Transverse propagation of action potentials between parallel chains of cardiac muscle and smooth muscle cells in PSpice simulations

Background  

We previously examined transverse propagation of action potentials between 2 and 3 parallel chain of cardiac muscle cells (CMC) simulated using the PSpice program. The present study was done to examine transverse propagation between 5 parallel chains in an expanded model of CMC and smooth muscle cells (SMC).     

Effect of transverse gap-junction channels on transverse propagation in an enlarged PSpice model of cardiac muscle

Background  

In previous PSpice modeling studies of simulated action potentials (APs) in parallel chains of cardiac muscle, it was found that transverse propagation could occur between adjacent chains in the absence of gap-junction (gj) channels, presumably by the electric field (EF) generated in the narrow interstitial space between the chains. Transverse propagation was sometimes erratic, the more distal chains firing out of order.     

Propagation of action potentials between parallel chains of cardiac muscle cells in PSpice simulation

Propagation of action potentials between parallel chains of cardiac muscle cells was simulated using the PSpice program. Excitation was transmitted from cell to cell along a strand of three or four cells not connected by low-resistance tunnels (gap-junction connexons) in parallel with one or two similar strands. Thus, two models were used: a 2 x 3 model (two parallel chains of three cells each) and a 3 x 4 model (three parallel chains of four cells each). The entire surface membrane of each cell fired nearly simultaneously, and nearly all the propagation time was spent at the cell junctions, thus giving a staircase-shaped propagation profile. The junctional delay time between contiguous cells in a chain was about 0.2-0.5 ms. A significant negative cleft potential develops in the narrow junctional clefts, whose magnitude depends on several factors, including the radial cleft resistance (Rjc). The cleft potential (Vjc) depolarizes the postjunctional membrane to threshold by a patch-clamp action. Therefore, one mechanism for the transfer of excitation from one cell to the next is by the electric field (EF) that is generated in the junctional cleft when the prejunctional membrane fires. Propagation velocity increased with elevation of Rjc. With electrical stimulation of the first cell of the first strand (cell A1), propagation rapidly spread down that chain and then jumped to the second strand (B chain), followed by jumping to the third strand (C chain) when present. The rapidity by which the parallel chains became activated depended on the longitudinal resistance of the narrow extracellular cleft between the parallel strands (Rol2). The higher the Rol2 resistance, the faster the propagation (lower propagation time) over the cardiac muscle sheet (2-dimensional). The transverse resistance of the cleft had no effect. When the first cell of the second strand (cell B1) was stimulated, propagation spread down the B chain and jumped to the other two strands (A and C) nearly simultaneously. When cell C1 was stimulated, propagation traveled down the C chain and jumped to the B chain, followed by excitation of the A chain. Thus, there was transverse propagation of excitation as longitudinal propagation was occurring. Therefore, transmission of excitation by the EF mechanism can occur between myocardial cells lying closely parallel to one another without the requirement of a specialized junction.     

Gap-junction channels inhibit transverse propagation in cardiac muscle

The effect of adding many gap-junctions (g-j) channels between contiguous cells in a linear chain on transverse propagation between parallel chains was examined in a 5 × 5 model (5 parallel chains of 5 cells each) for cardiac muscle. The action potential upstrokes were simulated using the PSpice program for circuit analysis. Either a single cell was stimulated (cell A1) or the entire chain was stimulated simultaneously (A-chain). Transverse velocity was calculated from the total propagation time (TPT) from when the first AP crossed a V_m of -20 mV and the last AP crossed -20 mV. The number of g-j channels per junction was varied from zero to 100, 1,000 and 10,000 (R_gj of ∞, 100 MΩ, 10 MΩ, 1.0 MΩ, respectively). The longitudinal resistance of the interstitial fluid (ISF) space between the parallel chains (R_ol2) was varied between 200 KΩ (standard value) and 1.0, 5.0, and 10 MΩ. The higher the R_ol2 value, the tighter the packing of the chains. It was found that adding many g-j channels inhibited transverse propagation by blocking activation of all 5 chains, unless R_ol2 was greatly increased above the standard value of 200 KΩ. This was true for either method of stimulation. This was explained by, when there is strong longitudinal coupling between all 5 cells of a chain awaiting excitation, there must be more transfer energy (i.e., more current) to simultaneously excite all 5 cells of a chain.     

Electrical impedance tomography imaging using a priori ultrasound data

Transverse propagation was previously found to occur in a two-dimensional model of cardiac muscle using the PSpice software program for electronic circuit design and analysis. Longitudinal propagation within each chain, and transverse propagation between parallel chains, occurred even when there were no gap-junction (g-j) channels inserted between the simulated myocardial cells either longitudinally or transversely. In those studies, there were pronounced edge (boundary) effects and end-effects even within single chains. Transverse velocity increased with increase in model size. The present study was performed to examine boundary effects on transverse propagation velocity when the length of the chains was held constant at 10 cells and the number of parallel chains was varied from 3 to 5, to 7, to 10, and to 20. The number of g-j channels was either zero, both longitudinally and transversely (0/0), or 100/100. Some experiments were also made at 100/0, 1/1, and 10/10. Transverse velocity and overall velocity (both longitudinal and transverse components) was calculated from the measured total propagation time (TPT), i.e., the elapsed time between when the first action potential (AP) and the last AP crossed the zero potential level. The transverse g-j channels were placed only at the ends of each chain, such that propagation would occur in a zigzag pattern. Electrical stimulation was applied intracellularly between cells A1 and A2. It was found that, with no g-j channels (0/0), overall velocity increased almost linearly when more and more chains were placed in parallel. In contrast, with many g-j channels (100/100), there was a much flatter relationship between overall velocity and number of parallel chains. The difference in velocities with 0/0 channels and 100/100 channels was reduced as the number of chains was increased. In conclusion, edges have important effects on propagation velocity (overall and transverse) in cardiac muscle simulations.     

Transverse propagation in an expanded PSpice model for cardiac muscle with gap-junction ion channels

Transverse propagation was previously found to occur in a two-dimensional model of cardiac muscle using the PSpice software program for electronic circuit design and analysis. Longitudinal propagation within each chain, and transverse propagation between parallel chains, occurred even when there were no gap-junction (g-j) channels inserted between the simulated myocardial cells either longitudinally or transversely. In those studies, there were pronounced edge (boundary) effects and end-effects even within single chains. Transverse velocity increased with increase in model size. The present study was performed to examine boundary effects on transverse propagation velocity when the length of the chains was held constant at 10 cells and the number of parallel chains was varied from 3 to 5, to 7, to 10, and to 20. The number of g-j channels was either zero, both longitudinally and transversely (0/0), or 100/100. Some experiments were also made at 100/0, 1/1, and 10/10. Transverse velocity and overall velocity (both longitudinal and transverse components) was calculated from the measured total propagation time (TPT), i.e., the elapsed time between when the first action potential (AP) and the last AP crossed the zero potential level. The transverse g-j channels were placed only at the ends of each chain, such that propagation would occur in a zigzag pattern. Electrical stimulation was applied intracellularly between cells A1 and A2. It was found that, with no g-j channels (0/0), overall velocity increased almost linearly when more and more chains were placed in parallel. In contrast, with many g-j channels (100/100), there was a much flatter relationship between overall velocity and number of parallel chains. The difference in velocities with 0/0 channels and 100/100 channels was reduced as the number of chains was increased. In conclusion, edges have important effects on propagation velocity (overall and transverse) in cardiac muscle simulations.     

Critical scale of propagation influences dynamics of waves in a model of excitable medium

Background  

Duration and speed of propagation of the pulse are essential factors for stability of excitation waves. We explore the propagation of excitation waves resulting from periodic stimulation of an excitable cable to determine the minimal stable pulse duration in a rate-dependent modification of a Chernyak-Starobin-Cohen reaction-diffusion model.     

Visualization of origins and propagation of excitation in canine gastric smooth muscle

The origin andspread of excitation were visualized with fluo 3 fluorescence intissues isolated from canine gastric antrum. Sheets of circular muscle(5 × 6 mm) had at least 1 (30%) and up to 3 discrete slow-wavepacing sites located near the longitudinal-circular muscle boundary,whereas similarly sized longitudinal sheets had an average of 5 sites(range 3-12 sites) that initiatedCa²⁺ waves. Superimposedfluorescent oscillations (circular muscle) and spikes (longitudinalmuscle) were seen to initiate and propagate as distinct events,separate from their underlying activities. Average propagationvelocities transverse (6-7 mm/s) and parallel (39-45 mm/s) tothe long axis of muscle fibers were similar for each type of event incircular and longitudinal tissues; however, distinct regions wherevelocities of some (but not all) events decreased by up to an order ofmagnitude were present. The distance propagated by individual eventswas limited by collisions with concurrent excitable events or recentlyactivated regions. Complex patterns of excitation in gastrointestinalsmooth muscle arise as a result of interactions between multiple pacingsites, heterogeneous conduction velocities, and the interplay ofadjacent pacemaker domains.

     

<Emphasis Type="Italic">In vitro</Emphasis> activity of telithromycin against <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> at epithelial lining fluid concentrations

Background  

Haemophilus influenzae is one of the main aetiological agents of community-acquired respiratory tract infections. The primary aim of this study was to evaluate the antibacterial activity of telithromycin against H. influenzae clinical isolates showing different pattern of resistance in comparison with azithromycin and clarithromycin at 1/4 ×, 1/2 ×, 1 ×, 2 ×, 4 × minimum inhibitory concentration (MIC) and to peak concentrations in epithelial lining fluid (ELF). The secondary aim was to determine the influence of CO₂ enriched atmosphere on bacterial susceptibility.     

Effect of umbilical cord mesenchymal stem cells on treatment of severe acute pancreatitis in rats

Background aimsThe aim of this study was to investigate the effect of umbilical cord mesenchymal stem cells (UCMSCs) on severe acute pancreatitis (SAP) in rats.MethodsSAP was established in rats by retrograde pancreatic duct injection of sodium taurocholate. In one group, 5 × 10⁶ cells/kg of UCMSC suspension was injected into the tail vein 0 h, 1 h, 6 h and 12 h after the induction of SAP. In other groups, different doses of UCMSC suspension (5 × 10⁴ cells/kg, 5 × 10⁵ cells/kg, 5 × 10⁶ cells/kg or 1 × 10⁷ cells/kg) were administered at 1 h. Serum amylase was assayed at 12 h. Mortality, ascites, serum tumor necrosis factor-α, interferon-γ (assayed using enzyme-linked immunosorbent assay) and the wet-dry weight of the pancreas gland were assessed at 48 h. Pathologic changes of pancreatic and pulmonary tissues were observed.ResultsMortality in rats receiving 5 × 10⁶ cells/kg of UCMSCs at 0 h was 10% compared with 58% in the SAP control group. Ascites, serum amylase and wet-dry pancreatic weight significantly decreased, and production of tumor necrosis factor-α and interferon-γ were reduced. Pathologic injuries of pancreatic and pulmonary tissues were markedly alleviated. Administration of UCMSCs (5 × 10⁵ cells/kg, 5 × 10⁶ cells/kg or 1 × 10⁷ cells/kg) at 1 h or 5 × 10⁶ cells/kg at 6 h significantly reduced the severity of SAP. The effect was less marked at 12 h and with lower concentrations of UCMSCs.ConclusionsUCMSCs significantly decreased pancreatic injury caused by SAP in a time-dependent and dose-dependent way.     

High speed sCMOS‐based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

Oblique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo‐4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure.

     

Characterization and flocculation properties of biopolymeric flocculant (glycosaminoglycan) produced by Cellulomonas sp. Okoh

Aims

Bioflocculant production potential of an actinobacteria isolated from a freshwater environment was evaluated and the bioflocculant characterized.

Methods and Results

16S rDNA nucleotide sequence and BLAST analysis was used to identify the actinobacteria and fermentation conditions, and nutritional requirements were evaluated for optimal bioflocculant production. Chemical analyses, FTIR, 1H NMR spectrometry and SEM imaging of the purified bioflocculant were carried out. The 16S rDNA nucleotide sequences showed 93% similarities to three Cellulomonas species (strain 794, Cellulomonas flavigena DSM 20109 and Cellulomonas flavigena NCIMB 8073), and the sequences was deposited in GenBank as Cellulomonas sp. Okoh (accession number HQ537132 ). Bioflocculant was optimally produced at an initial pH 7, incubation temperature 30°C, agitation speed of 160 rpm and an inoculum size of 2% (vol/vol) of cell density 1·5 × 10⁸ cfu ml^?1. Glucose (88·09% flocculating activity; yield: 4·04 ± 0·33 g l^?1), (NH₄)₂NO₃ (82·74% flocculating activity; yield: 4·47 ± 0·55 g l^?1) and MgCl₂ (90·40% flocculating activity; yield: 4·41 g l^?1) were the preferred nutritional source. Bioflocculant chemical analyses showed carbohydrate, protein and uronic acids in the proportion of 28·9, 19·3 and 18·7% in CPB and 31·4, 18·7 and 32·1% in PPB, respectively. FTIR and 1H NMR indicated the presence of carboxyl, hydroxyl and amino groups amongst others typical of glycosaminoglycan. SEM imaging revealed horizontal pleats of membranous sheets closely packed.

Conclusion

Cellulomonas sp. produces bioflocculant predominantly composed of glycosaminoglycan polysaccharides with high flocculation activity.

Significance and Impact of the Study

High flocculation activity suggests suitability for industrial applications; hence, it may serve to replace the hazardous flocculant used in water treatment.     

Is the development of falciparum malaria in the human host limited by the availability of uninfected erythrocytes?

Background  

The development and propagation of malaria parasites in their vertebrate host is a complex process in which various host and parasite factors are involved. Sometimes the evolution of parasitaemia seems to be quelled by parasite load. In order to understand the typical dynamics of evolution of parasitaemia, various mathematical models have been developed. The basic premise ingrained in most models is that the availability of uninfected red blood cells (RBC) in which the parasite develops is a limiting factor in the propagation of the parasite population.     

Biotic interactions influence the projected distribution of a specialist mammal under climate change

Aim

To measure the effects of including biotic interactions on climate‐based species distribution models (SDMs) used to predict distribution shifts under climate change. We evaluated the performance of distribution models for an endangered marsupial, the northern bettong (Bettongia tropica), comparing models that used only climate variables with models that also took into account biotic interactions.

Location

North‐east Queensland, Australia.

Methods

We developed separate climate‐based distribution models for the northern bettong, its two main resources and a competitor species. We then constructed models for the northern bettong by including climate suitability estimates for the resources and competitor as additional predictor variables to make climate + resource and climate + resource + competition models. We projected these models onto seven future climate scenarios and compared predictions of northern bettong distribution made by these differently structured models, using a ‘global’ metric, the I similarity statistic, to measure overlap in distribution and a ‘local’ metric to identify where predictions differed significantly.

Results

Inclusion of food resource biotic interactions improved model performance. Over moderate climate changes, up to 3.0 °C of warming, the climate‐only model for the northern bettong gave similar predictions of distribution to the more complex models including interactions, with differences only at the margins of predicted distributions. For climate changes beyond 3.0 °C, model predictions diverged significantly. The interactive model predicted less contraction of distribution than the simpler climate‐only model.

Main conclusions

Distribution models that account for interactions with other species, in particular direct resources, improve model predictions in the present‐day climate. For larger climate changes, shifts in distribution of interacting species cause predictions of interactive models to diverge from climate‐only models. Incorporating interactions with other species in SDMs may be needed for long‐term prediction of changes in distribution of species under climate change, particularly for specialized species strongly dependent on a small number of biotic interactions.     

Effects of temperature,illumination and node position on stem propagation of Miscanthus × giganteus

The sterile triploid Miscanthus × giganteus is capable of yielding more biomass per unit land area than most other temperate crops. Although the yield potential of M. × giganteus is high, sterility requires all propagation of the plant to be done vegetatively. The traditional rhizome propagation system achieves relatively low multiplication rates, i.e. the number of new plants generated from a single‐parent plant, and requires tillage that leaves soil vulnerable to CO₂ and erosion losses. A stem‐based propagation system is used in related crops like sugarcane, and may prove a viable alternative, but the environmental conditions required for shoot initiation from stems of M. × giganteus are unknown. A study was conducted to investigate the effect of temperature, illumination and node position on emergence of M. × giganteus shoots. Stems of M. × giganteus were cut into segments with a single node each, placed in controlled environments under varied soil temperature or light regimes and the number of emerged shoots were evaluated daily for 21 days. At temperatures of 20 and 25 °C, rhizomes produced significantly more shoots than did stem segments (P = 0.0105 and 0.0594, respectively), but the difference was not significant at 30 °C, where 63% of stems produced shoots compared to 80% of rhizomes (P = 0.2037). There was a strong positive effect (P = 0.0086) of soil temperature on emergence in the range of temperatures studied here (15–30 °C). Node positions higher on the stem were less likely to emerge (P < 0.0001) with a significant interaction between illumination and node position. Planting the lowest five nodes from stems of M. × giganteus in 30 °C soil in the light resulted in 75% emergence, which represents a potential multiplication rate 10–12 times greater than that of the current rhizome‐based system.     

The Double-Water-Film Electrode: A Device for Measuring the Resistance and the Capacitance of the Internode/Node Interface of Chara as Functions of Time and Temperature

Abstract A ``double-water-film electrode technique' has been developed for the long-term characterization of the electrical properties across the interface between the nodal (N) and internodal (A or B) cells and the vacuole along the length of an internode of Chara as a function of time and temperature. The electrode unit consisted of a pair of the water-film electrodes described elsewhere (Chilcott 1988; Chilcott and others 1983; Coster and others 1984; Lucas 1985; and Ogata 1983). The distance between two water-film probes was fixed at 1.0 cm. By scanning the electrode unit, the spatial variations in electrical resistance and capacitance along the longitudinal axis of Chara were observed. Analysis was performed by applying an electrical equivalent circuit for the biomembrane (Philippson 1921). Across the internode (−A or −B)/central nodal cells interface, the specific parallel resistance (Rm) and the parallel capacitance (Cm) at 20°C were 30 ± 5 × 10⁻³Ωm² and 1.5 ± 0.5 × 10⁻¹Fm⁻² (at 30 Hz), respectively. And the series resistance, corresponding to the vacuole of the internode was 8 × 10⁻³Ωm². Study of temperature dependencies of Rm and Cm suggested that a dynamic homeostatic regulation was operating at the interface where numerous plasmodesmata were observed with an electron microscope (Pickett-Heaps 1967; Spanswick and Costerton 1967). Assuming that the individual cylinder of plasmodesma was filled only with cytoplasm, the number of plasmodesma per interface was estimated at 2.6 × 10⁵. Received 19 January 2000; accepted 16 March 2000     

Non‐destructive allometric estimates of above‐ground and below‐ground biomass of high‐mountain vegetation in the Andes

Aim

Studies that monitor high‐mountain vegetation, such as paramo grasslands in the Andes, lack non‐destructive biomass estimation methods. We aimed to develop and apply allometric models for above‐ground, below‐ground and total biomass of paramo plants.

Location

The paramo of southern Colombia between 1°09′N and 077°50′W, at 3,400 and 3,700 m a.s.l.

Methods

We established 61 1‐m² plots at random locations, excluding disturbed, inaccessible and peat bog areas. We measured heights and basal diameters of all vascular plants in these plots and classified them into seven growth forms. Near each plot, we sampled the biomass from plants of abundant genera, after having measured their height and basal diameter. Hence, we measured the biomass of 476 plants (allometric set). For each growth form we applied power‐law functions to develop allometric models of biomass against basal diameter, height, height x basal diameter and height × basal area. The best models were selected using AIC_c weights. Using the observed and predicted plant biomass of the allometric set we calculated absolute percentage errors using cross‐validation. The biomass of a plot was estimated by summing the predicted biomass of all plants in a plot. Confidence limits around these sums were calculated by bootstrapping.

Results

For groups of <20 plants the biomass predictions yielded large (>15%) errors. Applying groups that resembled the 1‐m² plots in density and composition, the errors for above‐ground and total biomass estimates were <15%. Across all plots, we obtained an above‐ground, below‐ground and total plot biomass of 329 ± 190, 743 ± 486 and 1011 ± 627 g/m² (mean ± SD), respectively. These values were within the range of biomass estimates obtained destructively in the tropical Andes.

Conclusions

In new applications, if target vegetation samples are similar regarding growth forms and genera to our allometric set, their biomass might be predicted applying our equations, provided they contain at least 50–100 plants. In other situations, we would recommend gathering additional biomass measurements from local plants to evaluate new regression equations.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Human appropriation of net primary production as driver of change in landscape-scale vertebrate richness

Aim

Land use is the most pervasive driver of biodiversity loss. Predicting its impact on species richness (SR) is often based on indicators of habitat loss. However, the degradation of habitats, especially through land-use intensification, also affects species. Here, we evaluate whether an integrative metric of land-use intensity, the human appropriation of net primary production, is correlated with the decline of SR in used landscapes across the globe.

Location

Global.

Time period

Present.

Major taxa studied

Birds, mammals and amphibians.

Methods

Based on species range maps (spatial resolution: 20 km × 20 km) and an area-of-habitat approach, we calibrated a “species–energy model” by correlating the SR of three groups of vertebrates with net primary production and biogeographical covariables in “wilderness” areas (i.e., those where available energy is assumed to be still at pristine levels). We used this model to project the difference between pristine SR and the SR corresponding to the energy remaining in used landscapes (i.e., SR loss expected owing to human energy extraction outside wilderness areas). We validated the projected species loss by comparison with the realized and impending loss reconstructed from habitat conversion and documented by national Red Lists.

Results

Species–energy models largely explained landscape-scale variation of mapped SR in wilderness areas (adjusted R²-values: 0.79–0.93). Model-based projections of SR loss were lower, on average, than reconstructed and documented ones, but the spatial patterns were correlated significantly, with stronger correlation in mammals (Pearson's r = 0.68) than in amphibians (r = 0.60) and birds (r = 0.57).

Main conclusions

Our results suggest that the human appropriation of net primary production is a useful indicator of heterotrophic species loss in used landscapes, hence we recommend its inclusion in models based on species–area relationships to improve predictions of land-use-driven biodiversity loss.     

Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages

Mouse embryonic stem (ES) cells are widely used in early development studies and for transgenic animal production; however, a stable karyotype is a prerequisite for their use. We derived 32 ES cell lines of outbred mice (129 × BALB (1B), C57BL × 1B, and DD × 1B F1 hybrids). Pluripotency was assessed by utilizing stem-cell-marker gene expression, teratoma formation assays and the formation of chimeras. It was shown that only 21 of the 32 ES cell lines had a diploid modal number of chromosomes of 40. In these lines, the percentage of diploid cells varied from 30.3 to 78.9 %, and trisomy of chromosomes 1, 8 and 11 was observed in some cells in 16.7, 36.7 and 20.0 % of the diploid ES cell lines, respectively. Some cells had trisomy of chromosomes 6, 9, 12, 14, 18 and 19. In situ hybridization with an X chromosome paint probe revealed that 7 of the 11 XX-cell lines had X chromosome rearrangements in some cells. Analysis of the methylation status of the Dlk1-Dio3 locus showed that imprinting was altered in 4 of the 18 ES cell lines. Thus, mouse ES cell lines are prone to chromosome abnormalities even at early passages. Therefore, routine cytogenetic and imprinting analyses are necessary for ES cell characterization.