ARTICLE |
Correspondence to: Danielle Chassoux, Institut National de la Recherche Agronomique 806/EA 2703, IFR 63, Muséum National d'Histoire Naturelle, IBPC, 13 rue Pierre et Marie Curie, 75005 Paris, France. E-mail: chassoux@ibpc.fr
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Summary |
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We applied automatic quantitative fluorescence imaging of nuclear DNA to rat liver cells obtained from animals at various times after birth up to 3 months of age. We show that, in conditions best preserving the native cellular structures, DNA content measurements, performed on whole single cells in situ after Hoechst staining, were precise and accurate. Cells in the various ploidy and nuclearity classes could thus be identified correctly and their percentages were estimated on a total of 300 cells or more. DNA synthesis was shown to occur asynchronously in all ploidy and nuclearity classes around weaning time. Observation of the labeling patterns, after in vivo BrdU pulse and short-term culture (chase), showed that the cell cycle was shorter in diploid cells compared with cells undergoing polyploidization. These results show that the approach of fluorescence imaging is well suited to investigations on polyploidization mechanisms. (J Histochem Cytochem 51:319330, 2003)
Key Words: fluorescence microscopy, image analysis, computer assist, hepatocytes, polyploidization, DNA quantitation, Hoechst 33342, BrdU, double labeling
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Introduction |
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IMAGE ANALYSIS of cells in fluorescence to extract quantitative data from biological observations was proposed over 10 years ago. Several groups have described conditions for quantification of nuclear DNA (
Hepatocyte ploidy has been investigated in the past essentially through karyometry (
We set out to see if DNA quantitation could be precisely performed by fluorescence imaging on liver cell preparations so that quantitative analyses could be performed at the single-cell level in situ. For this we require well-separated whole flattened cells. These experiments are intended to serve in multiparametric analyses to study gene expression and protein localization (
The aim of this study was to demonstrate that nuclear DNA content could be precisely measured under the selected experimental conditions and that the cells in the various nuclearity and ploidy classes could be adequately identified within a total number of cells compatible with image analyses. In addition, we show that primary short-term culture of liver cells offers access to information on the speed of the cell cycle according to cell class.
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Materials and Methods |
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Preparations of Hepatocytes
Rats were purchased from IFFA-CREDO (Lyon, France). They were all fed the same standard diet. Hepatocytes were isolated from male Wistar rats by cannulating the portal vein and perfusing the liver with Liberase-calcium (Liberase Purified Enzyme Blend; BoehringerMannheim, Mannheim, Germany). The ages of animals ranged from birth up to 12 weeks. After isolation, cells were collected in L-15 medium enriched with 1 mg/ml bovine serum albumin (BSA) and left to sediment for 20 min at room temperature (RT). After three washes in the same medium, liver cells were seeded at 1.106 cells/cm2 on pretreated glass coverslips in Williams medium E, supplemented with 10% fetal bovine serum (FBS) and 1 mg/ml BSA at 37C in a 5% CO2 atmosphere (
After cell attachment (4 hr in medium with serum, referred to as Step 1 in the text), the medium was removed and replaced by fresh, serum-free Williams medium E containing 0.5 mg/ml BSA, 5 mg/ml bovine insulin, and 7 x 10-7 M hydrocortisone, hemisuccinate, and the cells were cultured for a further 12 hr (Step 2). All culture media used contained penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (250 ng/ml). These cell preparation conditions were designed to obtain flattened cells suitable for image analysis.
In some experiments, cells were seeded on collagen and kept in serum-free medium for 24 hr before being submitted to mitogenic stimulation by culturing them in medium supplemented with 50 ng/ml epidermal growth Factor (EGF) and 20 mM sodium pyruvate (
Cell Fixation
At the end of the culture period, the cells were washed three times in PBS and fixed in 4% PFA in PBS (PFA) for 10 min at RT. After extensive washing, cells were stored in 1% BSA in PBS or in 70% ethanol at 4C.
Appositions were fixed in PFA for 15 min at RT and washed in PBS before staining. For flow cytometry (FCM), cells suspensions (before seeding on coverslips) were fixed in 70% ethanol and kept at 4C.
DNA Staining
Cells were incubated in 2 µg/ml Hoechst 33342 (H42) (Riedel de Haen; Seele, Germany) in PBS for 30 min at RT before being washed and mounted in Permafluor (Immunotech; Marseille, France). The compound H42 has been shown to bind stoichiometrically to DNA when crosslinking fixatives are used (
For FCM, cells were treated with RNase and stained with 10 µg/ml propidium iodide (PI). FCM was performed on a FACStar-plus (BectonDickinson; Mountain View, CA).
BrdU Incorporation and Detection
Rats were injected intraperitoneally with bromodeoxyuridine (BrdU) (Sigma; St. Louis, MO) (30 mg/kg body weight) 1 hr before sacrifice. After hepatocyte isolation, cells were cultured either with (10 µM) or without BrdU. Both preparations were fixed at the same time, at the end of the culture period.
Double staining for BrdU and total DNA was performed in conditions that do not modify DNA content measurements (
DNA Content Measurements and Detection of BrdU Incorporation in the Same Cells Using Fluorescence Imaging
The cell preparations were examined under a Zeiss (Axiovert 35) (Carl Zeiss; Gottingen, Germany) inverted microscope equipped for epi-illumination (50-W mercury lamp). Zeiss Plan Neofluar objectives x20 (NA 0.5) and x40 (NA 0.75) were chosen, enabling the collection of light from the entire thickness of the nucleus, since these conditions are essential for adequate DNA content determinations (
Images were captured using a cooled CCD camera (Photometrics, Tucson, AZ; KAF 1400-G2, class 2), on 4056 gray levels as described (
Parameters such as integrated fluorescence (IntF) or area of selected objects were stored in computer files for analysis using Kaleidagraph. The IPLab software also provides a color code giving, from dark blue to red, the distribution of values from the lowest to the highest value. A total of 300400 cells were studied per timepoint, observed on 812 separate fields. This number is well in the range of other studies based on imaging (
For BrdU labeling, cells were inspected on the computer screen and scored as BrdU-positive by the operator when bright-green fluorescent dots were seen over the nuclear area, as identified by Hoechst stain. Cells scored as negative for BrdU displayed evenly dark nuclei, even after artificially increasing the contrast of the image. BrdU labeling patterns were recognized and classified according to the established nomenclature of the various stages of the S-phase, each recognized by typical distributions of the replication sites: early S-phase, many granules of small size distributed all over the nucleus, excluding nucleoli, not reaching the border ("early"); mid-S-phase, clustering of fluorescent signals around nucleoli and at the periphery of the nucleus ("mid"); late S-phase, fewer signals of larger size distributed in the interior of the nucleoplasm ("late") corresponding, in rodents, to heterochromatin-rich regions (
Statistical Analyses
These were performed using the Chi-squared test or the nonparametric test of MannWhitney (level of significance p< 0.05).
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Results |
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Ploidy Measurements by Fluorescence Imaging
Liver cell preparations from adult rats were fixed and stained for examination by fluorescence imaging. The analytical procedure is illustrated in Fig 1. Hoechst-stained nuclei appeared brightly fluorescent and could be assigned to a given cell by comparison with the corresponding brightfield image (Fig 1A and Fig 1B). Automatic quantitative measurement of Hoechst fluorescence intensity was performed after correction of the image and, depending on the value of this parameter, each nucleus was assigned a false color. As shown in Fig 1C, the color scale indicates that nuclei have integrated fluorescence (IntF) values centred around three values only. The smallest nuclei in mononucleated cells appear blue. Nuclei in binucleated cells, when considered as one object (by linking them artificially), appear to have the same color as larger nuclei in mononucleated cells (green) (Fig 1C and Fig 1D, right panel). When considered individually, each nucleus in a binucleated cell appears the same color as the smallest nuclei in mononucleated cells (blue) (Fig 1D, middle panel). The nucleus of one large mononucleated cell appears red. A typical histogram of data obtained is shown in Fig 2A. The first peak shows a coefficient of variation (CV = standard deviation/mean) of 5.8%. The second peak is at a position twice the value of the first peak. Considering mononucleated cells, DNA index (mean IntF of second peak/mean IntF of first peak) is 2.04. When the two nuclei in binucleated cells are analyzed as one object (hatched columns), there is a complete overlap of their IntF values with those of the mononucleated cells in the second peak (open columns). In contrast, when binucleated cells' nuclei are analyzed separately, their IntF values attain those of the mononucleated cells in the first peak (ratio of the means was 1.04 for 28 elements in this example). From these data we conclude that the first peak represents 2C cells nuclei and the second peak 4C cells nuclei [mononucleated 4C (4Cm) and binucleated 2x2C]. The CV (indicative of the precision of the measures) value as well as the DNA index (indicative of the accuracy) indicate the high probability of the separation of the first two peaks. In this field, a nucleus was found at twice the value of the second peak. This suggests that this cell belongs to a rare population of mononucleated 8C cells. Histogram representations of other DNA content measurements showed, without any exception, similar distributions of IntF values for each ploidy level, without intermediate values. The CV of 2C values ranged from 4% to 8% and DNA indexes ranged from 1.95 to 2.05 for the 812 fields examined in each of four rat preparations.
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Nuclear area values showed a wider dispersion than fluorescence intensity values. This applied to diploid cells as well as to tetraploid mononucleated and binucleated cells (Fig 2B). It was probably due to the different relative spreading of the cells within a group of cells and did not affect fluorescence intensity measurements. The mean area did increase with ploidy. In the example, the 4C cells' mean nuclear area increase over the 2C cells' mean nuclear area was 1.75.
These findings, several ploidy levels, absence of S-phase, binucleated cells, and increase in nuclear size with DNA content, are known features of adult rodent hepatocytes. This indicates that we were able, in the cell preparation and imaging conditions used here, to analyze correctly various parameters in the cell population. Regarding quantitative measurements, the size of the CVs of DNA content measures was satisfactory.
We then proposed to measure the DNA content of cells in S-phase. Adult rat liver cell suspensions spread on glass and cultured in mitogenic medium are known to re-enter S-phase with a characteristic timing (
Cells were fixed for 23 or 28 hr after mitogenic stimulation. BrdU, added to the medium 45 min before fixation, was revealed by indirect immunofluorescence and cells stained with Hoechst. "Early" and "mid" labeling patterns were recognized at both time points examined, indicating that cells are not truly synchronous in this experimental condition. Data were pooled, building up an overall level of proliferation of 13%. The DNA contents of doubly stained nuclei were found in between the ploidy peaks defined by BrdU-negative nuclei. This is illustrated in Fig 3A and Fig 3B, in which two fields were added. Nuclei displaying an "early" labeling pattern (discrete small foci distributed all over the nucleoplasm, excluding nucleoli) had IntF values to the right of the mean of the 2C, 4C, or 8C peak on the histogram. An increase in DNA content up to 23% above the mean value of the previous peak was measured in BrdU-positive nuclei (n=31). "Mid" labeling pattern (clustering of foci around nucleoli and at the nucleus periphery) corresponded to an increase in DNA content of 3087% (n=27) above 2C, 4C, or 8C mean value. These values also include rare nuclei with a "late" labeling pattern. A total of 563 cells were studied over 29 fields. In these experiments, the quantitative measurements were done at x40. The CV of 2C values of BrdU-negative nuclei ranged from 5% to 7% and DNA indexes ranged from 1.95 to 2.1. These results indicate that we can detect cells in S-phase by IntF measurements. BrdU-positive nuclei appeared as different colors from BrdU-negative nuclei on the color code for DNA content (not shown).
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Percentages of Cells in the Various Nuclearity and Ploidy Classes
Cells characterized in their ploidy and nuclearity could then be counted, collecting data from several microscopic fields to reach a number of total cells higher than 300.
Liver cell preparations from 6-day-old up to 12-week-old rats were analyzed. The proportions of cells in the nuclearity and ploidy classes are given in Table 1. Tetraploid cells were found as early as six days after birth at a frequency of 1%. They became the major population from 6 weeks onwards. Seventy percent tetraploid cells were found in 6-week-old rats by FCM, a similar percentage to that given by fluorescence imaging when adding up 2x2C and 4Cm populations. Binucleated 2x2C cells were also detected as early as 6 days of age, with a gradual increase in numbers up to 4 weeks and then a slow decline towards a value of around 20% at 3 months. When binucleated cells on appositions were counted, a lower percentage was found compared with fluorescence imaging. For example, at 21 days of age we found 6% 2x2C cells, whereas 4Cm cells numbers were similar to those found by fluorescence imaging. Octoploid cells arose as soon as 21 days after birth, with higher numbers for 2x4C than for 8C cells after 1 month of age.
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The percentages were established from a total of 300400 cells because we observed that no more significant information could be gained beyond 200 cells. This is indicated as follows. For each field, the total number of cells and the relative proportions in nuclearity and ploidy vary. As an indication of the inference of the sample size to the cell counts, we calculated the increase in precision provided by adding up fields. Data from a preparation of a rat just over 3 weeks old are presented as an example, studying nine fields (total number of cells 377). We calculated the cumulative percentages of 2C or 4C mononucleated cells, starting from any one of the nine fields (Fig 4). For frequent events (2C=60%) or less frequent events (4Cm=10%), the addition of five fields (mean number of cells n=196, range=187249) gives information that is statistically no different (Chi-squared test) from the addition of eight fields (n=363, range=293345).
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The limit of sensitivity of the assay can be placed at 1 in 300 because a minimum of 300 cells is analyzed at each age point and rare events are undoubtedly detected (Fig 1; Table 1).
Distributions of DNA Content Values at Various Ages
The DNA content histograms for liver cell preparations at given ages were established (Fig 5). These are representative of all the preparations presented in Table 1. The size of the CVs in the young did not appear to be larger than that in the adults. In each case, a discontinuous DNA content distribution was observed, suggesting that there was no cell in S-phase. This was surprising in a situation of rapidly expanding liver mass. The possibility that sampling could be a problem here was considered and we decided to search for cells entering the cell cycle by detecting incorporation of BrdU.
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Evidence for DNA Synthesis
BrdU was injected before sacrifice and also, after hepatocyte isolation, BrdU was added to the culture medium and left throughout the experimental time. The percentages of positive cells scored at days 13, 19, 28, and 42 were respectively 32%, 20%, 14%, and 1%. These values appeared surprisingly high and we wondered if there was S-phase induction during the culture step. We next designed experiments to investigate the contribution of each culture step to the observed level of BrdU incorporation. Cells were split into four groups, receiving BrdU for either one only of the subsequent culture steps (Step 1 or Step 2) or both of them and a control group that did not see BrdU in culture. Detection of incorporated BrdU was done, for each group, at the end of the culture period. Only in the youngest rats did the number of positive cells increase over that found by in vivo incorporation (50% increase, two experiments). Therefore, those cells ready to enter S-phase were able to do so during Step 1, indicating that their viability was not affected by the preparation procedure. In the absence of serum (Step 2), no further DNA synthesis took place so that the level of BrdU incorporation in the "Step 1 + Step 2" group was the same as for the "Step 1" group. Thus, mononucleated diploid cells, representing the vast majority of cells at this age of the animals (13 days), actively proliferate and engage in S-phase asynchronously, even in vitro, but only when serum is present. Fig 6 shows a BrdU-positive mitotic figure (late telophase) in a 13-day-old rat liver cell preparation taken from the "Step 1 + Step 2" group. With the possibility of the onset of S-phase taking place in vitro at that age, the observation of labeled mitoses suggest that the duration of S and G2+M is of 12 hr minimum. Therefore, the completion of a cell cycle may take place in vitro, giving rise to two labeled daughter cells, so that the labeling due to BrdU incorporation took place during the S-phase of the preceding cycle.
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Comparisons of DNA Content and DNA Synthesis
The apparent discrepancy between BrdU positivity and the corresponding DNA content measurements in young rat liver cells (Fig 5A5C) was addressed next in double labeling experiments in which total DNA content (stained with H42) was measured on the same cells in which incorporated BrdU was detected.
Pulse Labeling and BrdU Labeling Patterns. BrdU was given in vivo only (pulse) to get labeling patterns characteristic of the successive stages of S-phase. Fixation was done as usual at the end of the culture step. This situation is equivalent to a pulse chase experiment. BrdU-positive cells were found that displayed 2C, 4C, or 8C DNA content. This is indicated in Fig 7 by the color code in which blue is ascribed to 2C cells, green to 4C cells, and red to 8C cells. The various types of replication patterns were recognized in BrdU-positive nuclei. These were found in each of the nuclearity and ploidy classes, indicating that there was no selective loss of one of the classes nor loss of viability, during the observation period, of cells engaged in S-phase.
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Diploid Cells All three patterns were recognized, indicating asynchrony and cell cycle completion, regardless of the stage of diploid cell engagement in S-phase (labeled during S-phase, they were observed with a 2C DNA content). They were often seen as pairs of cells, whose nuclei exhibited similar labeling patterns (Fig 7A). Seventeen percent of the diploid cells were labeled at days 19 and 28, representing, respectively, 74% and 61% of all labeled cells.
Other Ploidy Classes
Among the mononucleated 4C cells, half of them were labeled at day 19, representing 25% of all labeled cells. At day 28, 12% were labeled for BrdU, representing 12% of all labeled cells. Most of them displayed an "early" labeling pattern (Fig 7B), although other patterns were also seen. Labeled at the onset of S-phase (of a 2C nucleus), they were observed with a nuclear DNA content of 4C. This demonstrates that they progressed through S-phase during the experimental time [the labeling pattern acquired on pulse was retained during the next S-phase stages (
Binucleated 2x2C hepatocytes positive for BrdU displayed a late pattern of staining. These cells were labeled during the preceding S-phase (of a 2C cell) and were likely formed during the culture step. This was a rare event (6% of labeled 2x2C versus total 2x2C cells at 19 and 28 days), suggesting that the binucleation process is a slower process than the division of a diploid cell into two daughter diploid cells. Binucleated 2x4C hepatocytes displaying "early", "mid," or "late" replication patterns were observed (Fig 7C and Fig 8), suggesting that, as in the case of mononucleated tetraploid hepatocytes, the cycle was arrested before mitosis. Most of the 2x4C cells (83%) were BrdU positive at day 28. Therefore, the absence of S-phase in the DNA content measurements is now explained by the fact that cells completed DNA synthesis during the culture step, with some cells pursuing the cycle towards mitosis and G1, as confirmed by the following observations.
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Continuous BrdU Labeling and Quenching of Hoechst.
In the experiments where BrdU was added continuously in culture, we noted that, in 2C cells positive for BrdU, DNA content measurements decreased by 1520% compared with 2C cells negative for BrdU present in the same field. This difference in IntF values was statistically significant (MannWhitney test). A shift to the left of the G1 peak, as shown in Fig 9, is typical of the classical phenomenon of quenching of Hoechst fluorescence by BrdU (indicative of a cell cycle completion after BrdU substitution during the entire preceding S-phase). A 20% quenching measured by FCM was reported with a 10 µM BrdU concentration (
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In these continuous labeling experiments, BrdU-labeled cells appeared brightly positive all over the nucleus, and labeling patterns could not be recognized any longer. This is another indication that the cells have continued in S-phase in vitro.
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Discussion |
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We have shown here that, under conditions compatible with optical microscopic image analysis, liver cells in the various ploidy and nuclearity classes are indeed correctly identified. The proportions that we observed in adult rats are in agreement with results obtained by others with larger numbers of cells. The preparation conditions used in previous studies were designed for ploidy alone or, in some instances, for ploidy and S-phase. Here we favored whole-cell preservation, with a view to future studies on gene expression and protein localization.
Analyzing ploidy on appositions of mouse liver,
Analyzing appositions, we found that binucleated cells (not assessed in
Ploidy classes for younger rats are little documented. Data obtained by karyometry on squashed tissue (
The relationship between binucleated and mononucleated polypoid cells is not clear. The largest numbers of binucleated cells are found earlier than that of 4Cm. This observation is compatible with the scheme according to which, in binucleated cells, a concomitant DNA synthesis takes place in the two nuclei (BrdU-positive 2x4C cells were found at day 28), and then there is constitution of a common spindle and mitosis, giving two mononucleated tetraploid cells (
An important point is that rare events (one cell in several hundreds) are unambiguously identified. This is permitted by the size of the CV and indicates that no information is lost due to the number of cells analyzed. It allows us to address situations such as physiological liver growth, presenting a low level of proliferation, with an assay keeping observations as close as possible to the in vivo situation. Pathological samples (fine needle aspirates, for instance) could also be analyzed by fluorescence imaging, in addition to (or in place of) FCM.
It should be stressed that static DNA content measures are not able, as such, to account for the events taking place in the young rat liver, because an active proliferation gives rise to G2 cells that cannot be discriminated, by DNA content alone, from mononucleated 4C polyploid hepatocytes. The short-term assay used in this study allowed rapidly cycling cells to proceed through S-phase to the G1 position of the next cycle, explaining why, in young rats, we found higher numbers of cells having incorporated BrdU compared with in vivo tritiated thymidine pulse experiments (
Double labeling experiments suggest that alteration in the length of the cell cycle may take place at the onset of polyploidization. Delayed entry into mitosis or re-replication without mitosis has been observed in a number of biological and experimental situations. DNA damage induced either by exogenous agents, such as ionizing radiation, or by pharmacological agents results in delayed entry into mitosis to allow for repair. Mutations affecting cell cycle molecules may lead to endoreplication. Whereas both situations are unlikely to take place in physiological liver growth, disruption of specific pathways indicates possible relevant mechanisms involving, e.g., p21 (
The main advantages of in situ fluorescence imaging lies in the relationship that can be established at the single-cell level between a given biological parameter of interest (subcellular localization) and the nuclearity and ploidy, which neither flow cytometry nor cytophotometry can achieve. The precision reached can be great, providing that critical attention is paid to material preparation and to image acquisition conditions. We propose that quantitative fluorescence imaging can be used to analyze liver polyploidization mechanisms.
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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Supported by INSERM and INRA.
We thank Catherine SenamaudBeaufort, Helene Mouly, and Olivier Bregerie for help with cell preparations, Chantal Desdouets, Christiane GuguenGuillouzo, and Pascale Mentré for discussions, and F. LeDiest (Hopital Necker) for FCM. We are indebted to J. Plumbridge for reading the manuscript.
Received for publication October 2, 2001; accepted October 16, 2002.
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