al., 2008; Ogawa et al., 2016). Large subunit of chromatin assembly factor-1 (CAF-1) is required for growth and larval viability, the depletion of the CAF-1 large subunit causes an accumulation of DNA damage, and an increased endoreplication, implying that endoreplication may be an adaptation to genomic damage (Klapholz et al., 2009), consistent with previous studies (Lazzerini Denchi et al., 2006; Mehrotra et al., 2008; Davoli et al., 2010). In Arabidopsis plants, the appearance of DNA damage led to G2–M arrest and ultimately triggered endoreplication, which is more tolerant to water deficit (Cookson et al., 2006). This means endoreplication can provide an advantage in adaptation to stress and improve growth in plants.

Tissue repair normally needs either cell growth or proliferation; when cell proliferation is limited, endoreplication ensues tissue repair can still continue. Forcing injured adult Drosophila abdominal epithelium to finish mitosis is unfavorable to wound repair because Drosophila epithelium can accumulate DNA damage, while mitotic errors occur when cells are compelled to proliferate (Grendler et al., 2019). In contrast, injury to Drosophila epithelium promotes cells to undergo endoreplication and cell fusion for wound healing (Losick et al., 2013; Grendler et al., 2019), indicating polyploidy can replace damaged cells and sustain normal cellular functions. This phenomenon also happens in the mouse model of Fuchs endothelial corneal dystrophy, where massive cell death drives pathology, and the remaining cells try to increase their ploidy status to compensate for cell loss and maintain size and synthetic capacity (Losick et al., 2016). Endoreplication is also critical to normal cell growth in some specific tissues. For example, reduced ploidy of ovarian nurse cells can lead to female sterility, as endoreplication is indispensable for normal oocyte development in Drosophila (Lilly and Spradling, 1996; Maines et al., 2004).

Cytokinesis failure

Cytokinesis is the last step in mitosis, promoting physical separation and driving the emergence of two daughter cells from one parental cell. Successful cytokinesis is required to distribute replicated genomes and other cellular components into subsequent daughter cells, while cytokinesis failure can give rise to binucleated polyploid cells and supernumerary centrosomes (Ganem et al., 2007; Rengstl et al., 2013). Although the reason for structural chromosome aberrations in polyploid cells remains unknown, improper chromosome segregation caused by extra centrosomes might be responsible for the aberrations (aneuploidy) often found in polyploid cells (Ganem et al., 2009). Cytokinesis failure occurs during the development of specific human organs, such as the heart or the liver (Fig. 2). Cytokinesis failure-induced polyploidy is also called endomitosis. Increased mechanical tension causes cytokinesis failure and generates polyploid cells in the epicardium, which are efficient enough to support epicardial regeneration (Cao et al., 2017).

Cell fusion

Cell–cell fusion is a special mechanism that creates polyploid cells without involving cell cycle progression. It is a crucial cellular process in which several mononuclear cells integrate to produce a multinucleated cell (Kemp et al., 2011, 2012). Cell fusion is also vital during tissue development and regeneration, as cytotrophoblasts fuse to generate placental syncytiotrophoblasts (Huppertz et al., 1998). In vivo, mouse retinal neurons can reach a precursor stage after spontaneously fusing with transplanted hematopoietic stem and progenitor cells via the activation of Wnt/β-catenin signaling, the newly formed cells subsequently proliferate and finally become differentiated neurons to rescue the damaged retinal tissue (Sanges et al., 2013). In our previous study, we observed that inhibition of bone marrow cells recruitment into the liver severely dampen its regeneration capacity following injury, where the mobilized hematopoietic stem and progenitor cells from resected liver were able to fuse with hepatocytes, and these hybrid cells proliferate earlier than hepatocytes (Pedone et al., 2017).

Mitotic slippage

During perturbed mitosis, when cells are subjected to spindle inhibitors, they are blocked at pro-metaphase by the spindle assembly checkpoint (SAC), which helps avoid chromosome mis-segregation and ensures each chromosome is properly attached to the mitotic spindle (Brito and Rieder, 2006; Mansilla et al., 2006). This blockage is caused by APC/C inhibition via the mitotic checkpoint complex, which is pivotal to regulate mitotic progression (Brito and Rieder, 2006). Normal cells have a robust SAC in which unattached chromosomes initiate signals to interfere with cellular APC/C activity and thereby hinder progression to anaphase (Brito and Rieder, 2006). When the SAC is weak, cells slip out of mitosis without undergoing anaphase or cytokinesis, known as mitotic slippage, which is prevalent in cells with prolonged mitotic arrest after exposure to spindle toxins treatment or in APC defective cells, and appear rarely spontaneous under physiological condition (Brito and Rieder, 2006; Dikovskaya et al., 2007). Microtubule-targeting agents that stimulate the SAC and disturb microtubule dynamics are able to cause cell death (Lamar et al., 2016; Kang et al., 2018). However, suppression of the SAC and subsequent slippage can lead to chromosome instability and is lethal to normal and tumor cells, demonstrating that its function is critical to cell survival (Kops et al., 2004; Michel et al., 2004).

Polyploidy in the heart

Cardiomyocyte polyploidy during development

The extent of cardiomyocyte polyploidy varies greatly in vertebrate species (Ostergaard et al., 2013; Bensley et al., 2016; Hirose et al., 2019). In endotherm mammalian and bird species, polyploid cardiomyocytes are the primary population (more than 90%), while most cardiomyocytes in zebrafish and newts are diploid (nearly 99%) (Bettencourt-Dias et al., 2003; Hirose et al., 2019). Of note, some fish species preserve substantial cardiomyocyte polyploidy (up to 85%) (Martynova et al., 2002). Noticeably, the nuclear number in polyploid cardiomyocytes varies among endotherm species (Adler et al., 1996; Anatskaya and Vinogradov 2004; Bensley et al., 2016). The majority of human adult cardiomyocytes only have one polyploid nucleus (about 60%) (Olivetti et al., 1996; Mollova et al., 2013). However, giraffe and bird cardiomyocytes are predominantly multinucleated, with 3–8 nuclei per cardiomyocyte, respectively (range from 50% to 99%; Anatskaya et al., 2001; Ostergaard et al., 2013; Hirose et al., 2019). Other mammalian species, such as mice, rabbits, sheep, and rats, displayed predominantly binucleated heart cells (range from 66% to 82%; Adler et al., 1996; Raulf et al., 2015; Bensley et al., 2016). In murine heart development, most cardiomyocytes (80%–95%) turn into polyploidy around 1 week after birth (Soonpaa et al., 1996). Interestingly, the presence of polyploid cardiomyocytes coincides with the change in thyroid hormone levels (Hirose et al., 2019). In sheep and mice, thyroid hormone plasma concentration increases several folds after birth, along with decreased cardiomyocyte proliferative capacity and increased cardiomyocyte polyploidy (Chattergoon et al., 2012a; Hirose et al., 2019). Additional thyroid hormone administration can significantly augment cardiomyocyte polyploidy; in contrast, inhibition of thyroid hormone receptors in cardiomyocytes of neonatal mice results in increased proliferative capacity and diploid cardiomyocyte number (Chattergoon et al., 2012a, 2012b; Hirose et al., 2019). This finding may explain why cardiomyocytes in some species become polyploid after birth.

During the perinatal period, cardiomyocytes are exposed to extensive oxidative stress, accompanied by increased DNA damage and cell cycle exit (Puente et al., 2014). DNA damage induced by oxidative stress causes mis-segregation of chromosomes during the M phase, which might be the reason for cardiomyocyte polyploidy (Puente et al., 2014; Aix et al., 2016). Besides, polyploidy in cardiomyocytes could be an adaptation to stress induced by increased hemodynamic demand in fetal or neonatal hearts (Barbera et al., 2000; Jonker et al., 2010). In the early stage of human heart development, most cardiomyocytes are diploid, where heart mass growth mainly depends on the proliferation of cardiomyocytes. However, shortly after birth, most cardiomyocytes lose the ability to complete cytokinesis and become binucleated, and the subsequent increase in heart size is independent of cardiomyocyte proliferation (Li et al., 1996; Soonpaa et al., 1996).

Interestingly, cardiomyocyte polyploidy appears even before birth in sheep (Jonker et al., 2007a). Thus, being born is unlikely to be the only reason for increased polyploid cardiomyocytes in mammals. Cell cycle regulators seem to be associated with cardiomyocyte polyploidy. Almost all cyclins are highly expressed during the embryonic stage, and subsequently decreases after birth (Kang et al., 1997), in particular, cyclin A and B expression levels are reportedly undetectable in adult hearts (Kang et al., 1997). The CDKs were also highly expressed during embryonic development while dropping to very low levels in the young hearts, consistent with the proliferating cell nuclear antigen (PCNA) pattern during this period (Kang et al., 1997), indicating that the decreased level of mitotic cyclins and CDKs probably trigger the withdrawal from cell cycle after birth, which ultimately triggers the occurrence of polyploid cardiomyocytes (Kang et al., 1997). Exogenous expression of mitotic cyclins seems a feasible way to overcome this hurdle. In the adult porcine heart, overexpression cyclin A2, a mitotic cyclin, in infarct myocardium caused increased cytokinesis and cardiomyocyte numbers with mature sarcomeric structure (Shapiro et al., 2014). Similarly, overexpression of cyclin B1-CDK1 complexes restarts cell division in adult rat cardiomyocytes (Bicknell et al., 2004). Conversely, CDK inhibition leads to the cell cycle exit of cardiomyocytes (Di Stefano et al., 2011; Tane et al., 2014). In addition, enhanced E2f1 and E2f2 can accelerate cell cycle and cardiomyocyte division via increased expression of cyclins B1 and B2. Meis1 was also identified as a critical regulator of the cardiomyocyte cell cycle. Meis1 silencing in mouse cardiomyocytes promotes extension of the postnatal proliferative window of cardiomyocytes and reactivates cardiomyocyte mitosis in the adult heart without detrimental effect on cardiac function. Instead, overexpression of Meis1 in cardiomyocytes dampens neonatal cardiomyocyte proliferation capacity (Mahmoud et al., 2013). Below, we summarize some crucial genes and conditions that were known to influence polyploidy in the heart (Table 1).

The centrosome is essential for separating duplicated DNA during cell division, acting as a critical hub for cell cycle regulation that determines if a cell should divide (Pascreau et al., 2011; Zebrowski et al., 2015). In adult newts and zebrafish, the structure of centrosomes within the heart muscle cells remains intact, while centrosomes in mammalian cardiomyocytes are disassembled shortly after birth, thereby potentially triggering P38-MAPK-mediated cell cycle arrest (Zebrowski et al., 2015). In contrast to this, P38-MAPK inhibition can promote the proliferation capacity of cardiomyocytes with split-centrioles, indicating that interference of P38-mediated stress-associated signal enhances cell cycle progression in cardiomyocytes that lack centriole cohesion (Zebrowski et al., 2015). Anillin is a cytokinesis-associated protein crucial for furrow construction and midbody formation during cytokinesis. P38-MAPK-mediated anillin localization defects create a barrier for midbody formation, leading to an incomplete cell cycle and binucleated cardiomyocytes, while P38-MAPK inhibition can repair the midbody formation failure (Engel et al., 2006). Similarly, Septins, a family of cytoskeletal GTPases, correlate with cytokinesis during heart development (Ahuja et al., 2006).

So far, the reason for cardiomyocyte polyploidy formation is still poorly defined. One hypothesis is that cardiomyocyte polyploidy is regulated as a response to cardiomyocytes’ physiological and functional demands. Cardiomyocytes may temporarily choose to undergo sarcomeres disassembly to meet the increased cardiac output. However, there are several potential drawbacks to this solution. First, it could be energy consuming and cause contractile dysfunction during cytokinesis. Consistent with this, upregulation of genes encoding contractile-related proteins was observed in polyploid heart tissues (Anatskaya and Vinogradov, 2007). Suppose the electric signal was conducted less homogenously and has to pass across more gap junctions; this could bring an increased risk of defective electrical coupling between newly developed cardiomyocyte and pre-existing cardiomyocyte (Gabisonia et al., 2019). Thus, cardiomyocyte polyploidy might be helpful as it contributes to heart growth and sustains myocardial contractile function. Furthermore, cardiomyocyte polyploidy contributes to coping with cellular stress, prolonging cell longevity by increased resistance to various stress, offering a faster transcriptional response to the changing physiological environments (Anatskaya and Vinogradov, 2007), and meeting increased protein synthesis requirements (Clubb and Bishop, 1984; Oparil et al., 1984; Soonpaa et al., 1996). In this case, cardiomyocyte polyploidy probably provides an alternative solution for increased heart mass from an energy-saving perspective. In brief, cardiac muscle structure stability, contractility capacity, and electrical coupling provide us with some viewpoints to comprehend why cardiomyocytes become polyploid during heart growth.

Cardiomyocyte polyploidy in heart pathology

Increased cardiomyocyte ploidy can be observed both in physiological and pathological conditions (Adler and Friedburg, 1986; Bergmann et al., 2015; Gilsbach et al., 2018). Cardiomyocyte ploidy in rodents and humans increases with age (Soonpaa et al., 1996; Li et al., 1996; Bergmann et al., 2009; Bergmann et al., 2015). Interestingly, cardiomyocyte ploidy level was elevated (up to 25%) after myocardial infarction, especially in the infarcted border zone (Meckert et al., 2005; Hesse et al., 2012). Infarction likely activated the remaining cells’ DNA synthesis, providing an adaptive response to the damaged myocardium (Herget et al., 1997). This implies that cardiomyocytes have the potential capacity to reenter the cell cycle and undergo DNA replication under pathological conditions. In addition, cardiomyocyte ploidy levels are also increased (up to 15–20n) in cardiac hypertrophy and in adults with congenital diseases (Erokhina et al., 1992; Brodsky et al., 1994). Pressure overload-induced cardiac hypertrophy appears to be correlated with polyploidy and is more apparent in the left than the right ventricle (Vliegen et al., 1995). In order to explore the relationship between cardiomyocyte proliferation, size, and polyploidy under hypertension, Jonker et al. (2007b) treated fetal sheep with chronic intravascular plasma infusions for 4 or 8 days to induce hypertension. The incidence of cell cycle activity is higher in the hypertension group than the control group. In the early phase, cell cycle activity increased as well as cardiomyocyte enlargement, whereas, in the later phase, binucleate cardiomyocyte number elevated (approximately 50%) with enhanced cell cycle activity, suggesting there are different physiological responses to hypertension in the fetal sheep heart (Jonker et al., 2007b).

A recent article shows that running exercise stimulates physiological cardiac hypertrophy and facilitates cell cycle activity, leading to increased diploid cardiomyocytes (nearly double) and newborn cardiomyocytes, even in injured mice that underwent myocardial infarction (Vujic et al., 2018). So far, no relevant research has investigated whether exercise-mediated physiological hypertrophy in humans can also increase the percentage of diploid cardiomyocytes.

Polyploidy in liver

Polyploidy in liver during development

The level of hepatocyte polyploidy varies among mammals, and polyploidy is seen as a critical feature of liver growth and physiological function (Anatskaya et al., 1994). Up to 90% of adult hepatocytes in murine and around 50% in adult humans are polyploid (Saeter et al., 1988; Duncan et al., 2010; Duncan et al., 2012b). Increased cell size appears to be an obvious result of an increase in ploidy, as it was usually accompanied by an increase in cell volume (Epstein, 1967). The volume of hepatocytes almost doubled with doubling DNA content. Interestingly, there is no dramatic difference between the volume of mononuclear and binuclear polyploid hepatocytes containing the same amount of DNA (2 × 2n and 4n cells) (Martin et al., 2002). One attractive question is how diploid hepatocytes become polyploid during physiological or pathological development. In the liver, polyploid cells appeared during suckling-to-weaning transition in which nutrition change is involved. This change will have an impact on liver metabolism, which might be involved in the formation of polyploid cells (Margall-Ducos et al., 2007; Celton-Morizur et al., 2009). During this period, diploid hepatocytes (2n) undergo cell cycle, producing two diploid hepatocytes or giving rise to one binucleate tetraploid hepatocyte because of failed cytokinesis. This process is a pivotal step for establishing polyploid hepatocytes during postnatal growth. When the binucleate tetraploid hepatocyte initiates the next cell cycle, it can initiate DNA replication again, leading either to successful cytokinesis with the generation of two mononucleate tetraploid hepatocytes (4n) or to defective cytokinesis with one binucleate octoploid hepatocyte generation (2 × 4n) (Guidotti et al., 1909). Polyploid hepatocytes contain extra centrosomes that can form multipolar spindles during mitotic division, thereby producing progeny with reduced ploidy. In this process, frequent chromosome mis-segregation occurs and causes structural rearrangements of chromosomes and the formation of aneuploid hepatocytes (Fig. 3). Guidotti et al. (1909) demonstrated that cytokinesis failure might derive from deficiencies of actin cytoskeleton reorganization. Consequently, astral microtubules cannot contact the cortex and convey molecular signals properly, interfering with the activation of the RhoA pathway, which causes cytokinesis defect and binucleated tetraploid hepatocyte formation (Margall-Ducos et al., 2007).

Insulin has been thought as a critical regulator in the appearance of binucleated polyploid hepatocytes. How does insulin regulate polyploidy development? Insulin signaling inhibition drastically hindered the formation of binuclear polyploid hepatocytes, whereas injection with insulin promoted the generation of polyploid cells (Nakatani et al., 1997; Gorla et al., 2001). Evidence shows failed cytokinetic events in hepatocytes after weaning might be modulated by insulin via PI3K-AKT pathway (Celton-Morizur et al., 2009; Celton-Morizur et al., 2010). AKT activity inhibition in hepatocytes boosts the actin cytoskeleton reorganization during mitosis and promotes successful localization of RhoA to the dividing area, which is required for cytokinesis (Celton-Morizur et al., 2009; Yoshida et al., 2009; Celton-Morizur et al., 2010). Although cytokinesis failure is a crucial mechanism for hepatocyte polyploidy, cell fusion was also observed in this process and can be one possible explanation for the formation of polyploid hepatocytes during postnatal liver growth (Faggioli et al., 2008; Faggioli et al., 2011). Further research suggests that hepatocyte–hepatocyte fusion in adult mice is negligible (Willenbring et al., 2004).

Oxidative damage may be another mechanism for hepatocytes polyploidization, a mechanism also discussed in heart ploidy control. Oxidative stress induced by pathological changes in nonalcoholic fatty liver activates G₂/M DNA damage checkpoint, eventually leading to highly polyploid hepatocytes. However, antioxidant treatment blocks the initiation of the G₂/M DNA damage checkpoint, restoring normal cell division and returning to a normal ploidy profile (Gentric et al., 2015). Oxidative DNA injury induced by radiation exposure and antioxidant depletion causes an increase in polyploid hepatocytes number and impair cellular proliferation capacity (Gorla et al., 2001; Malhi et al., 2002), in line with Nakatani’s study that endogenous antioxidant enzymes overexpression in transgenic mice significantly reduced hepatocyte polyploidy (Nakatani et al., 1997).

In addition to the insulin pathway and oxidative stress, other factors are also involved in the modulation of hepatic polyploidy. Table 2 summarizes key genes and stimuli that either promote or hinder polyploidy formation in the liver. In adults, hepatocyte polyploidy is also associated with other cellular stress, including metabolic overloads such as iron or copper (Madra et al., 1995; Muramatsu et al., 2000), telomere dysfunction (Lazzerini Denchi et al., 2006), and chronic hepatitis B/C infection (Toyoda et al., 2005; Toyoda et al., 2006). All these findings indicated a strong correlation between the genesis of polyploid hepatocytes and cellular stress, providing new insight for comprehending liver development and regeneration. Besides, E2f7/8 transcription factors are essential for hepatocyte polyploid. One astonishing finding is that loss of E2f7/8 has no impact on liver differentiation, metabolism, and regeneration (Pandit et al., 2012), raising the possibility that polyploid is dispensable for normal hepatocyte function. E2f7/8 also controls PIDDosome function, which is critical to hepatocyte polyploid regulation (Sladky et al., 2020a; Sladky et al., 2021). The PIDDosome is a protein complex containing the death domain-containing protein PIDD1 and the adaptor protein RAIDD, both required to activate CASP2 in response to centrosome amplification (Tinel and Tschopp, 2004; Sladky and Villunger, 2020). Extra centrosomes activate the PIDDosome–p53–p21 axis in hepatocytes after the failure of cytokinesis (Sladky et al., 2020a). After PIDDosome formation is initiated, CASP2 is activated which inactivates the E3 ligase MDM2 to stabilize P53 and its target gene P21. Consequently, this will lead to cell cycle arrest to limit hepatocyte polyploidy (Oliver et al., 2011; Fava et al., 2017; Sladky et al., 2020a). Consistently, the loss of CASP2, PIDD1, or RAIDD resulted in increased hepatocyte polyploidy (Sladky et al., 2020a), and protection from hepatocellular carcinoma, which indicates the PIDDosome might be a potential therapeutic target to control hepatocyte polyploidy for hepatocellular carcinoma prevention (Sladky et al., 2020b). Recent findings show that extra centrosomes are indeed the cue activating the PIDDosome, as hepatocytes lacking the master regulator of centriole biogenesis, Plk4, show also increased ploidy, as do hepatocytes that lack the connecting element between centrosomes and PIDD1, that is, Ankrd26 (Sladky et al., 2022).

Polyploidy produces genetic diversity in liver

Diploid hepatocytes can become polyploid by increasing their DNA content; however, polyploid hepatocytes can also develop multipolar mitotic spindles and form lower ploid hepatocytes. In some cases, cell division can produce offspring with lower ploidy states referred to as “ploidy reduction” (Guidotti et al., 1909; Duncan et al., 2010), which describes that hepatocytes can increase and then decrease their ploidy subsequently (Fig. 3). Although only a tiny proportion of polyploid hepatocytes might undergo ploidy reversal in vitro (Duncan et al., 2010), this seems to happen more extensively in vivo, where most polyploid hepatocytes experience ploidy reversal when forced to divide (Duncan et al., 2010). A multipolar mitotic spindle formation is often associated with chromosome segregation errors. In this case, the division of polyploid hepatocytes caused random and unbiased whole chromosome gains/losses followed by aneuploidy emergence (Duncan et al., 2010; Faggioli et al., 2011; Duncan et al., 2012b) (Fig. 3). This unique feature is relevant to cells continuously challenged by stress, which may lead to genomic instability (Faggioli et al., 2011). Therefore, ploidy reversal presumably creates genetic diversity and contributes to hepatocyte adaptation in response to a xenobiotic or nutritional injury (Duncan et al., 2010, 2012b), thereby reducing polyploid hepatocyte proportion to match the low level of the youthful liver and augmenting the proliferative capacity (Wang et al., 2014).

Genomic instability has long been considered a cause of cancer. For this reason, it was a surprise that the liver contains a significant number of aneuploid cells (Duncan et al., 2010, 2012b). Aneuploidy appeared randomly in healthy mice as all chromosomes were affected equally (Duncan et al., 2012a). Aneuploidy is also found in many healthy human tissues lacking evidence of malignancy, including skin, brain, ear, and kidney (Turker et al., 2004; McConnell et al., 2013; Knouse et al., 2014). Although aneuploidy is strongly correlated with cancer occurrence, Fujiwara et al. (2005) report that spontaneous liver cancer happens due to aneuploidy is quite rare (Chandra and Frith, 1992), challenging the concept of a causal link between aneuploid and cancer. Polyploid hepatocytes even facilitate liver regeneration after injury while undergoing ploidy reduction and subsequent re-polyploidy (Matsumoto et al., 2020). These findings further suggest that hepatocyte’s ploidy reversal and aneuploid were features of the normal liver. In some cases, aneuploidy seems to be a mechanism of adaptation to liver injury associated with the metabolic disorder (Duncan et al., 2012a). The loss of polyploidy in the liver resulted in decreased aneuploidy and impaired their ability to adapt to tyrosinemia-induced liver failure (Wilkinson et al., 2019a), indicating that polyploid hepatocytes are needed for the formation of aneuploidy offspring and is an important modulator that facilitates adaptation to chronic liver disease.

Up to now, it remains unclear how aneuploidy affects liver fate. Polyploid hepatocytes are less likely to be influenced by the gain/loss of one or more chromosomes, while aneuploid hepatocytes with two incomplete sets of chromosomes might change cellular function. For instance, losing one single homolog may create substantial genetic diversity. Some of the genes are monoallelically expressed, which might imply that chromosomal monosomies may result in the functional deprivation of many genes (Gimelbrant et al., 2007). Future studies are needed to explore the molecular mechanisms that control the ploid conveyor and how hepatocyte aneuploidy influences human health in physiological or pathological conditions.

Polyploidy enhances liver functions

What is the advantage of polyploidy over diploidy during liver growth and regeneration? Numerous hypotheses have been proposed to account for the functional significance of polyploidy in the liver. The first hypothesis was that hepatocyte polyploidy is relevant to terminal differentiation and aging (Sigal et al., 1995). However, hepatocytes can undergo ploidy reversal, indicating that hepatocyte polyploidy does not necessarily mean cellular senescence. For instance, tetraploid hepatocytes still sustain highly regenerative capacity after partial hepatectomy (Miyaoka et al., 2012), and FACS-isolated polyploid hepatocytes proliferated extensively in vivo transplantation and in vitro studies (Overturf et al., 1999; Weglarz et al., 2000; Duncan et al., 2009). Accordingly, aging and terminal differentiation fail to accurately describe the appearance of polyploid hepatocytes. The second consideration is that hepatocyte polyploidy might be a protective mechanism against oxidative stress (Lu et al., 2007). Antioxidants are linked to a lower degree of polyploidy in the liver (Sanz et al., 1995), while oxidative injury can directly induce hepatocyte polyploidy (Gorla et al., 2001). Additionally, a series of genes are related to hepatocyte polyploidy; most of these genes reflect metabolism changes and DNA damage responses to oxidative stress (Anatskaya and Vinogradov, 2007; Lu et al., 2007). This supports the view that hepatocyte polyploidy is a normal process that is helpful to oxidative stress resistance and maintains the detoxification capacity. However, a study in yeast suggested that increased ploidy does not provide an extra benefit for survival under the condition of DNA damage agents (Mable and Otto, 2001). Another assumption for hepatocyte polyploidy might result from a positive response to postnatal growth rate and metabolic load. For instance, hepatocyte polyploidy increased when the growth rate is stimulated by thyroid hormone additives (Torres et al., 1999). Thus, possibly when resources are limited, it can channel the energy that may flow into cell division toward other purposes (Anatskaya et al., 1994; Vinogradov et al., 2001). This might be particularly favorable to rapid-growing tissues as it could facilitate adaptation to different stresses. Correspondingly, high-energy consumption was found in parallel to the changes in hepatocyte polyploidy during the suckling–weaning period (Celton-Morizur et al., 2009).

Studies in the mammalian heart and liver show a link between polyploidy and coordination of functional gene expression, which could promote cell survival and tissue regeneration under stressful conditions (Sher et al., 2013). Polyploidy can alter the gene expression profile that may contribute to improving liver function as polyploidy might enhance translation, for example, providing twice as many genes might generate twice as many proteins. However, this is not always the case. Polyploidy can also promote nonuniform genome, transcriptome, and metabolome alterations, which probably get things complicated (Schoenfelder and Fox, 2015). On the other hand, polyploid cells are generally twice as big, so twice as many genes generate twice as many proteins in twice as much cellular volume. Therefore, this may not have any real impact compared to two diploid cells. Microarray analysis was conducted with the same amounts of RNA from diploid, tetraploid, and octoploid hepatocytes of mice and demonstrated that no major changes in gene expression patterns among different ploidy hepatocytes could be observed (Lu et al., 2007). Gene expression profiles vary between diploid and mononucleate polyploid hepatocytes but not between diploid and binucleate polyploid hepatocytes (Kreutz et al., 2017), indicating that nuclear ploidy in liver influences hepatocyte fate rather than the total ploidy within a cell. Intriguingly, low- and high-ploidy hepatocytes are equally sensitive to CCl4-induced cell death. Since hyperpolyploid hepatocytes are larger, their death results in a greater loss of functional parenchyma (Sladky et al., 2022).

Polyploid in other organs

Retinal pigmented epithelial cells

Nearly 3% retinal pigmented epithelial (RPE) cells in humans are binucleated, the majority are multinucleated, which is in line with rod and cone photoreceptor density (Starnes et al., 2016). In rodents, the proportion of multinucleated RPE cells is more than 80%, and the amount increases with age (Chen et al., 2016). Interestingly, the anatomical region seems to be the critical reason for the number of multinucleated RPE cells in mice rather than age. The multinucleated RPE is related to the rods’ ratio, which could be an adaption to nocturnal vision. Multinucleated RPE cells exhibited elevated ROS production and DNA damage following irradiation in rodents (Ke et al., 2022), in line with previous research that multinucleated human RPE1 cells presented more γH2Ax-marked DNA damage (Hart et al., 2021). So far, the potential mechanisms for increased DNA damage in multinucleated RPE remain unknown.

Evidence revealed that newly developed RPE cells are mainly derived from remaining peripheral RPE cells, similar to the observation in cardiomyocytes (Hanovice et al., 2019). The majority of diploid RPE cells are found in the periphery of the retinal eye cup, and this proportion gradually drops when it moves to the central area, where polyploid cells expand with age (Al-Hussaini et al., 2008; Chen et al., 2016). Interestingly, the renewal of RPE cells either in physiological conditions or after an injury is mostly confined to the peripheral territory in which diploid RPE cells reside instead of the central area where polyploid RPE cells exist, suggesting that there might be a correlation between DNA content and regenerative capacity (Al-Hussaini et al., 2008; Chen et al., 2016).

Pancreatic β-cells

The pancreas exerts a crucial role in regulating carbohydrate metabolism. Notably, polyploid in the pancreas occurs after weaning, coinciding with the adaption to hypertrophic growth (Finegood et al., 1995; Matondo et al., 2018). Pancreatic β-cells belong to endocrine cells and are surprisingly similar to cardiomyocyte division, as they are tightly interconnected and electrically excitable. Cell division in these cells probably impairs the interconnection and normal organ function (Meissner, 1976). As mentioned above, E2f7/8 are pivotal regulators of hepatocyte polyploidy. To determine if E2f7/8 can regulate the formation of polyploid cells in the pancreas, we used tamoxifen-inducible mouse models to assess the effect of postnatal E2f7/8 gene deletion. To our surprise, decreased polyploidy, caused by E2f7/8 genes deletion, did not show any striking consequence on postnatal growth and survival as well as the production of pancreatic hormones (Matondo et al., 2018). Instead, E2f1/2-deficient mice are more likely to have increased polyploidy, diabetes, and exocrine pancreas dysfunction (Iglesias-Ara et al., 2015). Loss of P53 inhibited cell apoptosis and improved pancreas dysfunction and diabetes in mice lacking E2f1/2, indicating that P53 may be required for tissue homeostasis and tumorigenesis prevention (Iglesias-Ara et al., 2015).

No convincing evidence revealed the link between DNA content and proliferative capacity in pancreatic cells. Nevertheless, a higher percentage of polyploid β-cells were observed in hyperglycemic states, which was interpreted as reflecting premature aging of β-cells (White et al., 1985). In addition, endoreplication-based hypertrophy without cell division was insufficient to maintain β-cell mass and compensate for increased metabolic demands (Zhong et al., 2007), suggesting that increased polyploid in β-cells may partly impair their proliferative capacity.

Keratinocytes

By investigating the underlying mechanism of the formation of keratinocyte polyploidy, researchers observed that oncogenic alterations such as P53 suppression, or genotoxic drugs can promote DNA damage that ultimately results in terminal differentiation and polyploidy (Di Micco et al., 2006; Freije et al., 2014). Continuous activation of c-Myc leads to mitosis block, endoreplication, and polyploidy in keratinocytes by inducing Cyclin E (Gandarillas et al., 2000; Freije et al., 2012). Inactivation of CDK1 or kinases of the mitosic spindle checkpoint, Aurora B or Aurora A, cell cycle kinases that can trigger keratinocyte terminal differentiation and polyploidy (Freije et al., 2012; Wen et al., 2012; Perez de Castro et al., 2013). Human papillomavirus type 16 proteins E6 and E7 can induce centrosome amplification and mis-segregation of chromosomes during mitosis, and eventually result in polyploidy in primary human keratinocytes, which may attribute to the disruption of the spindle checkpoint (Patel et al., 2004). The most attractive issue is why keratinocytes would accept to become polyploid and whether polyploidy endows additional functional support for the epidermis. Possibly, polyploid cells present stronger resistance than diploid cells in response to mechanical stress, and the rigidity given by keratins may restrict normal cytokinesis leading to cell division failure, similar to sarcomeric actin in the heart. As we know, keratinocytes are more exposed to harmful radiation. Therefore, it would not be surprising that keratinocytes attempt to rescue genomic instability by adopting a polyploid strategy.

Mammary gland cells

The mammary gland is a unique model to study how an organ alters its structure to match its physiological demands. At the beginning of gestation in mice, mitotic activity in the epithelial cells of the mammary gland is elevated, and the number of binucleated epithelial cells increases from the 18th day of gestation. From the 3rd to 18th day of lactation, the nuclear volume in mononucleated and binucleated epithelial cells increases, accompanied by higher output of mammary gland cells (Kriesten, 1984). Earlier findings (Banerjee et al., 1971; Banerjee and Wagner, 1972) indicated that DNA synthesis proceeds in the absence of commensurate cell division during late pregnancy and lactation, which is critical to the functional differentiation of the mammary epithelium. Milk production in nulliparous mouse mammary epithelium heavily relies on epithelial DNA synthesis capacity (Vonderhaar et al., 1978). Later studies suggested that mitotic events are irrelevant to the interdependence of DNA synthesis and milk production in nulliparous mouse mammary epithelium (Smith and Vonderhaar, 1981). Polyploid cells are also observed in lactating mammary glands in different species, including humans, cows, seals, and wallabies (Rios et al., 2016). Most of the secretory alveolar cells are binucleated, which occurs in the late stage of pregnancy because of cytokinesis failure. In mice, Aurora kinase A and Plk1 can induce the formation of polyploid cells via defective cytokinesis during the lactogenic switch. The number of polyploid cells decreases with the deprivation of Aurora kinase A signal resulting in decreased milk production (Rios et al., 2016), suggesting that polyploid mammary epithelial cells facilitate milk production and augment the survival of offspring, consistent with Smith’s study (Smith and Medina, 1988) that DNA synthesis and lactation are linked.

Subperineurial glia cells

Regulation of cell size is pivotal in organ development. The size of the nervous system increases dramatically during Drosophila larval development, while the number of SPG cells stays the same, most SPG cells become polyploid, and appear to be responsible for the growth of the nervous system (Unhavaithaya and Orr-Weaver, 2012). Inhibition of DNA replication could reduce SPG cell polyploidy and cell size, causing rupture of the septate junctions required for the blood–brain barrier formation, indicating that increased SPG cell size induced by polyploidy is needed to sustain a functional blood–brain barrier (Unhavaithaya and Orr-Weaver, 2012). miR-285 can decrease the expression of its downstream target cyclin E in SPG and triggers abnormal endoreplication, eventually leading to an aberrant DNA ploidy state and defective septate junctions (Li et al., 2017). Interestingly, SPG underwent two cell cycle changes during development, inducing endoreplication but restoring nuclear division capacity to increase nuclear content, which contributes to an increase in cell size (Von Stetina et al., 2018). Polyploid appears to be a tactic to coordinate tissue growth during organ development. The growing rate of DNA synthesis meets the increased metabolic demands as SPG gets larger during development (Unhavaithaya and Orr-Weaver, 2012). However, no clear relationship between cell size and DNA content was observed among different segmental nerves in SPG, indicating that this regulation might not be stringent as expected (Zulbahar et al., 2018).

Regeneration

Polyploid and heart regeneration

The adult mammalian heart is a postmitotic organ and incapable of regeneration after injury (Li et al., 1996; Soonpaa et al., 1996). However, some evidence challenged this dogma and showed a low level of endogenous turnover of cardiomyocytes, estimated at around 1% per year in both adult mice (Senyo et al., 2013; Ali et al., 2014) and humans (Bergmann et al., 2009), even displaying a slight increase after myocardial infarction (Beltrami et al., 2001). The adult mammalian cardiomyocyte can hardly divide, but some vertebrates can. Adult zebrafish and newts can regenerate damaged hearts via cardiomyocyte division (Oberpriller and Oberpriller, 1974; Poss et al., 2002; Wills et al., 2008). Cardiomyocytes in the embryonic heart of mice are diploid and proliferative, whereas neonatal mice can also produce new cardiomyocytes within the first week after birth through the division of pre-existing cardiomyocytes. Subsequently, most cardiomyocytes undergo cytokinesis failure, leading to increased binucleated polyploid cardiomyocytes, accompanied by the loss of regenerative ability in the heart (Soonpaa et al., 1996; Porrello et al., 2011; Senyo et al., 2013). One major difference between these species is the ploidy of cardiomyocytes. Zebrafish and newt cardiomyocytes are exclusively diploid throughout life (Patterson et al., 2017), whereas adult mammalian cardiomyocytes are primarily polyploid, which occurs 1 week after birth (Soonpaa et al., 1996). Taken together, it seems that the transition of mouse cardiomyocytes from diploid to polyploid coincides with the loss of heart regeneration potential, similar to the observations in the human heart (Mollova et al., 2013; Ye et al., 2016) (Fig. 4). Of course, many other cellular changes emerge in the same neonatal window, including a rapid metabolic switch from glycolysis to fatty acid oxidation, an increase in oxygen consumption (Bartelds et al., 2000). So far, little is known about how these events contribute to cell cycle arrest and eventually result in cellular polyploidy.

Regenerative properties of the heart in mice largely rely on the number of diploid cardiomyocytes based on the analysis of the regenerative potential in different mouse strains (Patterson et al., 2017), in line with previous observation (Bersell et al., 2009). These findings led to the assumption that ploidy may play a crucial role in cardiomyocyte proliferative potential, further confirmed by several experimental approaches. A transgenic strategy by transient cytokinesis inhibition, modulated by dominant-negative Ect2, was used to induce a high degree of polyploid cardiomyocytes in zebrafish. Cardiomyocytes remained polyploid even after removing Ect2 repression. Gonzalez-Rosa et al. (2018) demonstrated that conversion from diploid to polyploid in cardiomyocytes hindered regenerative capacity. Consequently, zebrafish are not able to regenerate injured hearts effectively or undergo ploidy reversal.

Similarly, in another study (Patterson et al., 2017), researchers observed that Tnni3k knockout mice presented a higher number of diploid cardiomyocytes as well as strengthened heart regeneration potential, while overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploid and compromised the capacity to regenerate, indicating that a critical proportion of diploid cardiomyocytes is needed to support heart regeneration. Inhibition of thyroid hormone receptors increased the number of diploid (nearly 2-folds) cardiomyocytes and improved myocardial regenerative ability, whereas animals treated with T3 hormone increased the percentage of binucleated cardiomyocytes (around 3-folds), followed by an interrupted cardiac regeneration (Hirose et al., 2019). All these results demonstrated compelling evidence that polyploidy creates a barrier to heart regeneration. However, ploidy is unlikely to be the only determining factor of heart regeneration because medaka fish could not achieve heart regeneration after myocardial injury with diploid cardiomyocytes (Lai et al., 2017).

Notably, these studies did not rule out the possibility that polyploid cardiomyocytes might also be able to support heart regeneration with appropriate stimulation. Several studies showed that binucleated cardiomyocytes can undergo complete cell division (Wang et al., 2017; Leone and Engel, 2019). Binucleated cardiomyocytes can be genetically driven to complete cytokinesis via β-catenin-induced expression of Ect2 (Jiang et al., 2019). Engel et al. (2005) demonstrated that binucleated cardiomyocytes can divide into two mononucleated after being stimulated by P38-MAPK inhibition. In his study, a cell-tracing experiment was performed by utilizing cultured adult newt cardiomyocytes, which revealed that multinucleated cells have the capacity for cytokinesis and mitotic division. Some of the cells that undergo one complete cycle can also progress through several rounds of successful cell division (Bettencourt-Dias et al., 2003). This was consistent with D’Uva’s findings which were based on live-cell imaging of neonatal mouse cardiomyocytes and showed that 3.9% of binucleated cardiomyocytes can form two mononucleated daughter cells via Erbb2 (D’Uva et al., 2015). However, this may challenge the Bersell et al.’s (2009) observation that only mononuclear cardiomyocytes could complete cytokinesis after Neuregulin 1 induction, rather than binucleated polyploid cardiomyocytes. All these evidences illustrate that binucleated cardiomyocytes still preserve proliferation potential. Nonetheless, it is still poorly understood if mononucleated polyploid cardiomyocytes can proliferate and how many binucleated cardiomyocytes can be induced to divide. Of note, these observations are based on an in vitro microscopy study, which is a reliable way to detect cell division through cytokinesis. However, cell culture lacks interaction with other cells and tissues, which may also affect cardiomyocytes’ senescence state, possibly making them behave differently when tested in vivo.

Mechanical stretching of epicardial cells can also induce endoreplication through tension and promotes regeneration of epicardial tissue (Cao et al., 2017). In these endoreplicating polyploid cells, the formation of cleavage furrow works normally, while tension causes defective cell abscission during cytokinesis, impairing midbody formation and abscission of the cleavage furrow, consistent with the previous observation that tension hinders cytokinetic abscission by perturbing the assembly of the ESCRT-III complex which is essential for successful abscission (Herget et al., 1997). Moreover, those polyploid cells appear to repair injured hearts more efficiently than diploid cells. In zebrafish, cardiomyocytes do not display polyploidy but upon surgical resection they disassemble their sarcomeric structure and detach from one another, attempting to facilitate cell cycle reentry (Duncan et al., 2012b). Genome-wide transcriptome profile analysis further confirmed this with the finding that the downregulation of sarcomeric-associated genes occurs after injury (Saeter et al., 1988). Newt hearts also undergo a massive downregulation of sarcomeric genes following amputation. As heart regeneration proceeds, the sarcomeric gene expression rises to normal levels 14 days after damage (Duncan et al., 2010). Although this disassembly process might cause mechanical uncoupling among cardiomyocytes and possibly impair the myocardial contractile power, the regenerative capacity in zebrafish and newts is still not compromised, which might attribute to their low blood pressure demands.

In conclusion, the biological function of cardiomyocyte polyploidy for heart development and regeneration is still unclear. Are the underlying mechanisms of polyploid cardiomyocytes the same during injury and homeostasis? What kind of characteristics are inherited by the daughter cells? Are there other different behaviors between polyploid and diploid cardiomyocytes? More investigations are therefore required to elucidate these issues in the future. One more important thing that needs to be noticed is, the traditional markers of cardiomyocyte proliferation applied in these studies, such as Ki67, pHH3. and Aurora B Kinase are inadequate to claim authentic cardiomyocyte proliferation. Ki67 is a marker indicating cell cycle progression (Gerdes et al., 1983; Duchrow et al., 1996) and used to be a compelling marker for assessing cellular proliferation. However, Ki67 might not be as reliable as we anticipated because cell cycle activity in adult cardiomyocytes is related to cell proliferation and polyploid state (Bersell et al., 2009). For instance, cardiomyocytes undergoing endoreduplication can also be detected with Ki67 positive without real cell division (Hesse et al., 2012), even in hypertrophy hearts (Drenckhahn et al., 2015). Thus, Ki67 can appear in both proliferative and non-proliferative cell cycles. pHH3 is a protein phosphorylated during chromatin condensation in mitosis and can evaluate mitotic activity (Hightower et al., 2012). Nevertheless, mitosis activity does not mean genuine cell division as it can result in polyploid because of cytokinesis defects (Meckert et al., 2005; Engel et al., 2006). Even Aurora B Kinase, involved in the formation of contractile rings at the region of cytoplasmic separation, is viewed as a convincing marker of cytokinesis (Crosio et al., 2002; Engel et al., 2005). Unfortunately, it is also not reliable for cell division, as various defects can still lead to cytokinesis failure and furrow regression with an Aurora B-positive midbody formation (Steigemann et al., 2009; Carlton et al., 2012). Given that no satisfying marker has yet been identified and can be used to reliably assess cell division, the published conclusions which were based on these immunohistology staining methods for evaluating different mitotic events in myocardial regeneration raise some concerns. More advanced approaches need to be developed to show authentic cardiomyocyte division for future clinical application.

Liver polyploid and proliferation

Hepatocytes are generally quiescent with a slow renewal rate (Zajicek et al., 1985). Hepatocytes seem not fully responsive to key cytokines and growth factors in their quiescent state, such as TNF, HGF, and TGF-α, which are potent stimulators of cell cycle reentry for hepatocytes (Webber et al., 1994, 1998). Although hepatocytes are generally quiescent, they demonstrated extremely high proliferative potential upon injury (Wang et al., 2015; Chen et al., 2019). Similar to the heart arena, newly developed hepatocytes are mainly derived from pre-existing hepatocytes instead of progenitor cells (Yanger et al., 2014).

Hepatocyte ploidy state is essential in regulating liver regeneration (Wilkinson et al., 2019a). Many observations supported the assumption that the polyploid state in hepatocytes restricts their regenerative capacity (Wilkinson et al., 2019b). For example, in normal physiological conditions, hepatocytes show robust proliferative ability before weaning and this capacity gradually goes down with time; by coincidence, polyploid hepatocytes occur at weaning in rodents and increase in parallel with age (Kudryavtsev et al., 1993; Margall-Ducos et al., 2007; Celton-Morizur et al., 2009; Duncan et al., 2010). Further, liver polyploid is highly structured and proceeds more rapidly in the mid-lobule zone than in the region close to the portal and central veins, which means that the mid-lobular zone presents a higher proportion of polyploid hepatocytes compared to the portal and central veins area (Tanami et al., 2017). Two studies based on lineage tracing strategies identified a small population of hepatocytes that is capable of homeostatic liver renewal and demonstrated that these cells were mostly proximal to the central or portal veins (Font-Burgada et al., 2015; Wang et al., 2015). Notably, Wnt-responsive hepatocytes adjacent to the central vein in the liver lobule seem to be primarily diploid, responsible for proliferation and homeostatic self-renewal in the uninjured liver (Wang et al., 2015). Wilkinson et al., (2019b) found that quiescent hepatocytes were predominantly polyploid, as diploid hepatocytes can reinitiate the cell cycle and proliferate faster than polyploid hepatocytes. They determined that all ploidy populations were capable of proliferation. However, diploid hepatocytes demonstrated around a 3-fold increase in BrdU⁺ percentage compared to polyploid hepatocytes. Sigal et al., (1999) revealed that two-thirds of partial hepatectomy in rats accelerated hepatocyte senescence, attenuated proliferative capacity, and increased hepatocyte polyploidy. Earlier studies (Post and Hoffman, 1965) using tritiated thymidine incorporation into cells showed that only a few polyploid cells were labeled, displaying a prolonged S phase, which was consistent with a study showing that diploid hepatocytes have a greater capacity for DNA synthesis compared to polyploid hepatocytes (Rajvanshi et al., 1998). Although polyploid hepatocytes still maintain DNA synthesis capacity, a key concern is their declining ability to produce new cells (Gerlyng et al., 1992), which is in line with a recent study that diploid hepatocytes show higher turnover than polyploid hepatocytes (Heinke et al., 2022). Polyploid hepatocytes isolated within 5 days of partial hepatectomy are less proliferative than diploid hepatocytes (Gorla et al., 2001), suggesting that partial hepatectomy-induced polyploid hampers cell proliferation. The notion that diploid cells proliferate faster than polyploid hepatocytes is also supported by several studies which observed that diploid hepatocytes are enriched in patients and rats with various disease states (Anti et al., 1994; Rua et al., 1996; Gandillet et al., 2003).

The molecular mechanisms of why diploid hepatocytes proliferate faster than polyploid is still not well defined. In 2007, Lu et al. (2007) studied gene expression patterns between quiescent diploid and polyploid hepatocytes from mice by using a high-density genome microarray. Surprisingly, no major changes in gene expression were found, indicating that hepatocyte ploidy subsets are similar at the gene expression level. In order to test if diploid and polyploid hepatocytes had different reactions in response to hepatic mitogens, researchers treated primary wild-type hepatocytes with various growth factors related to liver regeneration. They showed diploid hepatocytes can enter and progress through the cell cycle faster, both in vitro and during liver regeneration in vivo (Rua et al., 1996).

Many mitotic hepatocytes in highly polyploid liver display extra centrosomes, indicative of polyploid hepatocyte divisions (Faggioli et al., 2011; Knouse et al., 2018; Lin et al., 2020). Researchers designed a multicolor reporter system to trace polyploid cells in situ and found that polyploid hepatocytes maintain extensive proliferative capacity in different liver damage models and routinely undergo reductive mitoses during the proliferation process (Matsumoto et al., 2020). Polyploid hepatocytes also showed sufficient proliferation capacity to maintain the functional integrity of livers in which more than 97% of hepatocytes were polyploid following injury (Lin et al., 2020). These polyploid hepatocytes function as efficiently as diploid hepatocytes in repairing injured organs. In Fah-deprivation mouse models, a model for selective liver replacement (Overturf et al., 1996), diploid hepatocytes were demonstrated to share equivalent regenerative capacity with polyploid hepatocytes after transplantation (Duncan et al., 2010). A similar phenomenon was also observed in the E2f7/8-deficienct liver which is composed of predominately diploid hepatocytes, and no significant difference in regenerative capacity was found between the wild-type and E2f7/8-deficienct mice (Pandit et al., 2012). Blocking centriole-mediated ploidy control causes a significantly increased hepatocyte polyploidy, severe liver damage, and impaired liver function (Sladky et al., 2022).

In order to identify whether binucleated hepatocytes can be triggered to undergo cell division or not, a live-cell imaging experiment was applied and showed that binucleated hepatocytes can stimulate cell cycle reentry and give rise to two tetraploid mononucleated daughter cells (Guidotti et al., 1909), which is similar to previous observations that binucleated hepatocytes tend to divide into two mononucleated daughter cells and thus, eventually, binucleated polyploid hepatocytes decreases and polyploid hepatocytes increases (Gerlyng et al., 1993; Miyaoka et al., 2012). Further, they applied a genetic tracing strategy and determined that merely half of the hepatocytes that undergo DNA synthesis ultimately complete the cell cycle and generate two mononuclear daughter hepatocytes (Lu et al., 2007), implying that most cell cycle activity is not authentic proliferation. Wilkinson et al., (2019b) revealed that the overall ploidy spectrum keeps unchanged after full liver regeneration in a partial hepatectomy model, which indicated that either increased ploidy is only a temporary phenomenon, ultimately adjusts itself, or probably exists a conversion of binucleated cells (2 × 2n) to mononucleated tetraploid cells (1 × 4n) after injury. Sigal et al., (1999) found a sharp decrease in the fraction of polyploid hepatocytes upon partial hepatectomy, which might attribute to the apoptotic consequence that preferentially targets polyploid hepatocytes rather than diploid cells. Conversely, in another study, diploid hepatocytes are more sensitive to apoptosis signaling compared to polyploid cells (Kreutz et al., 2017).

It has to be noticed that, in the liver field, researchers widely apply the word “regeneration” to describe the recovery of tissue weight and function, but it does not mean this process is always totally accompanied by bona fide cell proliferation. As reported previously, liver regeneration is primarily achieved through hypertrophy growth by polyploid hepatocytes without cell division after 30% partial hepatectomy. However, proliferation and hypertrophy are equally important to liver regeneration after 70% hepatectomy. Although not all polyploid hepatocytes undergo cell division, binuclear hepatocytes can still divide to increase cell numbers (Miyaoka et al., 2012). Additionally, blocking mitosis through CDK1 deletion in mouse liver does not affect hepatic regeneration after 70% partial hepatectomy, indicating that regeneration can be accomplished by hypertrophic growth of polyploid cells (Diril et al., 2012). A close parallel was found between liver mitotic index and a decrease in polyploid hepatocytes (Wheatley, 1972). As an example, the mouse liver presents a lower mitotic index level accompanied by a higher degree of hepatocytes polyploid, while the opposite occurred in the rat liver. The liver is a fascinating organ that maintains robust regenerative capacity and allows the substantial presence of polyploid and aneuploid hepatocytes. Further investigations will help disclose the polyploid hepatocyte’s biological function during liver development and regeneration.

Conclusion

Polyploid is essential for cell growth and tissue regeneration. For instance, polyploid plays a vital role in mammal heart and liver regeneration via various cellular mechanisms and may be a promising strategy for tissue regeneration and functional recovery in clinical settings. To date, there are still several issues required to be addressed. One of the mysteries is why some polyploid cells, such as hepatocytes, can undergo successful division, whereas it is limited in others, like adult mammalian cardiomyocytes. Are there any differences in cellular levels between diploid and polyploid hepatocytes (including epigenetics, transcriptomics, and proteomics)? In addition, the advantages of multinucleated cells over mononucleated polyploid cells are still poorly understood. Perhaps an additional genome-to-nuclear surface ratio in multinucleated cells benefits cell growth or function. In the future, it will be critical to determine how the cell ploidy state is manipulated and how it affects cellular and tissue function, as a deeper understanding of polyploid will eventually benefit patients.

In short, by modulating some candidate genes or conditions, we can change polyploidy state and promote the repair and regeneration of organs such as liver and heart following injury in rodents. In this case, we believe these findings will provide a rationale for identifying therapeutic approaches to change organ ploidy state in future clinical trials, in particular in the human heart, as this might be a means to enhance heart regeneration in disease states.

References

[1] Adler CP, Friedburg H. Myocardial DNA content, ploidy level and cell number in geriatric hearts: post-mortem examinations of human myocardium in old age. J Mol Cell Cardiol 1986;18:39–53.

[2] Adler CP, Friedburg H, Herget GW et al. Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts. Virchows Arch 1996;429:159–164.

[3] Ahuja P, Perriard E, Trimble W et al. Probing the role of septins in cardiomyocytes. Exp Cell Res 2006;312:1598–1609.

[4] Aix E, Gutierrez-Gutierrez O, Sanchez-Ferrer C et al. Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation. J Cell Biol 2016;213:571–583.

[5] Al-Hussaini H, Kam JH, Vugler A et al. Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo. Mol Vis 2008;14:1784–1791.

[6] Ali SR, Hippenmeyer S, Saadat LV et al. Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice. Proc Natl Acad Sci U S A 2014;111:8850–8855.

[7] Anatskaya OV, Vinogradov AE. Paradoxical relationship between protein content and nucleolar activity in mammalian cardiomyocytes. Genome 2004;47:565–578.

[8] Anatskaya OV, Vinogradov AE. Genome multiplication as adaptation to tissue survival: evidence from gene expression in mammalian heart and liver. Genomics 2007;89:70–80.

[9] Anatskaya OV, Vinogradov AE, Kudryavtsev BN. Hepatocyte polyploidy and metabolism/life-history traits: hypotheses testing. J Theor Biol 1994;168:191–199.

[10] Anatskaya OV, Vinogradov AE, Kudryavtsev BN. Cardiomyocyte ploidy levels in birds with different growth rates. J Exp Zool 2001;289:48–58.

[11] Anti M, Marra G, Rapaccini GL et al. DNA ploidy pattern in human chronic liver diseases and hepatic nodular lesions. Flow cytometric analysis on echo-guided needle liver biopsy. Cancer 1994;73:281–288.

[12] Banerjee MR, Wagner JE. Gene amplification in mammary gland at differentiation. Biochem Biophys Res Commun 1972;49:480–487.

[13] Banerjee MR, Wagner JE, Kinder DL. DNA synthesis in the absence of cell reproduction during functional differentiation of mouse mammary gland. Life Sci II 1971;10:867–877.

[14] Barbera A, Giraud GD, Reller MD et al. Right ventricular systolic pressure load alters myocyte maturation in fetal sheep. Am J Physiol Regul Integr Comp Physiol 2000;279:R1157–R1164.

[15] Barnard DR, Lange B, Alonzo TA et al. Acute myeloid leukemia and myelodysplastic syndrome in children treated for cancer: comparison with primary presentation. Blood 2002;100:427–434.

[16] Bartelds B, Knoester H, Smid GB et al. Perinatal changes in myocardial metabolism in lambs. Circulation 2000;102:926–931.

[17] Beltrami AP, Urbanek K, Kajstura J et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med 2001;344:1750–1757.

[18] Bensley JG, De Matteo R, Harding R et al. Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. Sci Rep 2016;6:23756.

[19] Bergmann O, Bhardwaj RD, Bernard S et al. Evidence for cardiomyocyte renewal in humans. Science 2009;324:98–102.

[20] Bergmann O, Zdunek S, Felker A et al. Dynamics of Cell Generation and Turnover in the Human Heart. Cell 2015;161:1566–1575.

[21] Bersell K, Arab S, Haring B et al. Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury. Cell 2009;138:257–270.

[22] Bettencourt-Dias M, Mittnacht S, Brockes JP. Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes. J Cell Sci 2003;116:4001–4009.

[23] Bicknell KA, Coxon CH, Brooks G. Forced expression of the cyclin B1-CDC2 complex induces proliferation in adult rat cardiomyocytes. Biochem J 2004;382:411–416.

[24] Bogart JP. Evolutionary implications of polyploidy in amphibians and reptiles. Basic Life Sci 1979;13:341–378.

[25] Brito DA, Rieder CL. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr Biol 2006;16:1194–1200.

[26] Brodsky V, Sarkisov DS, Arefyeva AM et al. Polyploidy in cardiac myocytes of normal and hypertrophic human hearts; range of values. Virchows Arch 1994;424:429–435.

[27] Burigotto M, Mattivi A, Migliorati D et al. Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26. EMBO J 2021;40:e104844.

[28] Cao J, Wang J, Jackman CP et al. Tension creates an endoreplication wavefront that leads regeneration of epicardial tissue. Dev Cell 2017;42:600–615

[29] Carlton JG, Caballe A, Agromayor M et al. ESCRT-III governs the Aurora B-mediated abscission checkpoint through CHMP4C. Science 2012;336:220–225.

[30] Celton-Morizur S, Merlen G, Couton D et al. The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents. J Clin Invest 2009;119:1880–1887.

[31] Celton-Morizur S, Merlen G, Couton D et al. Polyploidy and liver proliferation: central role of insulin signaling. Cell Cycle 2010;9:460–466.

[32] Chandra M, Frith CH. Spontaneous neoplasms in aged CD-1 mice. Toxicol Lett 1992;61:67–74.

[33] Chattergoon NN, Giraud GD, Louey S et al. Thyroid hormone drives fetal cardiomyocyte maturation. FASEB J 2012a;26:397–408.

[34] Chattergoon NN, Louey S, Stork P et al. Mid-gestation ovine cardiomyocytes are vulnerable to mitotic suppression by thyroid hormone. Reprod Sci 2012b;19:642–649.

[35] Chen HZ, Ouseph MM, Li J et al. Canonical and atypical E2Fs regulate the mammalian endocycle. Nat Cell Biol 2012;14:1192–1202.

[36] Chen M, Rajapakse D, Fraczek M et al. Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis. Aging Cell 2016;15:436–445.

[37] Chen Y, Hata T, Rehman F et al. Visualization of Hepatocellular Regeneration in Mice After Partial Hepatectomy. J Surg Res 2019;235:494–500.

[38] Chipchase MD, O’Neill M, Melton DW. Characterization of premature liver polyploidy in DNA repair (Ercc1)-deficient mice. Hepatology 2003;38:958–966.

[39] Clubb FJ, Bishop SP. Formation of binucleated myocardial cells in the neonatal rat. An index for growth hypertrophy. Lab Invest 1984;50:571–577.

[40] Conner EA, Lemmer ER, Sanchez A et al. E2F1 blocks and c-Myc accelerates hepatic ploidy in transgenic mouse models. Biochem Biophys Res Commun 2003;302:114–120.

[41] Cookson SJ, Radziejwoski A, Granier C. Cell and leaf size plasticity in Arabidopsis: what is the role of endoreduplication? Plant Cell Environ 2006;29:1273–1283.

[42] Crosio C, Fimia GM, Loury R et al. Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol Cell Biol 2002;22:874–885.

[43] D’Uva G, Aharonov A, Lauriola M et al. ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation. Nat Cell Biol 2015;17:627–638.

[44] Davoli T, Denchi EL, de Lange T. Persistent telomere damage induces bypass of mitosis and tetraploidy. Cell 2010;141:81–93.

[45] De Santis Puzzonia M, Cozzolino AM, Grassi G et al. TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure. PLoS One 2016;11:e0167158.

[46] Dikovskaya D, Schiffmann D, Newton IP et al. Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis. J Cell Biol 2007;176:183–195.

[47] Di Micco R, Fumagalli M, Cicalese A et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 2006;444:638–642.

[48] Diril MK, Ratnacaram CK, Padmakumar VC et al. Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration. Proc Natl Acad Sci U S A 2012;109:3826–3831.

[49] Di Stefano V, Giacca M, Capogrossi MC et al. Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle. J Biol Chem 2011;286:8644–8654.

[50] Drenckhahn JD, Strasen J, Heinecke K et al. Impaired myocardial development resulting in neonatal cardiac hypoplasia alters postnatal growth and stress response in the heart. Cardiovasc Res 2015;106:43–54.

[51] Duchrow M, Schluter C, Wohlenberg C et al. Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67. Cell Prolif 1996;29:1–12.

[52] Duncan AW, Hickey RD, Paulk NK et al. Ploidy reductions in murine fusion-derived hepatocytes. PLoS Genet 2009;5:e1000385.

[53] Duncan AW, Taylor MH, Hickey RD et al. The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 2010;467:707–710.

[54] Duncan AW, Hanlon Newell AE, Bi W et al. Aneuploidy as a mechanism for stress-induced liver adaptation. J Clin Invest 2012a;122:3307–3315.

[55] Duncan AW, Hanlon Newell AE, Smith L et al. Frequent aneuploidy among normal human hepatocytes. Gastroenterology 2012b;142:25–28.

[56] Engel FB, Schebesta M, Duong MT et al. p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Genes Dev 2005;19:1175–1187.

[57] Engel FB, Schebesta M, Keating MT. Anillin localization defect in cardiomyocyte binucleation. J Mol Cell Cardiol 2006;41:601–612.

[58] Epstein CJ. Cell size, nuclear content, and the development of polyploidy in the Mammalian liver. Proc Natl Acad Sci U S A 1967;57:327–334.

[59] Erokhina IL, Selivanova GV, Vlasova TD et al. Ultrastructure and biosynthetic activity of polyploid atrial myocytes in patients with mitral valve disease. Acta Histochem Suppl 1992;42:293–299.

[60] Faggioli F, Sacco MG, Susani L et al. Cell fusion is a physiological process in mouse liver. Hepatology 2008;48:1655–1664.

[61] Faggioli F, Vezzoni P, Montagna C. Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes. PLoS One 2011;6:e26080.

[62] Fan C, Chen H, Liu K et al. Fibrinogen-like protein 2 contributes to normal murine cardiomyocyte maturation and heart development. Exp Physiol 2021;106:1559–1571.

[63] Fava LL, Schuler F, Sladky V et al. The PIDDosome activates p53 in response to supernumerary centrosomes. Genes Dev 2017;31:34–45.

[64] Finegood DT, Scaglia L, Bonner-Weir S. Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes 1995;44:249–256.

[65] Font-Burgada J, Shalapour S, Ramaswamy S et al. Hybrid periportal hepatocytes regenerate the injured liver without giving rise to cancer. Cell 2015;162:766–779.

[66] Freije A, Ceballos L, Coisy M et al. Cyclin E drives human keratinocyte growth into differentiation. Oncogene 2012;31:5180–5192.

[67] Freije A, Molinuevo R, Ceballos L et al. Inactivation of p53 in human keratinocytes leads to squamous differentiation and shedding via replication stress and mitotic slippage. Cell Rep 2014;9:1349–1360.

[68] Fujiwara T, Bandi M, Nitta M et al. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 2005;437:1043–1047.

[69] Gabisonia K, Prosdocimo G, Aquaro GD et al. MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature 2019;569:418–422.

[70] Gandarillas A, Davies D, Blanchard JM. Normal and c-Myc-promoted human keratinocyte differentiation both occur via a novel cell cycle involving cellular growth and endoreplication. Oncogene 2000;19:3278–3289.

[71] Gandillet A, Alexandre E, Royer C et al. Hepatocyte ploidy in regenerating livers after partial hepatectomy, drug-induced necrosis, and cirrhosis. Eur Surg Res 2003;35:148–160.

[72] Ganem NJ, Storchova Z, Pellman D. Tetraploidy, aneuploidy and cancer. Curr Opin Genet Dev 2007;17:157–162.

[73] Ganem NJ, Godinho SA, Pellman D. A mechanism linking extra centrosomes to chromosomal instability. Nature 2009;460:278–282.

[74] Gentric G, Maillet V, Paradis V et al. Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease. J Clin Invest 2015;125:981–992.

[75] Gerdes J, Schwab U, Lemke H et al. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 1983;31:13–20.

[76] Gerlyng P, Grotmol T, Erikstein B et al. Reduced proliferative activity of polyploid cells in primary hepatocellular carcinoma. Carcinogenesis 1992;13:1795–1801.

[77] Gerlyng P, Abyholm A, Grotmol T et al. Binucleation and polyploidization patterns in developmental and regenerative rat liver growth. Cell Prolif 1993;26:557–565.

[78] Gilsbach R, Schwaderer M, Preissl S et al. Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo. Nat Commun 2018;9:391.

[79] Gimelbrant A, Hutchinson JN, Thompson BR et al. Widespread monoallelic expression on human autosomes. Science 2007;318:1136–1140.

[80] Gonzalez-Rosa JM, Sharpe M, Field D et al. Myocardial polyploidization creates a barrier to heart regeneration in zebrafish. Dev Cell 2018;44:433–446

[81] Gorla GR, Malhi H, Gupta S. Polyploidy associated with oxidative injury attenuates proliferative potential of cells. J Cell Sci 2001;114:2943–2951.

[82] Grendler J, Lowgren S, Mills M et al. Wound-induced polyploidization is driven by Myc and supports tissue repair in the presence of DNA damage. Development 2019;146.

[83] Guc-Scekic M, Milasin J, Stevanovic M et al. Tetraploidy in a 26-month-old girl (cytogenetic and molecular studies). Clin Genet 2002;61:62–65.

[84] Guidotti JE, Bregerie O, Robert A et al. Liver cell polyploidization: a pivotal role for binuclear hepatocytes. J Biol Chem 1909278:5–19101.

[85] Gujabidze N, Rukhadze R. Influence of experimental hyperthyreosis on hepatocytes’ cell cycle in white mice. Georgian Med News 2006;131:112–115.

[86] Hagemann S, Wohlschlaeger J, Bertram S et al. Loss of Survivin influences liver regeneration and is associated with impaired Aurora B function. Cell Death Differ 2013;20:834–844.

[87] Han L, Choudhury S, Mich-Basso JD et al. Lamin B2 levels regulate polyploidization of cardiomyocyte nuclei and myocardial regeneration. Dev Cell 2020;53:42–59

[88] Hanovice NJ, Leach LL, Slater K et al. Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation. PLoS Genet 2019;15:e1007939.

[89] Hart M, Adams SD, Draviam VM. Multinucleation associated DNA damage blocks proliferation in p53-compromised cells. Commun Biol 2021;4:451.

[90] Heinke P, Rost F, Rode J et al. Diploid hepatocytes drive physiological liver renewal in adult humans. Cell Syst 2022;13:499–507

[91] Herget GW, Neuburger M, Plagwitz R et al. DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction. Cardiovasc Res 1997;36:45–51.

[92] Hesse M, Raulf A, Pilz GA et al. Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle. Nat Commun 2012;3:1076.

[93] Hightower E, Cabanillas ME, Fuller GN et al. Phospho-histone H3 (pHH3) immuno-reactivity as a prognostic marker in non-functioning pituitary adenomas. Pituitary 2012;15:556–561.

[94] Hirose K, Payumo AY, Cutie S et al. Evidence for hormonal control of heart regenerative capacity during endothermy acquisition. Science 2019;364:184–188.

[95] Hixon ML, Muro-Cacho C, Wagner MW et al. Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization. J Clin Invest 2000;106:1011–1020.

[96] Hsu SH, Delgado ER, Otero PA et al. MicroRNA-122 regulates polyploidization in the murine liver. Hepatology 2016;64:599–615.

[97] Huppertz B, Frank HG, Kingdom JC et al. Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem Cell Biol 1998;110:495–508.

[98] Iglesias-Ara A, Zenarruzabeitia O, Buelta L et al. E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution. Cell Death Differ 2015;22:1577–1589.

[99] Jacobs PA, Szulman AE, Funkhouser J et al. Human triploidy: relationship between parental origin of the additional haploid complement and development of partial hydatidiform mole. Ann Hum Genet 1982;46:223–231.

[100] Jiang YH, Zhu Y, Chen S et al. Re-enforcing hypoxia-induced polyploid cardiomyocytes enter cytokinesis through activation of beta-catenin. Sci Rep 2019;9:17865.

[101] Jiang YH, Wang HL, Peng J et al. Multinucleated polyploid cardiomyocytes undergo an enhanced adaptability to hypoxia via mitophagy. J Mol Cell Cardiol 2020;138:115–135.

[102] Jonker SS, Zhang L, Louey S et al. Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart. J Appl Physiol (1985) 2007a;102:1130–1142.

[103] Jonker SS, Faber JJ, Anderson DF et al. Sequential growth of fetal sheep cardiac myocytes in response to simultaneous arterial and venous hypertension. Am J Physiol Regul Integr Comp Physiol 2007b;292:R913–R919.

[104] Jonker SS, Giraud MK, Giraud GD et al. Cardiomyocyte enlargement, proliferation and maturation during chronic fetal anaemia in sheep. Exp Physiol 2010;95:131–139.

[105] Jonsdottir AB, Stefansson OA, Bjornsson J et al. Tetraploidy in BRCA2 breast tumours. Eur J Cancer 2012;48:305–310.

[106] Kang MJ, Kim JS, Chae SW et al. Cyclins and cyclin dependent kinases during cardiac development. Mol Cells 1997;7:360–366.

[107] Kang J, Hwang I, Yoo C et al. Nab-paclitaxel plus gemcitabine versus FOLFIRINOX as the first-line chemotherapy for patients with metastatic pancreatic cancer: retrospective analysis. Invest New Drugs 2018;36:732–741.

[108] Ke Q, Gong L, Zhu X et al. Multinucleated Retinal Pigment Epithelial Cells Adapt to Vision and Exhibit Increased DNA Damage Response. Cells 2022;11.

[109] Kemp K, Gordon D, Wraith DC et al. Fusion between human mesenchymal stem cells and rodent cerebellar Purkinje cells. Neuropathol Appl Neurobiol 2011;37:166–178.

[110] Kemp K, Gray E, Wilkins A et al. Purkinje cell fusion and binucleate heterokaryon formation in multiple sclerosis cerebellum. Brain 2012;135:2962–2972.

[111] Kent LN, Rakijas JB, Pandit SK et al. E2f8 mediates tumor suppression in postnatal liver development. J Clin Invest 2016;126:2955–2969.

[112] Kern L, Spreckels J, Nist A et al. Altered glycogen metabolism causes hepatomegaly following an Atg7 deletion. Cell Tissue Res 2016;366:651–665.

[113] Kim SH, Jeon Y, Kim HS et al. Hepatocyte homeostasis for chromosome ploidization and liver function is regulated by Ssu72 protein phosphatase. Hepatology 2016;63:247–259.

[114] Klapholz B, Dietrich BH, Schaffner C et al. CAF-1 is required for efficient replication of euchromatic DNA in Drosophila larval endocycling cells. Chromosoma 2009;118:235–248.

[115] Knouse KA, Wu J, Whittaker CA et al. Single cell sequencing reveals low levels of aneuploidy across mammalian tissues. Proc Natl Acad Sci U S A 2014 111:13409–13414.

[116] Knouse KA, Lopez KE, Bachofner M et al. Chromosome Segregation Fidelity in Epithelia Requires Tissue Architecture. Cell 2018;175:200–211

[117] Kops GJ, Foltz DR, Cleveland DW. Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint. Proc Natl Acad Sci U S A 2004;101:8699–8704.

[118] Kreutz C, MacNelly S, Follo M et al. Hepatocyte ploidy is a diversity factor for liver homeostasis. Front Physiol 2017;8:862.

[119] Kriesten K. [Relative incidence of mitosis and binucleated cells, nuclear volume and nucleolar rate per nucleus in the mammary gland eipithelium of the mouse during differentiation in the gestational and lactation phase]. Gegenbaurs Morphol Jahrb 1984;130:307–314.

[120] Kudryavtsev BN, Kudryavtseva MV, Sakuta GA et al. Human hepatocyte polyploidization kinetics in the course of life cycle. Virchows Arch B Cell Pathol Incl Mol Pathol 1993;64:387–393.

[121] Kurinna S, Stratton SA, Coban Z et al. p53 regulates a mitotic transcription program and determines ploidy in normal mouse liver. Hepatology 2013;57:2004–2013.

[122] Lai SL, Marin-Juez R, Moura PL et al. Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration. Elife 2017;6:1–20.

[123] Lamar ZS, Fino N, Palmer J et al. Dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) with or without rituximab as first-line therapy for aggressive non-Hodgkin lymphoma. Clin Lymphoma Myeloma Leuk 2016;16:76–81.

[124] Lazzerini Denchi E, Celli G, de Lange T. Hepatocytes with extensive telomere deprotection and fusion remain viable and regenerate liver mass through endoreduplication. Genes Dev 2006;20:2648–2653.

[125] Leitch AR, Leitch IJ. Genomic plasticity and the diversity of polyploid plants. Science 2008;320:481–483.

[126] Leone M, Engel FB. Pseudo-bipolar spindle formation and cell division in postnatal binucleated cardiomyocytes. J Mol Cell Cardiol 2019;134:69–73.

[127] Levkau B, Schafers M, Wohlschlaeger J et al. Survivin determines cardiac function by controlling total cardiomyocyte number. Circulation 2008;117:1583–1593.

[128] Li F, Wang X, Capasso JM et al. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996;28:1737–1746.

[129] Li D, Cen J, Chen X et al. Hepatic loss of survivin impairs postnatal liver development and promotes expansion of hepatic progenitor cells in mice. Hepatology 2013;58:2109–2121.

[130] Li D, Liu Y, Pei C et al. miR-285-Yki/Mask double-negative feedback loop mediates blood-brain barrier integrity in Drosophila. Proc Natl Acad Sci U S A 2017;114:E2365–E2374.

[131] Li W, Dai Y, Shi B et al. LRPPRC sustains Yap-P27-mediated cell ploidy and P62-HDAC6-mediated autophagy maturation and suppresses genome instability and hepatocellular carcinomas. Oncogene 2020;39:3879–3892.

[132] Lilly MA, Spradling AC. The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev 1996;10:2514–2526.

[133] Lin YH, Zhang S, Zhu M et al. Mice with increased numbers of polyploid hepatocytes maintain regenerative capacity but develop fewer hepatocellular carcinomas following chronic liver injury. Gastroenterology 2020;158:1698–1712

[134] Liu Z, Yue S, Chen X et al. Regulation of cardiomyocyte polyploidy and multinucleation by CyclinG1. Circ Res 2010;106:1498–1506.

[135] Losick VP, Fox DT, Spradling AC. Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium. Curr Biol 2013;23:2224–2232.

[136] Losick VP, Jun AS, Spradling AC. Wound-induced polyploidization: regulation by hippo and JNK signaling and conservation in mammals. PLoS One 2016;11:e0151251.

[137] Lu P, Prost S, Caldwell H et al. Microarray analysis of gene expression of mouse hepatocytes of different ploidy. Mamm Genome 2007;18:617–626.

[138] Mable BK, Otto SP. Masking and purging mutations following EMS treatment in haploid, diploid and tetraploid yeast (Saccharomyces cerevisiae). Genet Res 2001;77:9–26.

[139] Madra S, Styles J, Smith AG. Perturbation of hepatocyte nuclear populations induced by iron and polychlorinated biphenyls in C57BL/10ScSn mice during carcinogenesis. Carcinogenesis 1995;16:719–727.

[140] Mahmoud AI, Kocabas F, Muralidhar SA et al. Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature 2013;497:249–253.

[141] Maines JZ, Stevens LM, Tong X et al. Drosophila dMyc is required for ovary cell growth and endoreplication. Development 2004;131:775–786.

[142] Malhi H, Gorla GR, Irani AN et al. Cell transplantation after oxidative hepatic preconditioning with radiation and ischemia-reperfusion leads to extensive liver repopulation. Proc Natl Acad Sci U S A 2002;99:13114–13119.

[143] Mansilla S, Priebe W, Portugal J. Mitotic catastrophe results in cell death by caspase-dependent and caspase-independent mechanisms. Cell Cycle 2006;5:53–60.

[144] Margall-Ducos G, Celton-Morizur S, Couton D et al. Liver tetraploidization is controlled by a new process of incomplete cytokinesis. J Cell Sci 2007;120:3633–3639.

[145] Martin NC, McCullough CT, Bush PG et al. Functional analysis of mouse hepatocytes differing in DNA content: volume, receptor expression, and effect of IFNgamma. J Cell Physiol 2002;191:138–144.

[146] Martynova MG, Selivanova GV, Vlasova TD. Ploidy levels and the number of nuclei in cardiomyocytes of the lamprey and fish. Tsitologiia 2002;44:387–391.

[147] Matondo RB, Moreno E, Toussaint MJM et al. Atypical E2f functions are critical for pancreas polyploidization. PLoS One 2018;13:e0190899.

[148] Matsumoto T, Wakefield L, Tarlow BD et al. In vivo lineage tracing of polyploid hepatocytes reveals extensive proliferation during liver regeneration. Cell Stem Cell 2020;26:34–47

[149] Maurici D, Perez-Atayde A, Grier HE et al. Frequency and implications of chromosome 8 and 12 gains in Ewing sarcoma. Cancer Genet Cytogenet 1998;100:106–110.

[150] Mayhew CN, Bosco EE, Fox SR et al. Liver-specific pRB loss results in ectopic cell cycle entry and aberrant ploidy. Cancer Res 2005;65:4568–4577.

[151] McConnell MJ, Lindberg MR, Brennand KJ et al. Mosaic copy number variation in human neurons. Science 2013;342:632–637.

[152] Meckert PC, Rivello HG, Vigliano C et al. Endomitosis and polyploidization of myocardial cells in the periphery of human acute myocardial infarction. Cardiovasc Res 2005;67:116–123.

[153] Mehrotra S, Maqbool SB, Kolpakas A et al. Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress. Genes Dev 2008;22:3158–3171.

[154] Meissner HP. Electrophysiological evidence for coupling between beta cells of pancreatic islets. Nature 1976;262:502–504.

[155] Michel L, Diaz-Rodriguez E, Narayan G et al. Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells. Proc Natl Acad Sci U S A 2004;101:4459–4464.

[156] Minamishima YA, Nakayama K, Nakayama K. Recovery of liver mass without proliferation of hepatocytes after partial hepatectomy in Skp2-deficient mice. Cancer Res 2002;62:995–999.

[157] Miyaoka Y, Ebato K, Kato H et al. Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration. Curr Biol 2012;22:1166–1175.

[158] Mollova M, Bersell K, Walsh S et al. Cardiomyocyte proliferation contributes to heart growth in young humans. Proc Natl Acad Sci U S A 2013;110:1446–1451.

[159] Muramatsu Y, Yamada T, Moralejo DH et al. Increased polyploid incidence is associated with abnormal copper accumulation in the liver of LEC mutant rat. Res Commun Mol Pathol Pharmacol 2000;107:129–136.

[160] Nakatani T, Inouye M, Mirochnitchenko O. Overexpression of antioxidant enzymes in transgenic mice decreases cellular ploidy during liver regeneration. Exp Cell Res 1997;236:137–146.

[161] Nevzorova YA, Tschaharganeh D, Gassler N et al. Aberrant cell cycle progression and endoreplication in regenerating livers of mice that lack a single E-type cyclin. Gastroenterology 2009;137:691–703

[162] Oberpriller JO, Oberpriller JC. Response of the adult newt ventricle to injury. J Exp Zool 1974;187:249–259.

[163] Ogawa H, Takyu R, Morimoto H et al. Cell proliferation potency is independent of FGF4 signaling in trophoblast stem cells derived from androgenetic embryos. J Reprod Dev 2016;62:51–58.

[164] Oliver TG, Meylan E, Chang GP et al. Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop. Mol Cell 2011;43:57–71.

[165] Olivetti G, Cigola E, Maestri R et al. Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart. J Mol Cell Cardiol 1996;28:1463–1477.

[166] Oparil S, Bishop SP, Clubb FJ. Myocardial cell hypertrophy or hyperplasia. Hypertension 1984;6:III38–III43.

[167] Ortica S, Tarantino N, Aulner N et al. The 4 notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors. FASEB J 2014;28:603–614.

[168] Ostergaard KH, Baandrup UT, Wang T et al. Left ventricular morphology of the giraffe heart examined by stereological methods. Anat Rec (Hoboken) 2013;296:611–621.

[169] Overturf K, Al-Dhalimy M, Tanguay R et al. Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I. Nat Genet 1996;12:266–273.

[170] Overturf K, Al-Dhalimy M, Finegold M et al. The repopulation potential of hepatocyte populations differing in size and prior mitotic expansion. Am J Pathol 1999;155:2135–2143.

[171] Panattoni M, Maiorino L, Lukacs A et al. The COP9 signalosome is a repressor of replicative stress responses and polyploidization in the regenerating liver. Hepatology 2014;59:2331–2343.

[172] Pandit SK, Westendorp B, Nantasanti S et al. E2F8 is essential for polyploidization in mammalian cells. Nat Cell Biol 2012;14:1181–1191.

[173] Pascreau G, Churchill ME, Maller JL. Centrosomal localization of cyclins E and A: structural similarities and functional differences. Cell Cycle 2011;10:199–205.

[174] Patel D, Incassati A, Wang N et al. Human papillomavirus type 16 E6 and E7 cause polyploidy in human keratinocytes and up-regulation of G2-M-phase proteins. Cancer Res 2004;64:1299–1306.

[175] Patterson M, Barske L, Van Handel B et al. Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration. Nat Genet 2017;49:1346–1353.

[176] Pedone E, Olteanu VA, Marucci L et al. Modeling dynamics and function of bone marrow cells in mouse liver regeneration. Cell Rep 2017;18:107–121.

[177] Perez de Castro I, Aguirre-Portoles C, Fernandez-Miranda G et al. Requirements for Aurora-A in tissue regeneration and tumor development in adult mammals. Cancer Res 2013;73:6804–6815.

[178] Porrello ER, Mahmoud AI, Simpson E et al. Transient regenerative potential of the neonatal mouse heart. Science 2011;331:1078–1080.

[179] Poss KD, Wilson LG, Keating MT. Heart regeneration in zebrafish. Science 2002;298:2188–2190.

[180] Post J, Hoffman J. Further studies on the replication of rat liver cells in vivo. Exp Cell Res 1965;40:333–339.

[181] Puente BN, Kimura W, Muralidhar SA et al. The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell 2014;157:565–579.

[182] Rajvanshi P, Liu D, Ott M et al. Fractionation of rat hepatocyte subpopulations with varying metabolic potential, proliferative capacity, and retroviral gene transfer efficiency. Exp Cell Res 1998;244:405–419.

[183] Raulf A, Horder H, Tarnawski L et al. Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status. Basic Res Cardiol 2015;110:33.

[184] Reid BJ, Haggitt RC, Rubin CE et al. Barrett’s esophagus. Correlation between flow cytometry and histology in detection of patients at risk for adenocarcinoma. Gastroenterology 1987;93:1–11.

[185] Rengstl B, Newrzela S, Heinrich T et al. Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed-Sternberg cells. Proc Natl Acad Sci U S A 2013;110:9–20734.

[186] Rios AC, Fu NY, Jamieson PR et al. Essential role for a novel population of binucleated mammary epithelial cells in lactation. Nat Commun 2016;7:11400.

[187] Rua S, Comino A, Fruttero A et al. Flow cytometric DNA analysis of cirrhotic liver cells in patients with hepatocellular carcinoma can provide a new prognostic factor. Cancer 1996;78:1195–1202.

[188] Saeter G, Lee CZ, Schwarze PE et al. Changes in ploidy distributions in human liver carcinogenesis. J Natl Cancer Inst 1988;80:1480–1485.

[189] Sanges D, Romo N, Simonte G et al. Wnt/beta-catenin signaling triggers neuron reprogramming and regeneration in the mouse retina. Cell Rep 2013;4:271–286.

[190] Sanz N, Diez-Fernandez C, Fernandez-Simon L et al. Relationship between antioxidant systems, intracellular thiols and DNA ploidy in liver of rats during experimental cirrhogenesis. Carcinogenesis 1995;16:1585–1593.

[191] Schoenfelder KP, Fox DT. The expanding implications of polyploidy. J Cell Biol 2015;209:485–491.

[192] Senyo SE, Steinhauser ML, Pizzimenti CL et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature 2013;493:433–436.

[193] Shapiro SD, Ranjan AK, Kawase Y et al. Cyclin A2 induces cardiac regeneration after myocardial infarction through cytokinesis of adult cardiomyocytes. Sci Transl Med 2014;6:224ra227.

[194] Sher N, Von Stetina JR, Bell GW et al. Fundamental differences in endoreplication in mammals and Drosophila revealed by analysis of endocycling and endomitotic cells. Proc Natl Acad Sci U S A 2013;110:9368–9373.

[195] Sigal SH, Gupta S, Gebhard DF et al. Evidence for a terminal differentiation process in the rat liver. Differentiation 1995;59:35–42.

[196] Sigal SH, Rajvanshi P, Gorla GR et al. Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. Am J Physiol 1999;276:G1260–G1272.

[197] Sladky VC, Villunger A. Uncovering the PIDDosome and caspase-2 as regulators of organogenesis and cellular differentiation. Cell Death Differ 2020;27:2037–2047.

[198] Sladky VC, Knapp K, Soratroi C et al. E2F-family members engage the PIDDosome to limit hepatocyte ploidy in liver development and regeneration. Dev Cell 2020a;52:335–349

[199] Sladky VC, Knapp K, Szabo TG et al. PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis. EMBO Rep 2020b;21:e50893.

[200] Sladky VC, Eichin F, Reiberger T et al. Polyploidy control in hepatic health and disease. J Hepatol 2021;75:1177–1191.

[201] Sladky VC, Akbari H, Tapias-Gomez D et al. Centriole signaling restricts hepatocyte ploidy to maintain liver integrity. Genes Dev 2022;36:843–856.

[202] Smith GH, Medina D. A morphologically distinct candidate for an epithelial stem cell in mouse mammary gland. J Cell Sci 1988;90:173–183.

[203] Smith GH, Vonderhaar BK. Functional differentiation in mouse mammary gland epithelium is attained through DNA synthesis, inconsequent of mitosis. Dev Biol 1981;88:167–179.

[204] Soonpaa MH, Kim KK, Pajak L et al. Cardiomyocyte DNA synthesis and binucleation during murine development. Am J Physiol 1996;271:H2183–H2189.

[205] Soonpaa MH, Koh GY, Pajak L et al. Cyclin D1 overexpression promotes cardiomyocyte DNA synthesis and multinucleation in transgenic mice. J Clin Invest 1997;99:2644–2654.

[206] Starnes AC, Huisingh C, McGwin G et al. Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution. Vis Neurosci 2016;33:e001.

[207] Steigemann P, Wurzenberger C, Schmitz MH et al. Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 2009;136:473–484.

[208] Stopp S, Grundl M, Fackler M et al. Deletion of Gas2l3 in mice leads to specific defects in cardiomyocyte cytokinesis during development. Proc Natl Acad Sci U S A 2017;114:8029–8034.

[209] Tanami S, Ben-Moshe S, Elkayam A et al. Dynamic zonation of liver polyploidy. Cell Tissue Res 2017;368:405–410.

[210] Tane S, Ikenishi A, Okayama H et al. CDK inhibitors, p21(Cip1) and p27(Kip1), participate in cell cycle exit of mammalian cardiomyocytes. Biochem Biophys Res Commun 2014;443:1105–1109.

[211] Tinel A, Tschopp J. The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 2004;304:843–846.

[212] Tormos AM, Arduini A, Talens-Visconti R et al. Liver-specific p38alpha deficiency causes reduced cell growth and cytokinesis failure during chronic biliary cirrhosis in mice. Hepatology 2013;57:1950–1961.

[213] Tormos AM, Rius-Perez S, Jorques M et al. p38alpha regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging. PLoS One 2017;12:e0171738.

[214] Torres S, Diaz BP, Cabrera JJ et al. Thyroid hormone regulation of rat hepatocyte proliferation and polyploidization. Am J Physiol 1999;276:G155–G163.

[215] Toyoda H, Bregerie O, Vallet A et al. Changes to hepatocyte ploidy and binuclearity profiles during human chronic viral hepatitis. Gut 2005;54:297–302.

[216] Toyoda H, Kumada T, Bregerie O et al. Conserved balance of hepatocyte nuclear DNA content in mononuclear and binuclear hepatocyte populations during the course of chronic viral hepatitis. World J Gastroenterol 2006;12:4546–4548.

[217] Troadec MB, Courselaud B, Detivaud L et al. Iron overload promotes Cyclin D1 expression and alters cell cycle in mouse hepatocytes. J Hepatol 2006;44:391–399.

[218] Turker MS, Schwartz JL, Jordan R et al. Persistence of chromatid aberrations in the cells of solid mouse tissues exposed to 137Cs gamma radiation. Radiat Res 2004;162:357–364.

[219] Ullah Z, Kohn MJ, Yagi R et al. Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity. Genes Dev 2008;22:3024–3036.

[220] Unhavaithaya Y, Orr-Weaver TL. Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity. Genes Dev 2012;26:31–36.

[221] Vinogradov AE, Anatskaya OV, Kudryavtsev BN. Relationship of hepatocyte ploidy levels with body size and growth rate in mammals. Genome 2001;44:350–360.

[222] Vliegen HW, van der Laarse A, Cornelisse CJ et al. Myocardial changes in pressure overload-induced left ventricular hypertrophy. A study on tissue composition, polyploidization and multinucleation. Eur Heart J 1991;12:488–494.

[223] Vliegen HW, Eulderink F, Bruschke AV et al. Polyploidy of myocyte nuclei in pressure overloaded human hearts: a flow cytometric study in left and right ventricular myocardium. Am J Cardiovasc Pathol 1995;5:27–31.

[224] Vonderhaar BK, Smith GH, Pauley RJ et al. Difference between mammary epithelial cells from mature virgin and primiparous mice. Cancer Res 1978;38:4059–4065.

[225] Von Stetina JR, Frawley LE, Unhavaithaya Y et al. Variant cell cycles regulated by Notch signaling control cell size and ensure a functional blood-brain barrier. Development 2018;145.

[226] Vujic A, Lerchenmuller C, Wu TD et al. Exercise induces new cardiomyocyte generation in the adult mammalian heart. Nat Commun 2018;9:1659.

[227] Wang MJ, Chen F, Li JX et al. Reversal of hepatocyte senescence after continuous in vivo cell proliferation. Hepatology 2014;60:349–361.

[228] Wang B, Zhao L, Fish M et al. Self-renewing diploid Axin2(+) cells fuel homeostatic renewal of the liver. Nature 2015;524:180–185.

[229] Wang WE, Li L, Xia X et al. Dedifferentiation, proliferation, and redifferentiation of adult mammalian cardiomyocytes after ischemic injury. Circulation 2017;136:834–848.

[230] Webber EM, Godowski PJ, Fausto N. In vivo response of hepatocytes to growth factors requires an initial priming stimulus. Hepatology 1994;19:489–497.

[231] Webber EM, Bruix J, Pierce RH et al. Tumor necrosis factor primes hepatocytes for DNA replication in the rat. Hepatology 1998;28:1226–1234.

[232] Weglarz TC, Degen JL, Sandgren EP. Hepatocyte transplantation into diseased mouse liver. Kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes. Am J Pathol 2000;157:1963–1974.

[233] Wen Q, Goldenson B, Silver SJ et al. Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL. Cell 2012;150:575–589.

[234] Werle K, Chen J, Xu HG et al. Liver kinase B1 regulates the centrosome via PLK1. Cell Death Dis 2014;5:e1157.

[235] Wheatley DN. Binucleation in mammalian liver. Studies on the control of cytokinesis in vivo. Exp Cell Res 1972;74:455–465.

[236] White JW, Swartz FJ, Swartz AF. Excess glucose intake induces accelerated beta-cell polyploidization in normal mice: a possible deleterious effect. J Nutr 1985;115:271–278.

[237] Wilkinson PD, Alencastro F, Delgado ER et al. Polyploid hepatocytes facilitate adaptation and regeneration to chronic liver injury. Am J Pathol 2019a;189:1241–1255.

[238] Wilkinson PD, Delgado ER, Alencastro F et al. The polyploid state restricts hepatocyte proliferation and liver regeneration in mice. Hepatology 2019b;69:1242–1258.

[239] Willenbring H, Bailey AS, Foster M et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 2004;10:744–748.

[240] Wills AA, Holdway JE, Major RJ et al. Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish. Development 2008;135:183–192.

[241] Wu H, Wade M, Krall L et al. Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev 1996;10:245–260.

[242] Xiao G, Mao S, Baumgarten G et al. Inducible activation of c-Myc in adult myocardium in vivo provokes cardiac myocyte hypertrophy and reactivation of DNA synthesis. Circ Res 2001;89:1122–1129.

[243] Yanger K, Knigin D, Zong Y et al. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation. Cell Stem Cell 2014;15:340–349.

[244] Ye L, Qiu L, Zhang H et al. Cardiomyocytes in young infants with congenital heart disease: a three-month window of proliferation. Sci Rep 2016;6:23188.

[245] Yoshida S, Bartolini S, Pellman D. Mechanisms for concentrating Rho1 during cytokinesis. Genes Dev 2009;23:810–823.

[246] Zajicek G, Oren R, Weinreb M. The streaming liver. Liver 1985;5:293–300.

[247] Zanet J, Freije A, Ruiz M et al. A mitosis block links active cell cycle with human epidermal differentiation and results in endoreplication. PLoS One 2010;5:e15701.

[248] Zebrowski DC, Vergarajauregui S, Wu CC et al. Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes. Elife 2015;4.

[249] Zhang S, Zhou K, Luo X et al. The polyploid state plays a tumor-suppressive role in the liver. Dev Cell 2018a;44:447–459

[250] Zhang S, Nguyen LH, Zhou K et al. Knockdown of anillin actin binding protein blocks cytokinesis in hepatocytes and reduces liver tumor development in mice without affecting regeneration. Gastroenterology 2018b;154:1421–1434.

[251] Zhong L, Georgia S, Tschen SI et al. Essential role of Skp2-mediated p27 degradation in growth and adaptive expansion of pancreatic beta cells. J Clin Invest 2007;117:2869–2876.

[252] Zhou J, Ahmad F, Parikh S et al. Loss of adult cardiac myocyte GSK-3 leads to mitotic catastrophe resulting in fatal dilated cardiomyopathy. Circ Res 2016;118:1208–1222.

[253] Zielke N, Querings S, Rottig C et al. The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles. Genes Dev 2008;22:1690–1703.

[254] Zulbahar S, Sieglitz F, Kottmeier R et al. Differential expression of Obek controls ploidy in the Drosophila blood-brain barrier. Development 2018;145.

RIGHTS & PERMISSIONS

©The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.