Functional analysis of mouse hepatocytes differing in DNA content: volume,receptor expression,and effect of IFNgamma

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule‐1 (ICAM‐1, CD54) or IFNγR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNγ)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM‐1 increases with increasing DNA content and IFNγR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNγ‐induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro. J. Cell. Physiol. 191: 138–144, 2002. © 2002 Wiley‐Liss, Inc.     

Chromosome studies in southern species of Mimosa (Fabaceae,Mimosoideae) and their taxonomic and evolutionary inferences

In this work, chromosome numbers and karyotype parameters of 36 taxa of the genus Mimosa were studied, especially from the southern South America center of diversification. Results support that x = 13 is the basic chromosome number in the genus. Polyploidy is very frequent, ca. 56 % of the total of the studied species here are polyploid, confirming that polyploids are more frequent at higher latitudes. The most common ploidy levels found are 2x and 4x, but some species studied exhibit 6x and 8x. In different groups, several ploidy levels were found. Parameters of chromosome size show statistically significant differences between close species, and asymmetry index A ₂ exhibited low variation between them. It is possible to infer variations of chromosome size between diploids and tetraploids and between basal and derived taxa. The present studies confirm or reveal polyploidy in several groups of South America which are highly diversified in the southernmost area of distribution of the genus, such as sect. Batocaulon ser. Stipellares and sect. Calothamnos. Our data are discussed in a taxonomic context, making inferences about the origin of some polyploid taxa. Polyploidy could be an important phenomenon that increases the morphologic diversity and specific richness in southern South America. On basis of our data, it is possible to hypothesize hybridization between same-ploidy level or different ploidy level taxa. As already shown in the literature, our results confirm the importance of the polyploidy in the speciation of the genus.     

Ploidy Reductions in Murine Fusion-Derived Hepatocytes

We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH), and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ß-galactosidase and the Y-chromosome. Approximately 2–5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ß-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ß-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by ≥50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.     

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner.The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.     

Reproductive modes,ploidy distribution,and supernumerary chromosome frequencies of the flatworm Polycelis nigra (Platyhelminthes: Tricladida)

The hermaphroditic flatworm, Polycelis nigra, is characterized by two reproductive biotypes which differ with respect to ploidy; sexual individuals are diploid (n = 8, 2× = 16) and pseudogamous parthenogenetic individuals are polyploid (typically 3×). We have collected and karyotyped individuals from 15 sampling sites (13 in mid to northern Italy, one in Great Britain and one in The Netherlands). We found that biotypes can exist alone or in sympatry, and identified purely diploid, mixed diploid-polyploid, and purely polyploid populations. Karyotype data show that in addition to the normal autosome complement, B chromosomes of differing morphology as well as stable aneuploid chromosomes (extra-A) were found almost exclusively in polyploids (11 of 12 sites). We extensively sampled Lago di Toblino (northern Italy), a pure polyploid population characterized by a submetacentric to metacentric, mitotically stable B chromosome, as well as a stable extra-A chromosome. Here, individuals having 1–3 B chromosomes were more abundant (61%) than those having no B's, implying that B chromosome infection has little detrimental effect when occurring in low numbers. Furthermore, 66% of individuals from this population possessed extra-A chromosomes, although it is unclear whether these elements are aneuploid autosomes or B chromosomes of different morphology. The ubiquity of these chromosomes, within asexuals in particular, is suggestive of a correlation between the origination of the elements and the evolution of polyploidy, or may reflect increased tolerance of parthenogenetic genomes to aneuploidy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic basis for dosage sensitivity in Arabidopsis thaliana

Aneuploidy, the relative excess or deficiency of specific chromosome types, results in gene dosage imbalance. Plants can produce viable and fertile aneuploid individuals, while most animal aneuploids are inviable or developmentally abnormal. The swarms of aneuploid progeny produced by Arabidopsis triploids constitute an excellent model to investigate the mechanisms governing dosage sensitivity and aneuploid syndromes. Indeed, genotype alters the frequency of aneuploid types within these swarms. Recombinant inbred lines that were derived from a triploid hybrid segregated into diploid and tetraploid individuals. In these recombinant inbred lines, a single locus, which we call SENSITIVE TO DOSAGE IMBALANCE (SDI), exhibited segregation distortion in the tetraploid subpopulation only. Recent progress in quantitative genotyping now allows molecular karyotyping and genetic analysis of aneuploid populations. In this study, we investigated the causes of the ploidy-specific distortion at SDI. Allele frequency was distorted in the aneuploid swarms produced by the triploid hybrid. We developed a simple quantitative measure for aneuploidy lethality and using this measure demonstrated that distortion was greatest in the aneuploids facing the strongest viability selection. When triploids were crossed to euploids, the progeny, which lack severe aneuploids, exhibited no distortion at SDI. Genetic characterization of SDI in the aneuploid swarm identified a mechanism governing aneuploid survival, perhaps by buffering the effects of dosage imbalance. As such, SDI could increase the likelihood of retaining genomic rearrangements such as segmental duplications. Additionally, in species where triploids are fertile, aneuploid survival would facilitate gene flow between diploid and tetraploid populations via a triploid bridge and prevent polyploid speciation. Our results demonstrate that positional cloning of loci affecting traits in populations containing ploidy and chromosome number variants is now feasible using quantitative genotyping approaches.     

Prognostic importance of the DNA ploidy pattern in malignant mesothelioma of the pleura.

Malignant mesotheliomas often raise a difficult diagnostic problem: once the diagnosis is made, the possibilities of predicting the biologic activity and prognosis of the tumor are limited. DNA ploidy patterns have been used as a prognostic instrument for other tumors, and this pattern was therefore studied in 37 cases of verified mesothelioma. The measurements were made on Feulgen-stained smears from pleural effusions using a Leica Miamed computer microscope. When a highly aneuploid tumor was defined as a condition with greater than 5% of the cells within defined intervals outside the ranges of the stem-line and the peaks representing polyploidization thereof, a near-diploid/polyploid pattern was obtained in 41% of the cases, while the remaining 59% were classified as highly aneuploid. The prognosis was significantly better among the near-diploid/polyploid cases. This estimate of the outcome could not be improved by using alternative algorithms for high-grade aneuploidy.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Spontaneous and induced aneuploidy,considerations which may influence chromosome malsegregation

Aneuploidy plays a major role in the production of human birth defects and is becoming increasingly recognised as a critical event in the etiology of a wide range of human cancers. Thus, the detection of aneuploidy and the characterisation of the mechanisms which lead to chromosome malsegregation is an important area of genotoxicological research. As an aid to aneuploidy research, methods have been developed to analyse the mechanisms of chromosome malsegregation and to investigate the role of aneuploidy in tumour progression. The presence of aneuploid cells is a common characteristic of many of tumour cell types as illustrated by the wide range of chromosome number changes detected in post-menopausal breast tumours. To investigate the time of occurrence of aneuploidy during tumour progression, we have studied the chromosome number status of Syrian hamster dermal (SHD) cells cultures progressing to morphological transformation. The production of both polyploid and aneuploid cells is a common feature of progressing cells in this model. The elevation of both progression to morphological transformation and aneuploid frequencies can be produced by exposure to a diverse range of carcinogens and tumour promoters. Analysis of the genotoxic activity of the hormone 17-beta oestradiol demonstrated its ability to induce both chromosome loss and non-disjunction in human lymphoblastoid cells implicating aneugenic activity in hormone related cancers. Mutations in the p53 tumour suppressor gene introduced into human fibroblasts produced modifications in chromosome separation at mitosis which may lead to the production of both aneuploidy and polyploid cells. Our studies indicate that the production of aneuploid cells can be influenced by both endogenous and exogenous factors and occur throughout the progression of normal cells to a malignant phenotype.     

Mutagenesis and plant phylogenesis

There are two types of genetic mutations - nuclear and cytoplasmic. We consider genic, chromosomic and genomic nuclear mutations in several seed plant families characterized by different evolutionary age and life forms. "Atlas of chromosome numbers of flowering plants" published in our laboratory in 1969 and containing information about 35 000 species and periodical "Index to plant chromosome numbers" (USA) covering in all about 150 000 species were used for comparative study of chromosome numbers. Gymnosperms originated approximately 300 000 000 years ago and represented predominantly by arboreous and shrub forms are characterized by practically total lack of polyploidy and rare aneuploidy, thus the evolutionary progress in this group has been provided by genic mutations. The morphology of chromosomes in Gymnosperms is much more uniform as compared with Angiosperms - all 200 species of Conifers have 24 large meta- and submetacentric chromosomes Angiosperms. This group originated twice later includes 300 000 species with wide range of living forms - from initial arboreous to ephemeric ones. Therefore, the dominating type of mutations for some groups of Angiosperms as Fagaceae, Aceraceae, Aquifoliaceae, Caricaceae and Lauraceae is genic one. The major part of arboreous Angiosperms has clear polyploid series like 2n = 28, 56, 84 (Betula) and 2n = 38, 76, 114 (Magnolia). Polyploidy is the prevalent type of mutagenesis because of the advantage consisting in amplification of total number of genes against a background of preservation of the genome integrity. The chromosomic type of mutations prevalent in groups with asexual reproduction provides the flow of genes between genomes as a result of aneuploidy. Genomic mutations are observed mostly in herbaceous plants. In such groups as Poaceae, Cyperaceae, Ranunculaceae and Rosaceae we observe up to 90 % of polyploid species. Due to such ploidy restrictions like the size of karyon, and the size and the number of chromosomes numerous shorter polyploid series are observed in this group. Hence primitive mutations are prevalent in ancient Gymnosperms. Chromosomic and genomic mutations arose later providing gene flow without functional changes of source genomes.     

A quantitative study of the second meiotic metaphase in male mice (Mus musculus).

Over 11,000 second meiotic metaphase spreads stained for the pericentromeric region have been studied quantitatively in male mice of 14 strains. The sex-chromosome constitution of a cell could be judged objectively if X and Y chromosomes and ploidy were all scored. A bias arose if only Y chromosomes and ploidy were scored but could be corrected statistically. There was no sign of other forms of bias. The original contiguity of X and Y second metaphases in vivo was very occasionally evident in the preparations. Most of the subhaploid aneuploid counts were assumed to be artifactual. The incidence of truly aneuploid second metaphases in 13 strains was estimated as 0.38+/-0.12%. The estimated average rate per chromosome was 0.019+/-0.006%, with a comparable order of magnitude for the sex chromosomes alone. Simultaneous aneuploidy of two or more chromosomes of the haploid set was estimated to be very rare. Of the spreads from 13 strains, 9.6% were polyploid (2N, 3N, 4N) and showed most of the possible combinations of sex chromosomes. Nearly all the polyploid spreads were considered to arise by artifactual cell fusion at the time of second metaphase during the preparative technique, especially of the X and Y daughter-cell products of the first meiotic division. Other modes of origin (true polyploidy, accidental superposition of cells during preparation) were unlikely. The data could be accommodated by a statistical model with only four parameters. It allowed for artifactual fusion mainly between daughter cells but also between non-daughter cells, bias in one scoring method, and bias in the numbers of cells with given ploidy successfully mounted. Current techniques of chromosome preparation were thought to be wholly unsuitable for the recognition of true polyploidy. The artifactual origin of polyploid spreads was borne out by an absence of polyploid spermatozoa in 14 strains. There appeared to be a virtually constant transmission rate of paternal X and Y chromosomes from early meiosis to late blastocyst. The estimated rate of 49.05+/-0.67% with a Y chromosome also estimated the primary sex ratio. There was evidence of polymorphism in autosomal pericentromeric staining in 3 strains. No measure of the numbers of autosomes or sex chromosomes varied significantly between duplicate preparations or between duplicate males of a strain.     

Aneuploidization under segmental allotetraploidy in rice and its phenotypic manifestation

Key message

We report a repertoire of diverse aneuploids harbored by a newly synthesized segmental allotetraploid rice population with fully sequenced sub-genomes and demonstrate their retention features and phenotypic consequences.

Abstract

Aneuploidy, defined as unequal numbers of different chromosomes, is a large-effect genetic variant and may produce diverse cellular and organismal phenotypes. Polyploids are more permissive to chromosomal content imbalance than their diploid and haploid counterparts, and therefore, may enable more in-depth investigation of the phenotypic consequences of aneuploidy. Based on whole-genome resequencing, we identify that ca. 40% of the 312 selfed individual plants sampled from an early generation rice segmental allotetraploid population are constitutive aneuploids harboring 55 distinct aneuploid karyotypes. We document that gain of a chromosome is more prevalent than loss of a chromosome, and the 12 rice chromosomes have distinct tendencies to be in an aneuploid state. These properties of aneuploidy are constrained by multiple factors including the number of genes residing on the chromosome and predicted functional connectivity with other chromosomes. Two broad categories of aneuploidy-associated phenotypes are recognized: those shared by different aneuploids, and those associated with aneuploidy of a specific chromosome. A repertoire of diverse aneuploids in the context of a segmental allotetraploid rice genome with fully sequenced sub-genomes provides a tractable resource to explore the roles of aneuploidy in nascent polyploid genome evolution and helps to decipher the mechanisms conferring karyotypic stabilization on the path to polyploid speciation and towards artificial construction of novel polyploid crops.

     

Nondiploid cancer cells: Stress,tolerance and therapeutic inspirations

Aberrant ploidy status is a prominent characteristic in malignant neoplasms. Approximately 90% of solid tumors and 75% of haematopoietic malignancies contain aneuploidy cells, and 30%–60% of tumors undergo whole-genome doubling, indicating that nondiploidy might be a prevalent genomic aberration in cancer. Although the role of aneuploid and polyploid cells in cancer remains to be elucidated, recent studies have suggested that nondiploid cells might be a dangerous minority that severely challenges cancer management. Ploidy shifts cause multiple fitness coasts for cancer cells, mainly including genomic, proteotoxic, metabolic and immune stresses. However, nondiploid comprises a well-adopted subpopulation, with many tolerance mechanisms evident in cells along with ploidy shifts. Aneuploid and polyploid cells elegantly maintain an autonomous balance between the stress and tolerance during adaptive evolution in cancer. Breaking the balance might provide some inspiration for ploidy-selective cancer therapy and alleviation of ploidy-related chemoresistance. To understand of the complex role and therapeutic potential of nondiploid cells better, we reviewed the survival stresses and adaptive tolerances within nondiploid cancer cells and summarized therapeutic ploidy-selective alterations for potential use in developing future cancer therapy.     

Ploidy variation in multinucleate cells changes under stress

Ploidy variation is found in contexts as diverse as solid tumors, drug resistance in fungal infection, and normal development. Altering chromosome or genome copy number supports adaptation to fluctuating environments but is also associated with fitness defects attributed to protein imbalances. Both aneuploidy and polyploidy can arise from multinucleate states after failed cytokinesis or cell fusion. The consequences of ploidy variation in syncytia are difficult to predict because protein imbalances are theoretically buffered by a common cytoplasm. We examined ploidy in a naturally multinucleate fungus, Ashbya gossypii. Using integrated lac operator arrays, we found that chromosome number varies substantially among nuclei sharing a common cytoplasm. Populations of nuclei range from 1N to >4N, with different polyploidies in the same cell and low levels of aneuploidy. The degree of ploidy variation increases as cells age. In response to cellular stress, polyploid nuclei diminish and haploid nuclei predominate. These data suggest that mixed ploidy is tolerated in these syncytia; however, there may be costs associated with variation as stress homogenizes the genome content of nuclei. Furthermore, the results suggest that sharing of gene products is limited, and thus there is incomplete buffering of ploidy variation despite a common cytosol.     

The function of leaf meristematic cells in Cotinus coggygria Scop

Proliferation of leaf meristem cells and formation of multicellular glands and unicellular hairs in Cotinus coggygria were studied under conditions of complex stressors exerting influence on this species when introduced and grown in such a big industrial city as Voronezh. Variability in the length of dividing meristematic cells (from 7.5 to 30.0 microns) was detected. The chromosome number was calculated, and the mean length of chromosomes (1 micron) was determined. The modal chromosome number in the leaf meristem cells in 2 n = 30. However, along with these cells, aneuploid (2.7%), uninucleate polypoid (1.9%), binuclear (10%), and polynuclear (3%) cells were also observed. The length of trichomes initials, formed in the epidermis, was near 40 microns. These have a large nucleus and may have, presumably, a polyploid nature. It is assumed that a heterogeneous meristematic cell population, at the expense of selection into such a population, is able to provide an optimum combination of cells with different level ploidy and aneuploidy for more effective realization of gene function in the extremal environment conditions.     

The kinetics of the cell population of human liver parenchyma at different periods of life]

Processes of polyploidization in the liver parenchyma were investigated in the course of postnatal organism growth, stabilization of growth and ageing, using cytophotometry on the slides of isolated hepatocytes from normal livers of 140 donors aged from 1 day to 92 years. In addition, livers of human embryos (4, 5, 6 and 7 month old) were investigated. It is concluded that polyploid cells in the human liver appear in individuals aged from 1 to 5 years. However, during the postnatal development their relative number increases insignificantly. At the end of the intensive postnatal growth period the share of polyploid human liver cells is less than 3%. Binuclear cells with diploid nuclei are seen as early as in the embryonic liver. After birth their number increases slowly to reach 7.1% in the 16-20 year age group. The postnatal growth of human liver is due mainly to mitotic divisions of mononuclear diploid hepatocytes whose relative number is more than 90% during the postnatal growth. During the period of maturity (from 21 to 50 years), when the liver practically stops to grow, the levels of hepatocyte ploidy are changed insignificantly: part of 2c-hepatocytes decreases slowly (up to 84.8% by the end of period) and (2c x 2)-hepatocyte number increases slowly too. The number of polyploid cells increases by several times, but is equal only to 6.6% of all the hepatocytes counted. Under ageing, on the background of human liver atrophy, acceleration of hepatocyte polyploidization takes place. In the age group of 86-92 years parts of 2c- and (2c x 2)-hepatocytes reach 60.3 and 14.3%, resp., and the total share of polyploid cells is as much as near 25%, calculated from the cell population of liver parenchyma. The maximum ploidy levels in hepatocytes of normal human liver during ageing is becoming 16c and 8c x 2 for mononuclear and binuclear cells, resp. Transition rates among hepatocytes of different ploidy classes (2c--2c, 2c--2c x 2, 2c x 2--4c, 2c--4c) were calculated in addition to the coefficient of changing of the hepatocyte proliferative activity with the increase in its ploidy and cell death rate in different periods of human life. A rather high hepatocyte proliferative activity in the early postnatal period of human life was seen to lower during the following years of life. In maturity it is the lowermost to make less than 5% of that in newborns. During ageing the hepatocyte DNA-synthesizing activity being almost 1.6-1.7 times as much as in maturity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Parasexual Ploidy Reduction Drives Population Heterogeneity Through Random and Transient Aneuploidy in Candida albicans

The opportunistic pathogen Candida albicans has a large repertoire of mechanisms to generate genetic and phenotypic diversity despite the lack of meiosis in its life cycle. Its parasexual cycle enables shifts in ploidy, which in turn facilitate recombination, aneuploidy, and homozygosis of whole chromosomes to fuel rapid adaptation. Here we show that the tetraploid state potentiates ploidy variation and drives population heterogeneity. In tetraploids, the rate of losing a single heterozygous marker [loss of heterozygosity (LOH)] is elevated ∼30-fold higher than the rate in diploid cells. Furthermore, isolates recovered after selection for LOH of one, two, or three markers were highly aneuploid, with a broad range of karyotypes including strains with a combination of di-, tri-, and tetrasomic chromosomes. We followed the ploidy trajectories for these tetraploid- and aneuploid-derived isolates, using a combination of flow cytometry and double-digestion restriction-site-associated DNA analyzed with next-generation sequencing. Isolates derived from either tetraploid or aneuploid isolates predominately resolved to a stable euploid state. The majority of isolates reduced to the conventional diploid state; however, stable triploid and tetraploid states were observed in ∼30% of the isolates. Notably, aneuploid isolates were more transient than tetraploid isolates, resolving to a euploid state within a few passages. Furthermore, the likelihood that a particular isolate will resolve to the same ploidy state in replicate evolution experiments is only ∼50%, supporting the idea that the chromosome loss process of the parasexual cycle is random and does not follow trajectories involving specific combinations of chromosomes. Together, our results indicate that tetraploid progenitors can produce populations of progeny cells with a high degree of genomic diversity, from altered ploidy to homozygosis, providing an excellent source of genetic variation upon which selection can act.     

Culture expansion induces non-tumorigenic aneuploidy in adipose tissue-derived mesenchymal stromal cells

Background aimsAdipose tissue-derived mesenchymal stromal cells (ASCs) are of interest as a cell therapeutic agent for immunologic and degenerative diseases. During in vitro expansion, ASCs may be at risk for genetic alterations, and genetic screening is a prerequisite. We examined the presence of aneuploidy in ASCs and its origin and development during culture and evaluated the implications of aneuploidy for therapeutic use of ASCs.MethodsAdipose tissue of healthy individuals was used for isolation and expansion of ASCs. Chromosome copy numbers were studied using fluorescence in situ hybridization analysis. Aneuploidy was studied in freshly isolated ASCs, in ASCs cultured for 0–16 passages and in senescent cultures. To evaluate the plasticity of ploidy, ASCs were cloned, and the variation of ploidy in the clones was examined. Tumorigenicity was studied by subcutaneous injection of aneuploid ASCs in immunodeficient NOD/SCID mice.ResultsNo aneuploidy was detected in freshly isolated ASCs. In low passages (passages 0–4), aneuploidy was detected in 3.4% of ASCs. Prolonged culture expansion of ASCs (passages 5–16) resulted in a significant increase of aneuploidy to 7.1%. With senescence, aneuploidy increased further to 19.8%. Aneuploidy was observed in clones of diploid ASCs, demonstrating the de novo development of aneuploidy. No transformation of ASCs was observed, and in contrast to cancer cell lines, aneuploid ASCs were incapable of tumor formation in immunodeficient mice.ConclusionsASC cultures contain a stable percentage of aneuploid cells. Aneuploidy was not a predecessor of transformation or tumor formation. This finding indicates that aneuploidy is culture-induced but unlikely to compromise clinical application of ASCs.     

FISH analysis reveals aneuploidy and continual generation of chromosomal mosaicism in Leishmania major

The protozoan parasite Leishmania is generally considered to be diploid, although a few chromosomes have been described as aneuploid. Using fluorescence in situ hybridization (FISH), we determined the number of homologous chromosomes per individual cell in L. major (i) during interphase and (ii) during mitosis. We show that, in Leishmania, aneuploidy appears to be the rule, as it affects all the chromosomes that we studied. Moreover, every chromosome was observed in at least two ploidy states, among monosomic, disomic or trisomic, in the cell population. This variable chromosomal ploidy among individual cells generates intra-strain heterogeneity, here precisely chromosomal mosaicism. We also show that this mosaicism, hence chromosome ploidy distribution, is variable among clones and strains. Finally, when we examined dividing nuclei, we found a surprisingly high rate of asymmetric chromosome allotments, showing that the transmission of genetic material during mitosis is highly unstable in this 'divergent' eukaryote: this leads to continual generation of chromosomal mosaicism. Using these results, we propose a model for the occurrence and persistence of this mosaicism. We discuss the implications of this additional unique feature of Leishmania for its biology and genetics, in particular as a novel genetic mechanism to generate phenotypic variability from genomic plasticity.     

Elevated Tolerance to Aneuploidy in Cancer Cells: Estimating the Fitness Effects of Chromosome Number Alterations by In Silico Modelling of Somatic Genome Evolution

An unbalanced chromosome number (aneuploidy) is present in most malignant tumours and has been attributed to mitotic mis-segregation of chromosomes. However, recent studies have shown a relatively high rate of chromosomal mis-segregation also in non-neoplastic human cells, while the frequency of aneuploid cells remains low throughout life in most normal tissues. This implies that newly formed aneuploid cells are subject to negative selection in healthy tissues and that attenuation of this selection could contribute to aneuploidy in cancer. To test this, we modelled cellular growth as discrete time branching processes, during which chromosome gains and losses were generated and their host cells subjected to selection pressures of various magnitudes. We then assessed experimentally the frequency of chromosomal mis-segregation as well as the prevalence of aneuploid cells in human non-neoplastic cells and in cancer cells. Integrating these data into our models allowed estimation of the fitness reduction resulting from a single chromosome copy number change to an average of ≈30% in normal cells. In comparison, cancer cells showed an average fitness reduction of only 6% (p = 0.0008), indicative of aneuploidy tolerance. Simulations based on the combined presence of chromosomal mis-segregation and aneuploidy tolerance reproduced distributions of chromosome aberrations in >400 cancer cases with higher fidelity than models based on chromosomal mis-segregation alone. Reverse engineering of aneuploid cancer cell development in silico predicted that aneuploidy intolerance is a stronger limiting factor for clonal expansion of aneuploid cells than chromosomal mis-segregation rate. In conclusion, our findings indicate that not only an elevated chromosomal mis-segregation rate, but also a generalised tolerance to novel chromosomal imbalances contribute to the genomic landscape of human tumours.