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Keywords:

  • image processing;
  • rare phenotype;
  • confocal microscopy;
  • stem cells;
  • myogenic

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Background:

Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines.

Methods:

The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis.

Results:

The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy.

Conclusion:

Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation. © 2007 International Society for Analytical Cytology

The understanding of cell differentiation and development of complex biological systems is largely facilitated by the use of in vitro cell culture systems of primary cells or cell lines capable of undergoing differentiation under artificial conditions. The qualitative and quantitative analysis of the rare phenotypic variants in such systems is a major challenge. On one hand, the study of cellular patterns and cell to cell interactions requires morphological, typical microscopic analysis of the undisturbed cell culture. On the other hand, quantitative analysis of various phenotypic characteristics of a large number of individual cells typically requires invasive methods, such as flow cytometry (FCM), that disrupt the spatial patterns of the population. Especially, collecting quantitative measures on cells with rare phenotype representing only a few percent of the whole population requires the analysis of a large number of cells. Slide-based or microscopy-based cytometric methods associate the advantage of the FCM analysis and the microscopic observation (1, 2). Technically, this approach requires acquisition, treatment, and analysis of images of cell cultures. Since individualization of nuclei or cells in aggregates or complex tissue organization is a prerequisite for subsequent image analysis, the segmentation method is one of the critical steps of the image treatment. Previous efforts made available efficient image segmentation methods for cell nuclei and whole cells into two and even three dimensional biological objects. Most of them were obtained by combining image treatment with confocal microscopy technology (3–8).

Automated acquisition and segmentation of the images, measurement of the fluorescence intensity of each cell or cell nucleus, and the recording of their position in the plate made possible to associate the resolution of microscopic analysis with the potential of FCM. In the present paper, we have adapted the principles of the microscopy-based cytometry to a specific biological problem, the identification of rare cells with stem-cell like phenotype in culture. Pluripotential cells can be identified in many cell lines, primary cell cultures established directly from tissues or even from tumors on the basis of their high activity of the ATP-binding cassette (ABC) transporters, which allows the efficient exclusion of the fluorescent dye Hoechst 33342 (9, 10). As a result of their high dye exclusion capacity, the fluorescence intensity of the nuclei in these cells is lower than that in the nuclei of the population. This property allows the identification and isolation of these so called “Side Population” (SP cells) versus the “Main Population” (MP) (10). Whereas, SP cells are routinely isolated for molecular analysis from a cell suspension using a cell sorter, their in situ examination in culture could help to elucidate what interactions with their neighbors they have. The aim of our work was to develop a method that allows the rapid and reliable identification of stem-cell like SP cells in culture. The method was set up and validated on cultures of the myogenic C2C12 cell line as a model system. The populations of the C2C12 cells contain a small subpopulation of cells with stem cell-like phenotype (11), which represent only 1–2% of the whole culture. By using the principles of slide-based cytometry our method overcomes the difficulties associated with the standardization of Hoechst labeling in adherent cells, the precise measurement of the fluorescence intensities and the identification and localization of the SP cells in culture.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Cell Culture

C2C12, a sub clone derived from the original C2 mouse myoblast cell line was obtained from the American Type Culture Collection (CRL 1772). C2C12 cells were routinely propagated in proliferation Dulbecco's Modified Eagle's Medium (DMEM, Gibco BRL, Gaithersburg, MD) with 4.5 g/ml of glucose, supplemented with 20% (v/v) foetal calf serum (FCS, Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin. The cultures were performed at 37°C under a humidified atmosphere of air with 7% CO2. Initial plating density was between 2 × 103 and 3 × 103 cell/cm2 and the cells were cultured for 6 days. Thirty-five millimeter glass bottom culture dishes (Mat Tek Corporation, Ashland, MA) were used for cell culture and confocal analyses.

Hoechst 33342 Staining and Cell Sorting

This protocol was adapted from the staining of haematopoietic originating cell populations (9). C2C12 cells were trypsinized (Gibco BRL – batch 3101673) and suspended at a working concentration of 106 cells/ml in ice-cold PBS containing 2% FCS, or kept adherent in tissue culture dishes in their proliferation medium. In each case, the DNA dye Hoechst 33342 (Sigma-Aldrich B-2261, batch 123K4080, St. Louis, MO) was added to a final concentration of 11 μg/ml and cells were incubated 90 min at 37°C under mild shaking. The controls were incubated in the presence of 100 μM of verapamil (Sigma-Aldrich, V-106 batch 28 H4699). After staining, adherent cells were trypsinized. Both cultures were spun down and cell suspensions were suspended in fresh 2% FCS–PBS with 2 μg/ml propidium iodide (PI, Sigma) for the partition of dead vs. living cells. Samples were kept on ice until flow cytometry (FCM) analysis.

Analysis and sorting were performed on a dual-laser MoFlo flow cytometer (Dako, Glostrup, Denmark). An argon laser tuned to 350–360 nm (100 mW) was used to excite the Hoechst dye. Fluorescence emission was collected with a 450/65 band pass (BP) filter for the Hoechst ‘blue’ and a 570/40 BP filter for the Hoechst ‘red’. A 510 DMLP was used to separate the emission wavelengths. A second 488 nm argon laser (100 mW) was used to excite PI fluorescence. Fluorescence emission was then measured with a 630/30 BP filter. PI-positive dead cells were excluded from the analysis. Results were analyzed using the Summit v3.1 software (Cytomation, Dako, Glostrup, Denmark). The ‘blue’ emission = f(‘red’ emission) diagram revealed a side population (SP) displaying low staining with Hoechst and a main population (MP) of cells that were more brightly stained.

Microscopy and Image Processing

The two-photon confocal microscope consisted of a Radiance 2100 MP scan head (Bio-Rad, Hercules, CA) equipped with a mode-locked Titanium–Sapphire laser system (Coherent Verdi-Mira) pumped at 5 W and tuned to a 800 nm excitation with 100 fs pulses at 76 MHz. On-line checking of the excitation parameters was done through the use of beam conditioning and Pockel Cell units (Bio-Rad) that enabled pulse length to be controlled and output power levels to be attenuated below a range so that no damage can be observed on the tissue (typically 10–46 mW depending on the objective lens). Power measurements exiting the microscope objective were made with a Coherent LaserMate model 33-0191 power meter. Two-photon fluorescence was detected by a nondescanned detector (Bio-Rad) after passing optical filters. The Dual-emission of Hoescht dye used a dichroic mirror 500DCLPXR (Bio-Rad) and two band pass emission filters: HQ450/80 for the blue emission and 575D150 for the red emission (FRET filter; Omega Optical, Brattleboro, VT). The detector gain settings were fixed to 50% for each detector and the laser power with a 10× objective was set to 10 mW. The microscope was an inverted Nikon TE300 (Nikon Instech Co., Kanagawa, Japan) with Nikon objectives, dry CFI Plan APO, 10×, NA0.50. The acquired images were analyzed by our own software written in C language.

Two images are collected simultaneously by a two photon confocal microscope. They correspond respectively to the blue and red fluorescence emitted by the Hoechst located in the nucleus of the C2C12 cells (Figs. 2A and 2C). The first image process used is a basic threshold (Figs. 2B and 2D), which leaves a noise characterized by clusters of a few pixels. This noise is removed by means of a median filter with a kernel of 3 × 3 pixels (Figs. 2E and 2G). The nuclei in contact of the frame borders on the filtered image were removed (Figs. 2F and 2H). An algorithm settled by Serafini is then used to attribute a label to each cell of the treated binary image (12). The user set a minimal size so that the objects smaller to this size are recognized, selected, and eliminated (Figs. 2G and 2H). A distance map is then obtained as described by Shih and Wu (13). Each white pixel is replaced by a grey pixel, where value represents the square of the Euclidian distance from the nearest black pixel (Figs. 2I and 2K). This distance map emphasizes the maxima regions: pixels within the cells that are the far form the borders (not shown). These maxima are used as labels for the segmentation. We used the unbiased watershed algorithm with the “waiting queue” data structure developed by Meyer (14) and improved by Beucher (15) (http://cmm.ensmp.fr/∼beucher/publi/LPE_sans_biais_V2.pdf). It enables a correct individualization of the cellular nuclei by tracing a watershed line at the border between the connected nuclei (Figs. 2J and 2L). Finally, to prevent over-segmentation, the size of each segmented object is calculated and the objects with a size smaller to the user-defined size are considered as being a watershed line.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

SP/MP Profile of C2C12 on Adherent Cells

First, we have set up the conditions for the labeling of adherent C2C12 cells with the Hoechst 33342 dye so that fluorescence profiles given by flow cytometry are equivalent to those obtained when cells are stained in suspension. To avoid the misidentification of the SP cells (16), the optimal Hoechst staining conditions were determined by using different concentrations of the dye. A Hoechst concentration of 11 μg/ml for 90 min, was found to be optimal for both adherent cells and cells in suspension. Controls incubated with verapamil, an inhibitor of the Mdr1 activity, were also analyzed to check the “specificity” of the SP population.

The Figure 1 shows the profiles obtained by flow cytometry of cells labeled under “in situ” or “in suspension” conditions. There were no significant differences between the SP/MP profiles obtained in the two conditions (Figs. 1A and 1C). When the exclusion of the dye is inhibited by verapamil, the SP cells cannot be discriminated from the MP cells (Figs. 1B and 1D). This result indicates that the capacity of the SP fraction of C2C12 cells to exclude the Hoechst 33342 is not affected by their adhesion to the substrate of the culture plate.

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Figure 1. Hoechst-profile of the C2C12 cells labeled in suspension (A, B) or attached to the culture dish (C, D). Both labeling methods give identical profiles at 11 μg/ml of Hoechst 33342 concentration. When the cells are treated with verapamil (B and D), the fluorescence of the SP cell fraction is shifted to a higher level that specifies the Mdr activity of the SP population.

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Image Acquisition

The next step was to adapt the method for the SP cell identification based on the measurement of the fluorescence intensity of the cell nuclei by the use of a non invasive imaging system. We chose two-photon confocal microscopy, because it allows quantification of the fluorescence under conditions of Hoechst excitation similar to that of our flow cytometer. Moreover, two-photon imaging reduces substantial damage in the living cells (17). Images of cell nuclei collected with a two-photon confocal microscope were then processed. As the confocal instrument produces optical sections, we checked that the Z position did not affect the relative fluorescence intensity of the nuclei.

Image Processing

The different steps of the image processing are shown on Figure 2. The segmentation algorithm described here allowed us to individualize the majority of the nuclei in a dense cell culture without over-segmentation and eliminate artifacts corresponding to the background noise on the original image. The efficiency of the algorithm was evaluated by manual counting of the over- and under-segmented nuclei of an image containing ∼1,000 nuclei. The number of successfully individualized cell nuclei was 90% with our algorithm at the threshold of 33/255 of grey level and 94% with a threshold of 50/255. Both threshold levels were far below the minimal fluorescent intensity of the nuclei.

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Figure 2. Major steps of the image treatment. The images of the red and blue fluorescent cell nuclei are acquired on a two-photon confocal microscope (A). After binarization and threshold selection (B and E) the background noise is removed from the image by a median filter (E). Cells in contact with the border are removed by conditional dilatation (F). A distance map is constructed by replacement of the white pixels by a grey (I) leaving to the individualization of the cell nuclei (J). A zoom of the same part of the global image is represented on the side (C, D, G, H, K, and L of the A, B, E, F, I, and J panels, respectively). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Validation of the Method on Sorted Populations

To validate the method, we isolated the subpopulations of the SP and MP cells by cell sorting, then they were put in a culture dish and maintained at 4°C. The two subpopulations were imaged separately. The images were processed as described. The results show that the SP cell nuclei can be differentiated from those of the MP cells (Figs. 3A and 3B). Therefore, our method that associates a specific staining procedure, standardized image acquisitions and a new software development allows us to detect weak differences between SP and MP cells as by FCM.

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Figure 3. SP/MP profile reconstructed from segmented images. Sorted SP and MP cells are plated at equal density. The SP nuclei (A) are less fluorescent than MP nuclei (B) as also shown on the red and blue fluorescence intensity plots (C and D, respectively). Some intensely fluorescent cells in the SP preparation are likely to be MP contaminants. E is the superposition of C and D.

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Identification and localization of SP Cells in an In Vitro Growing C2C12 Cell Culture

We have evaluated the capacity of our system to detect SP cells in a growing population of C2C12 cells. The low incidence of SP cells expected in a growing cell population required the acquisition of a large number of images for collecting the information from thousands of cell nuclei. We have acquired series of up to 30 images of 800–1,200 nuclei of each cell culture and analyzed them with our method. The comparison of the plots (Fig. 4) of fluorescence values from the normal and verapamil-treated cell populations revealed that a fraction of cells had low fluorescence due to the high Hoechst 33342 exclusion activity inhibited by verapamil. The proportion of these cells—on average of 1%—is not different from those usually obtained by flow cytometry for the SP cells by similar criteria. We consider therefore the cells with low fluorescence values as bona fide SP cells.

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Figure 4. The SP/MP profil of a typical C2C12 population. Six day cell cultures untreated (A) or treated (B) with verapamil were labeled as above. The average fluorescence intensity of the nuclei treated by verapamil is higher. The superposition of the two plots (C) reveals the SP cell fraction in the untreated culture (A). The plot is similar of that obtained by FCM.

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Since the graphical coordinates of each cell are recorded we could localize the rare SP cells dispersed within the culture (Fig. 5). Therefore, it is possible to obtain quantitative information on a large number of cells as by flow cytometry, but also to localize the cells in the culture and make direct observations in their in vitro environment by microscopy.

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Figure 5. Localization of the SP cells in vitro. Series of images treated by the segmentation software (A) or in phase contrast (B). The weaker fluorescent intensity of the SP nuclei is evident on the high power pictures shown on C and D with the SP nuclei indicated by an arrow. Low power bright field fluorescent (E, F) and phase contrast (G and H) images with the SP cells (in yellow, indicated by an arrow).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The method reported here follows the principles of the slide-based cytometry by joining the advantage of the flow cytometry to obtain quantitative information on a large number of individual cells with the morphological analysis of the cell cultures by microscopy. Our method represents a specific application of these principles for the identification via Hoechst efflux of putative stem cells in cultures of adherent cells of a model cell line. This approach required the adaptation of the cell labeling method to adherent cells and the development of efficient image acquisition and processing methods specific to the biological material investigated. The application of our method to other culture systems—as primary cell populations containing stem cells or neoplastic cell lines with cancer stem cells—may help elucidate the nature of interactions the putative stem cells have with the surrounding other cells in the culture.

The precise localization of the SP cells in a cell culture is for a great part due to the efficiency of the computer tool to treat dozens of thousands of events. With our algorithm we could achieve segmentation of the original images, individualizing each cell nucleus, even those from complex cell clusters. The successful reproduction of the SP/MP profiles similar to those typically obtained by flow cytometry provides a proof of concept of our method. The software developed here is fully automated and does not require user initiation except for setting up the background noise level, which can vary according to the condition of staining or acquisition, as well as the determination of the minimal size of the events to be considered. This setting is greatly facilitated by a real time viewer that displays new images when changes are operated in threshold or minimum size levels.

The time required to process an image of 1,024 × 1,024 pixels is about 20 min, when using a “mean Pc configuration”, and allows a large number of nuclei to be examined and quantified so that statistical accuracy can be achieved in a reasonable period of time.

The capacity to identify rare cellular phenotypes by microscopy opens the way toward the analysis of rare biological events as interactions in the microenvironment of the stem cell-like cells in heterogeneous cultures of large cell populations of primary cells isolated from normal or neoplastic tissues without the disruption of the population morphology and intercellular interactions.

Future extensions of the method will include the use of fluorescently labeled markers, as antibodies or direct fluorescent labels of various cellular phenotypes. Quantitative data can first be obtained from the recorded images with the same statistical robustness as usually provided by flow cytometry analyses. Then, the cells with rare phenotypes can be studied by microscopy in their original microenvironment in the cell culture. Our method also opens the way to the time-lapse analysis of cells with rare phenotypes. Such a method will largely contribute to the understanding of dynamic phenomena like cell differentiation or emergence of heterogeneity in cell cultures.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED