Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures
N.M. Ocarino*, A. Bozzi**, R.D.O. Pereira*, N.M. Breyner**, V.L. Silva*, P. Castanheira**, A.M. Goes** and R. Serakides*
* Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária da Universidade Federal de Minas Gerais, Brazil.
** Departamento de Bioquímica e Imunologia do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais,
Brazil.
Address correspondence to: Dr. R. Serakides. Departamento de Clínica e Cirugia Veterinárias, Escola de Veterinária da Universidade Federal de Minas Gerais, Avenida Presidente Antonio Carlos, 6627, CEP 30.161-970, Belo Horizonte, Minas Gerais, BRASIL. E-mail: serakide@dedalus.lcc.ufmg.br
ABSTRACT: 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells. The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.
Keywords: Bone marrow; Cell cultures; Rat; Osteogenic differentiation
Introduction
The interaction of 4',6-diamidino-2-phenylindole
dihydrochloride (DAPI) with DNA has been the subject
of numerous studies (Kubista et al., 1987; Blaisdell
and Wallace, 2007; Shin et al., 2007). This substance
was developed as a diamidino compound, analogous to
either diminazene aceturate (Berenyl) or stilbamidine,
to be used as a trypanocidal agent. DAPI however
showed a variety of biological effects, including antibiotic,
anti-trypanosomal and antiviral activity, and it is
now an important tool in molecular biology and
citochemistry. As such, DAPI is a fluorescent substance
which exhibits several binding modes to DNA (Wilson
et al., 1990; Eriksson et al., 1993), being transferred to
descendant cells during cell proliferation. Studies onapoptosis utilize DAPI staining to verify the morphology
of apoptotic bodies and the chromatin condensation
(Kim et al., 2006).
Mesenchymal stem cells are pluripotent stem cells
mostly residing within the adult bone marrow. In contrast
to hematopoietic cells, these cells are adherent and
can be expanded in culture. They may differentiate not
only into osteoblasts, neurons, and skeletal muscle
cells, but also into vascular endothelial cells and
cardiomyocytes (Jiang et al., 2002).
With the rapid increase of reported uses of stem
cells to treat cardiovascular (Li et al., 2006), retinal,
neurological (Mayhall et al., 2004) and bone diseases
(Kraus and Kirker-Head, 2006; Wang et al., 2006), it
has become important to track stem cells in vivo during
clinical trials. For such purposes, the labeling agent associated
with a stem cell must be biocompatible, safe,
non toxic and should allow the location and quantification
of stem cells in the organism (Frangioni and Hajjar,
2004). DAPI apparently meets these criteria and is being
widely used to mark and trace stem cells used in
therapy (Thompson et al., 2004; Niagara et al., 2004;
Richardson et al., 2005; Tang et al., 2006).
Studies in human leukemic marrow cells showed
that DAPI doesn't modify their proliferation and it is
not cytotoxic for these cells in vitro (Park et al., 1985;
Tarnowski et al., 1993). Also, Niu et al. (2004) and Tang
et al. (2006) have shown that in vivo DAPI marking
does not show negative effects on mesenchymal stem
cells used in the treatment of injured rat hearts. However,
in vitro studies to verify the effect of DAPI on
mesenchymal stem cells in osteogenic and non osteogenic
cultures appeared necessary, and this was the aim
of the present study.
Materials and Methods
Cell harvesting and culture
Adult, 130-140 g male rats were used. They were
housed under a 12-hour- ligth/dark cycle and were fed
ad libitum with a commercial rat chow containing 22%
of crude protein, 1.4% of calcium and 0.6% of phosphorus.
Water was also permanently available. To obtain
bone marrow samples, rats were killed with an overdose
of anesthesia (Tionembutal 2.5%), as approved by
the Ethical Committee of the Federal University of
Minas Gerais.
The femurs and tibias were dissected from attached
muscle and connective tissue under aseptic conditions
and the epiphyses were removed. Bone marrow was
flushed out with Dulbecco's modified Eagle's medium-DMEM (Gibco, Grand Island, NY). The released cells
were suspended in DMEM supplemented with 10% fetal
bovine serum (Gibco) plus antibiotics (60 μg/L gentamicin,
+ 25 μg/L amphotericin B, 10.000 U/mL penicillin,
and 10.000 μg/mL streptomycin, (Merck,
Germany), and collected in a 75 cm2 culture flask containing
10 mL culture medium. The cells were grown at
37ºC and 95% O2 + 5% CO2 for 3 days. The nonadherent
cell population was removed and the adherent layer
washed once with fresh media. Culture medium was
changed twice weekly. After 3 weeks of culture, the
adherent cells were detached by trypsin and seeded for
experiments.
Phenotypic characterization (experiment 1 and 2)
Cells at passage fourth were harvested with trypsin/EDTA, centrifuged at 1400 rpm for 10 min, and resuspended at 1x106 cell/well in phosphate buffered saline (PBS, 0.01 M phosphate buffer, pH=7.4, and 0.9% NaCl). Exposure of cell suspensions to a 1: 50 dilution of a single primary antibody (either against CD45, CD90, CD73 or CD54, BD Biosciences, San Jose, CA USA) were made for 30 min at 4ºC, while control cell suspensions with no antibody were simultaneously run. Afterwards, the cells were washed in PBS and incubated with fluorescent conjugated secondary antibody for 30 min at 4ºC. At least 20.000 events per sample were acquired into a FACScan cytometer (Becton Dickinson Immunocytometry System, San Jose, CA) with the CELLQuest software (Becton Dickinson). Data were analyzed by WinMDI program through histogram graphics.
Experiment 1: Mesenchymal stem cells stained by DAPI in DMEM
DAPI staining: Cells were counted in a Neubauer
camera and adjusted to a density of 1x105 cells/well.
Cells were incubated in DAPI (Sigma, St. Louis, MO)
solution (50 nM) for 30 min in the dark, and were observed
under a fluorescence microscope (Olympus, Japan).
Cells were allocated to two experimental groups:
untreated (control) and DAPI-treated cells, and were
cultured for 0, 3, 6 or 9 days.
Cell counting: The number of cells was quantified
in of material obtained from each well (3 microscope
fields), under a light microscope (Olympus, Japan) with
a 4x objective. Results are expressed as the mean and standard error of triplicate determinations made for each
group and for each experimental period. Also, the number
of DAPI-marked cells was determined by counting
marked cells in 7 microscope fields per well, under a
fluorescence microscope with green filter (Olympus,
Japan) and a 10x objective. Also, results were expressed
as the mean and standard error of triplicate determinations
for each experimental period.
Viability assay: Cytotoxicity was measured by 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (Sigma) and trypan blue exclusion assays.
In brief, control and stained cells were incubated
with MTT for 2 h at 37ºC. After incubation they were
treated for 12 h with the solubilization solution (1%
sodium dodecyl sulfate -SDS- and 10% HCl) and the
absorbance at 595 nm of solubilized MTT formazan
product was measured. For trypan blue exclusion assay,
control and stained cells were exposed to trypan blue
for 10 min. The number of viable cells (by dye exclusion)
was counted under the light microscope using a
Neubauer camera and expressed as a percentage of total
cells.
Alkaline phosphatase activity: Control and DAPIstained
cells were incubated with BCIP/NBT (5-bromo,
4-chloro, 3 indolylphosphate nitroblue tetrazolium) solution
(Gibco) for 2 h at 37ºC. After incubation, they
were treated for 12 h with the solubilization solution.
The absorbance at 595 nm was measured using a
microtiter plate reader.
MitoTracker Green (MTG) staining and flow
cytometry analysis: MTG fluorescence has been used
as a measure of mitochondrial mass (Pendergrass et al.,
2004; Lugli et al., 2005). To study the mitochondrial
mass, stem cells were analyzed after zero, three, six and
nine days of culture with DMEM medium, with or without
DAPI (1:50). The adhered stem cell was washed by
trypsin action and collected by centrifugation (1400 rpm
at 10 min). The pellet of cell was resuspended in DMEM
medium and the cell concentration was adjusted to 3,0
x 105 cells/well and stained, in 96-well U-bottomed
plates with MitoTracker Green (MTG) dye diluted 1:10
in PBS 0,15M, pH 7,0 and incubated by 30 min at 4ºC.
Non-marked stem cells were used as controls. At least
20.000 events per sample were acquired into a FACScan
cytometer (Becton Dickinson Immunocytometry System,
San Jose, CA) with the CELLQuest software
(Becton Dickinson). Data were analyzed by WinMDI
program through histogram graphics.
Experiment 2: Mesenchymal stem cell stained by DAPI in an osteogenic medium
The same cells were counted in a Neubauer camera and adjusted to a density of 1 x 105 cells/well were incubated in DAPI (Sigma, St. Louis, MO) solution (50 nM) for 30 min in the dark. Cells were allotted to two experimental groups: untreated (control) and treated with DAPI. After an attachment period of 24 h, the cells were cultured in an osteogenic medium consisting of DMEM supplemented with 10% fetal bovine serum (Gibco) plus antibiotics (60 μg/L gentamicin, 25 μg/L amphotericin B, 10.000 U/mL penicillin, and 10.000 μg/mL streptomycin, 10 mM ß-glycerophosphate, 50 μg/ mL ascorbic acid, (Merck, Germany), 10 nM dexamethasone (Sigma). The cells were grown at 37ºC and 95% O2 + 5% CO2 for 21 days. The cells were observed under a fluorescence microscope along the culture period (Olympus, Japan). Cell proliferation and viability were determined as described above. At the end of the culture period, osteoblastic differentiation and mineralization were assessed by determining alkaline phosphatase activity (as describe above) and by counting the number of mineralized nodules formed. For the latter purpose, cultures were rinsed in PBS, fixed in 70% ethanol and rinsed with deionized water. After the addition of 5% silver nitrate solution, the wells were exposed to light for 2 h. The plates were rinsed with deionized water, and the residual silver nitrate was neutralized by 5% sodium thiosulfate. The number of nodules was quantified in 35 microscope fields with a 10x objective, and the mean and standard error of triplicate determinations was calculated for each group.
Cellular morphology and cell counting: Control and stained DAPI cells were cultured in 24-well plates with coverslips. The cells were fixed in 70% ethanol solution for 30 min and hydrated in deionized water for 5 min, and then stained with hematoxylin and eosin. The number of cells was estimated in both groups by cell counting in 35 microscope fields with a 10x objective. Mean and standard error were determined in triplicate for each group.
Statistical analysis: Delineation was entirely at random in both experiments with a 2 x 3 factorial (two groups x three periods) in experiment 1. The data were subjected to analysis of variance (ANOVA), and means were compared by Student Newman Keuls Test (SNK) (Instat, version 3.00, 32 Win 95/NT; GraphPad Software San Diego, CA, USA). Differences were considered significant if p< 0.05.
Results
The phenotypic characterization of bone marrow MSCs demonstrated absence of expression of CD45 in 97% of the obtained cells. But there was expression of CD73, CD54 and CD90 in 94%, 95.10% and 87% of the obtained cells, respectively.
Experiment 1
Proliferation and viability of mesenchymal stem cells after DAPI treatment.
In both groups: The number of stem cells/field increased progressively and significantly up to the ninth day of culture. There were no significant differences between groups (Fig. 1).
FIGURE 1. Number of cells per field (mean±SE) in
groups without DAPI (control) and with DAPI treatment.
However, the number of fluorescent cells/field, i.e., cells with DAPI-marked nucleus, increased significantly up to the sixth day only. Then the number of fluorescent cells (Fig. 2) and fluorescence intensity decreased (Fig. 3). Cells in mitotic division and with two or more nuclei, were clearly visualized by DAPI-staining. DAPI was present in nucleus and cytoplasm.
FIGURE 2. Number of DAPI-marked
cells per field (mean±SE).
FIGURE 3. Mesenchymal stem cells stained with DAPI. Bar = 187 μm. a) Number of cells with DAPI-marked nucleus
on day 0. b) on day 3, c) on day 6, and d) on day 9. The number of cells with DAPI-marked nucleus increased
significantly up to day 6. On day 9, both the number of fluorescent cells and the fluorescence intensity were reduced.
Although the number of DAPI-treated cells increased progressively up to the ninth day, MTT assay showed a significant reduction of the spectrophotometric absorption of formazan in this group from day 3 (Fig. 4). However, trypan blue assay showed 99% of cell viability in both the control and in the DAPI-treated groups. The trypan blue assay showed no significant differences between groups, at none of the studied periods.
FIGURE 4. Cell viability (mean±SE) in groups
without DAPI (control) and with DAPI treatment.
Alkaline phosphatase activity: In the control group, alkaline phosphatase activity increased significantly on the ninth day as compared to the previous days. DAPI significantly reduced mesenchymal stem cells alkaline phosphatase activity both at the sixth and ninth days (Fig. 5).
FIGURE 5. Alkaline phosphatase activity (mean±SE) in
groups without DAPI (control) and with DAPI treatment.
MTG staining and flow cytometry analysis: In all of the studied times, i.e., zero, three, six and nine days in culture, we found a similar mitochondrial mass in stem cells cultured with or without DAPI (Fig. 6). Nevertheless, with MTG staining, mesenchymal stem cell morphology differed between groups. On the sixth and ninth day of culture, DAPI-treated group most of the times, presented round cells with fine and short extensions. Control cells were mainly fusiform with several long extensions linked to neighboring cells. On the 0 day and third day, there was not significant difference between groups (Fig. 7).
FIGURE 6. Mitochondrial mass evaluation in stem cells from rats by MitoTracker Green (MTG) staining
and flow cytometry. The histograms show the frequency of mitochondria in stem cells cultured for
0, 3, 6, or 9 days, with or without DAPI treatment. The fluorescence scale (X axis) is considerably
positive when the peak of cells is above 101. The full black peak refers to unmarked stem cells. The
empty green peak refers to stem cells cultured with DAPI and the empty black peak refers to stem
cells cultured without DAPI treatment. The same results are presented in bars in the bottom graph.
FIGURE 7. Mesenchymal stem cells stained with MitoTracker Green. Bar = 67 μm. Panels a and c: control
groups on days 0 and 3, respectively; panels b and d: DAPI-treated groups on days 0 and 3, respectively,
showing mostly round cells, with fine and short extensions, and with no differences between groups. Panels
e and g: control group on day 6 and 9, respectively, showing fusiform cells with many long extensions,
apparently linking to neighboring cells. Panels f and h: DAPI-treated groups on days 6 and 9, respectively,
showing mostly round cells with fine and short extensions.
Experiment 2
The number of cells/field did not differed statistically between the control and the DAPI- marked groups. Fluorescence intensity was reduced along the experimental period in the DAPI-treated group. In the ninth day there were not DAPI-marked cells left. As opposed to experiment 1 the MTT assay showed no significant difference between groups, i.e., the number of cells/field, the viability assay, the activity of alkaline phosphatase and the number of mineralized nodules/field were similar in both groups (Table 1).
TABLE 1.
Proliferation, viability, mineralization and alkaline
phosphatase activity of mesenchymal stem cells
grown during 21 days in an osteogenic medium, with
and without DAPI treatment.
Discussion
The bone marrow is known to contain mesenchymal
stem cells, hematopoietic cells (Bianco et al., 2001;
Bobis et al., 2006) and fibroblasts (Ishii et al., 2005a).
The cells used in the current study have been characterized
as being positive for CD73, CD54 and CD90, and
negative for CD45. The latter antigen is expressed by
hematopoietic cells (Pittenger et al., 1999; Bobis et al.,
2006) and some fibroblasts (Ishii et al., 2005b), while
CD54 and CD90 are typical of mesenchymal stem cells
(Covas et al., 2003; Bobis et al., 2006). On its part, CD73
may be expressed by both fibroblasts and mesenchymal
stem cells (Ishii et al., 2005a; Bobis et al., 2006). It
is concluded, therefore, that the bone marrow cells used
in this study are indeed mesenchymal stem cells.
A potentially interesting finding of the current study
is the diminishing effect of DAPI staining on formazan
formation after MTT treatment from the third day on,
even though cell viability (as detected by the trypan blue
method remained high) and the proliferation ability of
DAPI stained cells seemed not impaired. The MTT test
is a sensitive and quantitative colorimetric method,
which measures the ability of mitochondrial succinate
dehydrogenase enzymes of viable cells, to convert MTT
into formazan crystals (Altman, 1976; Holt et al., 1987).
At least two hypotheses may explain DAPI interference
with the MTT assay: (1) that DAPI may reduce the mitochondrial
mass present in cells; and (2) that DAPI may
affect the mitochondrial reduction of MTT. The first
hypothesis was rejected since a similar mitochondrial
mass was determined by MTG staining and flow
cytometry in cells that were cultured either with or without
DAPI during 3-9 days.
As expected, DAPI treatment did not affect the increase
in the number of both DAPI-marked cells and
mitosis up to the sixth day in culture. Ideally, a stem
cell marker must be transferred to descending cells with
minimal or no dilution during subsequent cell divisions
(Frangioni and Hajjar, 2004). DAPI was transferred to
descendant cells in the current study; however, both the
number of DAPI-marked cells and the intensity of DAPI
labeling on the day nine of culture were lower than on
day 6 in non-osteogenic cultures. Dissapearing of DAPI
marking occurred even faster in osteogenic cultures. This
is in contrast to in vivo studies in which mesenchymal
stem cells stained by DAPI were observed up to 10 weeks
after inoculation and differentiation in cardiomyocytes
(Niu et al., 2004).
Cells with more than two nuclei were observed in
the DAPI-treated group, which may suggest that they
were undergoing nuclear division with no cytokinesis.
This apparent failure in cell division, together with the
observed morphological changes of stem cells after
DAPI treatment, may suggest that this dye may be affecting
the cytoskeleton.
The activity of alkaline phosphatase by cells may
be estimated through BCIP-NBT solution (5-bromo,4-chloro,3-indolylphosphate-nitroblue tetrazolium).
BCIP-NBT is used to as substrate for alkaline phosphatase
enzyme (De Jong et al., 1985). Alkaline phosphatase
cleaves phosphate group of BCIP producing
bluish staining at the same time that NBT is reduced
and produces a dark red insoluble deposit. DAPI-treated
cells present a low activity of alkaline phosphatase in
non osteogenic cultures. But DAPI treatment did not
alter either the activity of alkaline phosphatase or the
synthesis of mineralized nodules in those osteogenic
cultures, up to day 21.
X-Ray-based methods, optical imaging, ultrasound,
single-photon emission computed tomography, positron
emission tomography, and magnetic resonance imaging
are contrast methods for in vivo stem cell tracking.
At present no imaging technology presents all the appropriate
characteristics for clinical stem cell trials.
DAPI is being used thoroughly to mark and trace stem
cells route which will be used in the therapy of diseases
(Thompson et al., 2004; Niagara et al., 2004; Richardson
et al., 2005; Tang et al., 2006). Although stem cells are
being used to treat bone diseases (Kraus and Kirker-Head, 2006; Wang et al., 2006), this is the first study in
which effects of DAPI on mesenchymal stem cells were
shown in osteogenic cultures.
It is concluded that in non osteogenic cultures,
DAPI changes the morphology of mesenchymal stem
cells and reduces the production of alkaline phosphatase
and formazan crystals, probably by changing mitochondrial
enzymatic activity and not by changing the mitochondrial
mass. But, DAPI did not affect cell viability,
proliferation and the process of osteogenic differentiation.
The fact that the cells loose their mark after day 9
in culture in osteogenic cultures suggests that DAPI may
not be a good marker for long term studies of mesenchymal
stem cells implanted in bone tissue.
Acknowledgements
This work was supported by grants from Fundação de Amparo a Pesquisa de Minas Gerais (Fapemig) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Received on August 24, 2007.
Accepted on May 15, 2008.