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Cannabinoids modulate cell survival in embryoid bodies

Authors:
  • Food Safety Authority of Santa Catarina State (CIDASC)
  • National Institute of Traumatology and Orthopedics

Abstract and Figures

ESCs (embryonic stem cells) are potentially able to replace damaged cells in animal models of neural pathologies such as Parkinson's disease, stroke and spinal cord lesions. Nevertheless, many issues remain unsolved regarding optimal culturing procedures for these cells. For instance, on their path to differentiation in vitro, which usually involves the formation of EBs (embryoid bodies), they may present chromosomal instability, loss of pluripotency or simply die. Therefore, finding strategies to increase the survival of cells within EBs is of great interest. Cannabinoid receptors have many roles in the physiology of the adult body, but little is known about their role in the biology of ESCs. Herein, we investigated how two cannabinoid receptors, CB1 and CB2, may affect the outcome of ESCs aggregated as EBs. RT-PCR (reverse transcriptase-PCR) revealed that EBs expressed both CB1 and CB2 receptors. Aggregation of ESCs into EBs followed by 2-day incubation with a CB1/CB2 agonist reduced cell death by approximately 45%, which was reversed by a CB1 antagonist. A specific CB2 agonist also reduced cell death by approximately 20%. These data indicate that both cannabinoid receptors, CB1 and CB2, are involved in reducing cell death in EBs mediated by exogenous cannabinoids. No increase in proliferation, neural differentiation or changes in chromosomal stability was observed. This study indicates that cannabinoid signalling is functionally implicated in the biology of differentiating ESCs, being the first to show that activation of cannabinoid receptors is able to increase cell viability via reduction of cell death rate in EBs.
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Cannabinoids modulate cell survival in embryoid bodies
Jader Nones, Tania C.L.S. Spohr
1
, Daniel R. Furtado
1
, Rafaela C. Sartore, Bruna S. Paulsen, Marilia Z.P. Guimara˜ es and
Stevens K. Rehen
2
Instituto de Cie
ˆncias Biome
´dicas, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil
Abstract
ESCs (embryonic stem cells) are potentially able to replace damaged cells in animal models of neural pathologies such as
Parkinson’s disease, stroke and spinal cord lesions. Nevertheless, many issues remain unsolved regarding optimal
culturing procedures for these cells. For instance, on their path to differentiation in vitro, which usually involves the
formation of EBs (embryoid bodies), they may present chromosomal instability, loss of pluripotency or simply die.
Therefore, finding strategies to increase the survival of cells within EBs is of great interest. Cannabinoid receptors have
many roles in the physiology of the adult body, but little is known about their role in the biology of ESCs. Herein, we
investigated how two cannabinoid receptors, CB1 and CB2, may affect the outcome of ESCs aggregated as EBs. RT-PCR
(reverse transcriptase-PCR) revealed that EBs expressed both CB1 and CB2 receptors. Aggregation of ESCs into EBs
followed by 2-day incubation with a CB1/CB2 agonist reduced cell death by approximately 45%, which was reversed by a
CB1 antagonist. A specific CB2 agonist also reduced cell death by approximately 20%. These data indicate that both
cannabinoid receptors, CB1 and CB2, are involved in reducing cell death in EBs mediated by exogenous cannabinoids.
No increase in proliferation, neural differentiation or changes in chromosomal stability was observed. This study indicates
that cannabinoid signalling is functionally implicated in the biology of differentiating ESCs, being the first to show that
activation of cannabinoid receptors is able to increase cell viability via reduction of cell death rate in EBs.
Keywords: CB1; CB2; cell signalling; differentiation; embryonic stem cell; neural development; neural differentiation; pluripotency; stem cell
1. Introduction
ESCs (embryonic stem cells) are pluripotent cells isolated from the
inner cell mass of preimplanted blastocysts (Evans and Kaufman,
1981; Martin, 1981; Thomson et al., 1995, 1998; Cowan et al.,
2004). Owing to their pluripotency and self-renewal properties,
they are ideal candidates for use in basic research and drug
screening (Draper and Fox, 2003; Ameen et al., 2008).
Additionally, their ability to differentiate into any cell type gives
ESCs the potential to replace damaged cells in several patho-
logical conditions for which no effective treatments or cure are
currently available (Goya et al., 2007; Coutts and Keirstead, 2008;
Joannides and Chandran, 2008).
mESCs (mouse embryonic stem cells) can be expanded
continuously when in co-culture with a monolayer of MEFs
(murine embryonic fibroblasts), but their ability to remain
pluripotent requires the addition of the LIF (leukaemia inhibitory
factor). When both MEFs and LIF are removed from mESCs grown
in suspension, these cells spontaneously differentiate and form
three-dimensional clusters known as EBs (embryoid bodies) (Zhou
et al., 2005). EBs recapitulate many temporal and spatial aspects
of early embryogenesis and originate the three germ layers. In
general, differentiation of ESCs is commonly associated with an
increase in EBs cell death (Evans and Kaufman, 1981; Martin,
1981). Therefore, finding strategies to increase the survival of EBs
is of great interest, since this would augment the number of cells
undergoing differentiation available for potential treatments.
Cannabinoids were first isolated from the plant Cannabis
sativa, popularly known as marijuana. These substances act by
stimulating G protein-coupled receptors (Howlett et al., 1986), the
first of which was identified in the brain and named CB1
(cannabinoid receptor 1) (Matsuda et al., 1990). Subsequently, a
second cannabinoid receptor (CB2) was isolated from a promye-
locytic cell line (Munro et al., 1993). The identification of these
receptors led to a search for their endogenous ligands. The first
endogenous ligand identified for a cannabinoid receptor was
anandamide (arachidonoyl ethanolamide), followed by 2-arachy-
donoyil-glycerol. Both endocannabinoids were shown to be
synthesized in vivo from phospholipids (Piomelli, 2003).
It is known that cannabinoid signalling modulates a range of
physiological functions in the adult body. However, our under-
standing of its role in the early embryo is still limited. A few reports
suggested that endocannabinoids play a role in embryonic
development (Das et al., 1995; Paria et al., 1995). The uterine
wall, for instance, produces anandamide, which is important for
embryo implantation (Das et al., 1995; Paria et al., 1995). More
recently, it has been shown that cannabinoids stimulate neural
progenitor proliferation (Aguado et al., 2005; Molina-Holgado et al.,
2007) and glial differentiation in vitro (Aguado et al., 2006).
Endocannabinoids also play an important role in protecting the
central nervous system from cerebral injuries such as ischaemia
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email srehen@anato.ufrj.br).
Abbreviations: 5-HT, serotonin; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; DAPI, 4,6-diamidino-2-phenylindole; DMEM/F12, Dulbecco’s
modified Eagle’s medium with nutrient mixture F-12; EBs, embryoid bodies; ESC, embryonic stem cells; mESC, mouse embryonic stem cells; LIF, leukaemia
inhibitory factor; MEF, murine embryonic fibroblasts; PFA, paraformaldehyde; RA, retinoic acid; RT-PCR, reverse transcriptase-PCR; TCA, trichloroacetic acid;
TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labelling; WIN, WIN 55212-2.
Cell Biol. Int. (2010) 34, 399–408 (Printed in Great Britain)
Research Article
EThe Author(s) Journal compilation E2010 Portland Press Limited Volume 34 (4) Npages 399–408 Ndoi:10.1042/CBI20090036 Nwww.cellbiolint.org 399
(Mechoulam et al., 2002). This protective effect seems to involve
oligodendrocyte progenitors (Molina-Holgado et al., 2002).
Despite these apparent benefits, the use of marijuana has been
correlated with a low birthweight (Zuckerman et al., 1989;
Sherwood et al., 1999), premature birth (Fried et al., 1984),
intrauterine growth retardation and congenital abnormalities
(Gibson et al., 1983). It has also been associated with perinatal
death and delay in the onset of newborn breathing (Gibson et al.,
1983).
Recently, it has been suggested that activation of CB1 and
CB2 receptors modulate the survival of mESC (Jiang et al., 2007).
However, this potential role of CB1 and CB2 receptors was based
on a qualitative observation that the viability of mESC colonies
seemed to be impaired by incubation with cannabinoid receptor
antagonists. The actual effect, if any, of CB1 and CB2 receptors
on either the survival or cell proliferation within EBs have not been
quantified.
Herein, we used cannabinoid agonists and antagonists to
investigate their effects on EBs expressing cannabinoid receptors.
Our results indicate that cannabinoid signalling is implicated in the
biology of ESCs, in which it may act by reducing the cell death
rate. A better understanding of how this signalling mechanism
works may allow the use of cannabinoid agonists in the
production of differentiated ESCs for therapy.
2. Materials and methods
2.1. Cell culture
Mouse ES cells R1 and USP1 (kindly provided by Dr Lygia V.
Pereira and Irina Kerkis, University of Sa
˜o Paulo – USP) were
grown on top of MEF feeder cells that had been inactivated by
treatment with 10 mg/ml mitomycin C (Sigma). The growth medium
consisted of DMEM/F12 (Dulbecco’s modified Eagle medium with
nutrient mixture F-12; Gibco), supplemented with 15% knockout
serum (Gibco), 1% nonessential amino acid solution (Gibco), 2
mM L-glutamine (Gibco), 0.1 mM 2-mercaptoethanol (Gibco, cat.
no. 21985-023), 50 mg/ml gentamicin sulfate (Schering-Plough)
and 0.2% of conditioned medium from LIF-producing Chinese
hamster ovary cells (Moreau et al., 1988).
USP1 cells were tested for maintenance of pluripotency.
Figure 1(a) shows that mESC colonies are positive for Oct4
(Nichols et al., 1998), by immunostaining. To further characterize
the self-renewal capacity of USP1 mESCs, the expression of Oct4
(Nichols et al., 1998) and two more markers of undifferentiated
embryonic stem cells, Nanog (Chambers et al., 2003) and Sox2
(Avilion et al., 2003), were detected by RT-PCR (reverse
transcriptase-PCR) (Figure 1b) as described below.
2.2. RT-PCR assay
Total RNA was extracted from mESC colonies and EBs with trizol
reagent (Invitrogen) according to the manufacturer’s instructions.
cDNA was synthesized by reverse transcription (Promega). cDNA
samples were amplified by PCR using the following primers:
Oct4-F (59-AGAGCAGTGACGGGAACAGAG-39), Oct4-R (59-
CCAACGAGAAGAGTATGAGGC-39); Sox2-F (59-GAGAGCAA-
GTACTGGCAAGACCG-3); Sox2-R (59-TAT ACATGGATT-
CTCGCCAGCC-39); Nanog-F (59-GTGCATATACTCTCTCCTT-
CCC-39); Nanog-R (59-AGCTACCCTCAAACTCCTGGT-39);
GAPDH-F (59-ATCACCATTTCCAGGAGCG-39); GAPDH-R (59-
CCTGCTTCACCACCTTCTTG-39); CB1-F (59-CGTGGGCA-
GCCTGTTCCTCA-39); CB1-R (59-CATGCGGGCTTGGTCTGG-
39); CB2-F (59-CTGCTGAGCGCCCTGGAGAAC-39), CB2-R (59-
CCGGAAAAGAGGATGGCAATGAAT-39). These primer
sequences were obtained from previously published paper
(Jiang et al., 2007). The PCR reaction consisted of 30 cycles of
45 s at 94˚C, 30 s at 55˚C (Oct4, Sox2, Nanog), or 60˚C (GAPDH,
CB1 and CB2), and 1 min and 30 s at 72 ˚C, followed by 5 min at
72˚C in the last cycle. PCR was done using a Mastercycler
Personal Thermal Cycler (Eppendorf). The expected sizes for
Oct4, Nanog, Sox2, GAPDH, CB1 and CB2 amplicons were 177,
213, 335, 571, 402 and 478 bp, respectively.
Colonies (Figure 1a) cultivated on a feeder layer of MEFs were
dissociated, transferred to bacteriological Petri dishes and
allowed to aggregate into EBs (Figure 2a). After 2 days in culture,
total RNA was extracted and used to detect cannabinoid
receptors by RT-PCR.
2.3. Establishment and treatment of EBs
To isolate mESCs from cells of the feeder layer, 1610
6
mESCs
were cultivated for 2 days on tissue culture plates coated with 2%
gelatin (Sigma). EBs were obtained by treating mESC colonies
Figure 1 mESCs are pluripotent
mESC colonies stained with DAPI (top) mESC colonies immunostained with an anti-
Oct4 antibody (bottom) (a). RT-PCR showing the expression of the undifferentiated ES
cell markers Oct4, Sox2 and Nanog by mESCs (b). GAPDH was used as an internal
control. –RT, negative control (no reverse transcriptase added). Scale bar in (a)
corresponds to 200 mm.
Cannabinoids in embryoid bodies
400 www.cellbiolint.org NVolume 34 (4) Npages 399–408 EThe Author(s) Journal compilation E2010 Portland Press Limited
with 0.025% trypsin (Sigma) for 5 min at 37˚C and subsequently
resuspending the cells in EB medium, prepared as follows:
DMEM/F12, 15% fetal bovine serum (Gibco), 1% non-essential
amino acid solution, 2 mM L-glutamine and 0.1 mM 2-mercap-
toethanol, 50 mg/ml gentamicin sulfate and LIF (1%, prepared as
above). Spherical cell aggregates (EBs) were formed within 2 days
(Figure 2a). EBs were then treated for 2 days with the cannabinoid
agonist WIN (WIN 55212-2; Axxora), at concentrations ranging
from 0.1 to 100 nM). In order to analyse the cannabinoid actions
via CB1, EBs were incubated with 300 nM AM251 (Tocris), a
CB1-specific antagonist, for 15 min, followed by the addition of
10 nM WIN and incubation for 2 days. To evaluate the involve-
ment of the CB2 receptor, EBs were incubated for 2 days with 200
nM JWH 015 (Cayman Chemical Company), a CB2-specific
agonist.
2.4. [
3
H]Thymidine incorporation assay
EBs, exposed to 10 or 100 nM WIN for 48 h, were incubated
with [
3
H]thymidine (1 mCi/ml; Amersham) in the last 6 h of the
treatment. EBs were washed with PBS pH 7.4 and homogenized
in 500 ml 0.4 M NaOH. Aliquots of homogenates were precipitated
with 10% TCA (trichloroacetic acid) and filtered. Filters were
washed with 10% TCA and ethanol subsequently, dried at 100 ˚C
in an oven and counted in a Packard model 1600TR liquid-
scintillation analyser.
2.5. Detection of cell death in EBs
After treatment with drugs (WIN 5512-2, AM251 and JWH 015) or
vehicle, EBs were fixed in 4% PFA (paraformaldehyde) for 30 min
and embedded in increasing concentrations of sucrose in PBS,
followed by OCT (optimal cutting temperature; Tissue-Tek),
frozen and cut into 10 mm histological cryosections. A TUNEL
(terminal deoxynucleotidyl-transferase-mediated dUTP nick
end-labelling) (Promega) assay was used to identify DNA
fragmentation. EB slides were treated with proteinase K
(20 mg/ml) for 8 min at room temperature (25˚C), then rinsed in
PBS, pH 7.4, and incubated in 16equilibration buffer for 10 min.
The sections were incubated with terminal deoxynucleotidyl
transferase for 1 h and 30 min at 37˚C, blocked with stop buffer
(26SSC) at 25˚C for 15 min and washed with PBS. Nuclear
staining was performed by incubating with a 1.0-mg/ml solution of
DAPI (4,6-diamidino-2-phenylindole). Percentages of TUNEL-
positive cells were determined with an optical fluorescence
microscope (Nikon T300). Approximately 30 EBs from each
experiment were analysed. Each experiment had its internal
control. The percentage of TUNEL-positive cells in controls varied
from 15% to 30% on average within each experiment.
2.6. EBs size analysis
To measure the area of EBs, they were first cultivated for 2 days
and, afterwards, incubated with 10 nM WIN for 2 days, all 4 days
in suspension. Images were obtained with an optic microscope
(Nikon T300). EBs were manually delimited, and the correspond-
ing areas were quantified with ImageJ software. Analyses were
made in three distinct experiments, and at least 225 EBs were
measured in each experimental group.
2.7. Immunofluorescence analysis
For immunocytochemistry, mESC colonies or adherent EBs were
fixed in 4% PFA for 15 min, whereas EBs cultivated in suspension
were fixed in 4% PFA for 30 min and embedded in OCT as
described above to obtain 10 mm cryosections. mESC colonies,
adherent EBs or EB sections were washed twice in PBS,
treated with 0.3% Triton (Isofar) for 5 min followed by 5% BSA
for 5 min. A monoclonal antibody anti-Oct4 (1:100, Santa Cruz)
was used to stain mESC colonies to confirm the pluripotency
of these cells. EB sections were incubated with a polyclonal
antibody anti-phospho-H3 (1:500, Upstate Cell Signaling
Solutions), a proliferation marker. After washing three times in
PBS, the cells were incubated with secondary antibodies:
goat anti-mouse IgG (1:400, Molecular Probes) for Oct4 or goat
anti-rabbit IgG (1:400, Molecular Probes) following anti-
phospho-H3, for 1 h. The samples were washed three times in
PBS, and then, DAPI staining was used to label the nuclei. At least
30 EBs from each experiment were analysed by fluorescence
microscopy (Nikon T300). The percentages of anti-phospho-H3-
positive cells among the total number of cells were determined by
counting.
2.8. Ploidy assay
EBs were incubated with colcemid 0.1 mg/ml (Karyo Max, Gibco)
for 6 h to arrest cells at metaphase. After dissociation (trypsin/
EDTA 0.05%), the isolated cells were treated with a hypotonic
solution consisting of 75 mM KCl at 37˚C for 15 min. Cells were
then fixed in methanol/acetic acid 3:1 (v/v) and stored at 4 ˚C
overnight. Next day, the cell suspension was washed three times
with the fixative solution and then spread onto clean glass slides.
The slides were quickly exposed to water steam, to cause cells to
burst. Chromosomes were stained with DAPI and mounted with
glass coverslips in N-propyl gallate. Metaphase images were
Figure 2 EBs express cannabinoid receptors
mESC colonies, cultivated on top of a layer of non-dividing MEFs, were transferred to
non-adherent culture dishes and allowed to aggregate into EBs (a). RT-PCR showing
the expression of both the CB1 and the CB2 by EBs (b). Total mRNA from mouse brain
was used as a positive control. –RT, negative control (no reverse transcriptase added).
Scale bar in (a) corresponds to 100 mm.
Cell Biol. Int. (2010) 34, 399–408
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acquired using a Zeiss 6100 objective on a fluorescence
microscope and analysed with the Axio Vision software.
2.9. Neural differentiation assay
To induce neuronal differentiation, EBs were treated with 2 mM
retinoic acid for 2 days, followed by two more days in the
presence of 10 nM WIN. Subsequently, EBs were placed on
adhering cover glasses. These cover glasses were prepared as
follows. First they were incubated in 1.5 mg/ml poly-ornithine
(Sigma) for 4 h. Then cover glasses were rinsed three times with
sterile milli-Q water and incubated with 1 mg/ml fibronectin
(Invitrogen) and 1 mg/ml laminin (Invitrogen). The cover slides
were maintained in this solution overnight. EBs, prepared as
described, were transferred to a conical tube and, upon
decantation, had their culture media replaced by Neurobasal
medium, consisting of DMEM/F12, supplemented with 20 ng/ml
bFGF (basic fibroblast growth factor) (R&D), N2 (16, Gibco),
40 mg/ml gentamicin sulfate and 0.1 mM 2-mercaptoethanol.
About 25 EBs were placed on top of each 13 mm coverslip and
allowed to grow for 2 days, after which they were fixed with 4%
PFA and processed for immunostaining.
2.10. Cell quantification in situ
Sections of at least 30 different EBs were photographed and
magnified to be counted. The entire section was analysed. All
results are expressed as percentage of stained cells relative to the
total (DAPI positive cells).
2.11. Statistical analyses
Statistical analyses of ploidy number was performed with
GraphPad Prism 4 and validated by applying Student’s ttests.
Other comparisons were obtained with one-way ANOVA (analysis
of variance), followed by Tukey’s post-tests. Confidence intervals
were calculated at the 95% confidence level (P,0.05 was
considered to be statistically significant), unless otherwise
indicated.
3. Results
3.1. EBs express cannabinoid receptors
To confirm cannabinoid signalling during the differentiation of
embryonic stem cells, we examined the expression of the
cannabinoid receptors, CB1 and CB2, in mESCs. Indeed, we
were able to detect the expression of both CB1 and CB2
receptors in EBs by RT-PCR (Figure 2b). Expression of these
two receptors indicates that they are likely to be involved in
cannabinoid signalling in mESCs.
3.2. Activation of cannabinoid receptors reduces cell
death and increases EBs average size
Considering that EBs expressed both CB1 and CB2 receptors, we
asked whether their activation could affect the survival and growth
of those cell aggregates. EBs were incubated for 2 days with
Figure 3 Activation of cannabinoid receptors reduces cell death and increases EBs average size
EBs were incubated for 2 days with: vehicle (control) (a) or WIN, a cannabinoid receptor agonist (b,c). Following treatment, EBs were fixed and subjected
to a fluorescent TUNEL assay. Insets: DAPI staining. Quantification of cell death by the TUNEL assay relative to the total DAPI-stained cells, n53
experiments, with five EBs each (d). Activation of cannabinoid receptors with 10 nM WIN increases the size of EBs (e). n53 experiments. Significantly
different from the control, *P,0.05, **P,0.01, ***P,0.001. Scale bar in (ac) corresponds to 50 mm.
Cannabinoids in embryoid bodies
402 www.cellbiolint.org NVolume 34 (4) Npages 399–408 EThe Author(s) Journal compilation E2010 Portland Press Limited
increasing concentrations of WIN, which is a potent synthetic
agonist of cannabinoid receptors and stained for DNA fragmenta-
tion by the TUNEL assay (Figures 3a–3c). At a small concentration
(10 nM), WIN was capable of decreasing cell death by
approximately 45%. We also observed a significant reduction in
cell death when EBs were incubated with 50 nM WIN, albeit this
effect was less substantial (Figure 3d). Incubation with 100 nM
WIN no longer reduced EBs cell death. The mESC line, R1, was
also evaluated regarding cannabinoid-induced decrease in cell
death. Indeed, 10 nM WIN treatment diminished TUNEL staining
by approximately 30%, confirming that this phenomenon is not
cell line specific (Supplementary Figure S1 at http://www.cellbiolint.
org/cbi/034/cbi0340399add.htm).
Since incubation of EBs with 10 nM WIN reduced cell death,
we expected that EBs would, in turn, increase in size. Indeed, EBs
treated with 10 nM WIN exhibited a slightly larger cross-sectional
area (10%) if compared with untreated EBs. This increase
represented an actual augmentation in the EBs average size/
volume (Figure 3e).
3.3. Reduction in cell death is mediated by both
receptors
Since CB1 and CB2 are expressed in EBs, and WIN is able to
activate both cannabinoid receptors, we performed the following
experiments to dissect out the contribution of each receptor type
to the cell death reduction effect. First, EBs were incubated with
300 nM AM251, which is a specific CB1 receptor antagonist, with
or without 10 nM WIN. Next, EBs were stained for DNA
fragmentation using the TUNEL assay (Figures 4a–4d), and cell
death rate was quantified (Figure 4e).
Incubation of EBs with AM251 alone did not produce any
reduction in cell death when compared with control untreated
EBs. However, AM251 was able to reverse WIN-induced cell
death reduction. Hence, blockade of CB1 receptors significantly
reduces WIN effects on cell survival. Although CB1-specific
receptor inhibition virtually eliminated WIN survival effects, we
investigated whether CB2 receptors were also involved in cell
death regulation by cannabinoids. EBs were then treated with
200 nM JWH 015, a specific CB2 receptor agonist (Figure 5b).
Indeed, incubation of EBs with JWH 015 reduced cell death by
20% (Figure 5c). These data indicate that both CB1 and CB2
receptors are implicated in a reduction in cell death mediated by
cannabinoids (Figures 4 and 5).
3.4. Activation of cannabinoid receptors does not
increase cell proliferation in EBs
Cannabinoids are known to modulate cell proliferation in a number
of cell types (Sacerdote et al., 2000; Song and Zhong, 2000;
Kishimoto et al., 2003; Walter et al., 2003; Jorda
`et al., 2004;
Kishimoto et al., 2005; He et al., 2007). Therefore, we asked
whether incubation of EBs with WIN alone would have any effect
on the proliferation rate of cells within EBs (Figures 6a–6c).
Immunostained procedures with anti-phospho-H3 antibodies
revealed no significant difference in immunostaining between
control and WIN-treated EBs at any concentration tested
(Figure 6c). The [
3
H]thymidine incorporation method was also
employed to confirm this finding (Supplementary Figure S2 at
http://www.cellbiolint.org/cbi/034/cbi0340399add.htm). Again,
WIN treatment did not cause an increase in proliferation.
3.5. Activation of cannabinoid receptors does not
affect neural differentiation in EBs
Incubation with all-trans RA (retinoic acid) is a canonical method
to induce EBs to differentiate into cells of the neural lineage (Bibel
et al., 2004). We asked whether WIN treatment would affect RA-
induced neural differentiation. When the number of b-tubulin III-
labelled cells per area was determined, there was no difference
Figure 4 Reduction of cell death is mediated by the CB1 receptor
EBs were incubated for 2 days with: vehicle (control) (a); 300 nM AM251, a CB1 receptor antagonist (b); 10 nM WIN, a cannabinoid receptor agonist (c)or
both (d). Following treatment, EBs were fixed and subjected to a fluorescent TUNEL assay. Insets: DAPI staining. Quantification of cell death by the TUNEL
assay (e). n53 experiments. Significantly different from each other, ***P,0.001. Scale bar corresponds to 50 mm.
Cell Biol. Int. (2010) 34, 399–408
EThe Author(s) Journal compilation E2010 Portland Press Limited Volume 34 (4) Npages 399–408 Nwww.cellbiolint.org 403
between WIN-treated EBs and controls (Supplementary Figure S3
at http://www.cellbiolint.org/cbi/034/cbi0340399add.htm). The
percentage of b-tubulin III-positive cells does not change between
WIN and control. These results indicate that WIN does not
increase the number of cells within EBs prone to differentiate into
neurons. Therefore, cannabinoid treatment increases the number
of cells without affecting their capacity to differentiate.
3.6. Activation of cannabinoid receptors does not
interfere with chromosome stability
The existence of chromosomal abnormalities in cultured ESCs,
such as aneuploid cells, may prevent their use in regenerative
medicine (Mantel et al., 2007). Given that activation of cannabi-
noid receptors reduced cell death in EBs, we asked if this
activation could promote the survival of aneuploid cells.
Figure 5 Reduction in cell death is also mediated by the CB2 receptor
EBs were incubated for 2 days with: vehicle (control) (a) or 200 nM JWH 015, a CB2 receptor agonist (b). Following treatment, EBs were fixed and
subjected to a fluorescent TUNEL assay. Insets: DAPI staining. Quantification of cell death by the TUNEL assay (c). n53 experiments. Significantly different
from the control, ***P,0.001. Scale bar in (aand b) corresponds to 50 mm.
Figure 6 Activation of cannabinoid receptors does not increase cell proliferation in EBs
EBs were incubated for 2 days with: vehicle (control) (a) or WIN, a cannabinoid receptor agonist (b). Following treatment, EBs were immunostained
with anti-phospho-H3 antibodies. Insets: DAPI staining. Quantification of the phosphor-H3-positive cells (c). n53 experiments. Scale bar in (aand b)
corresponds to 50 mm.
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Incubation of EBs with 10 nM WIN did not alter chromosome
stability (Table 1). This indicates that survival promoted by
cannabinoid receptor activation in EBs was not a consequence
of adaptive advantages generated by an increase in aneuploidy.
4. Discussion
It is known that cannabinoid signalling modulates a range of
physiological functions in the adult organism. For instance,
activation of CB1 receptors in the nervous system reduces pain,
gastrointestinal motility and emesis, while it enhances appetite
(Piomelli, 2003; Sanger, 2007). Activation of CB2 receptors, in
turn, results in the reduction of inflammatory responses (Sanger,
2007).
It has been shown that CB1 and CB2 receptors are both
expressed by the preimplanted mouse embryo (Paria et al., 1995),
whereas the CB1 receptor is expressed throughout Xenopus
laevis development (Migliarini et al., 2006). Additionally, activation
of the CB1 receptor by anandamide is necessary for a successful
implantation and maintenance of the embryo (Wang et al., 1999).
Nevertheless, our understanding on the expression of cannabi-
noid receptors throughout the successive embryonic devel-
opmental stages, including stem cells, is still limited.
Recent work showed that activation of the CB1 and CB2
receptors stimulates the proliferation of neural stem cells (Aguado
et al., 2005; Palazuelos et al., 2006; Molina-Holgado et al., 2007)
and induces their differentiation both in vitro (Aguado et al., 2006)
and in vivo (Aguado et al., 2007). This information agrees with the
observation that CB1 receptor knockout mice exhibit a defective
adult neurogenesis (Jin et al., 2004).
Together, these data suggest that signalling via cannabinoid
receptors might play a role in the biology of embryonic stem cells.
Expression of CB1 and CB2 receptors was detected by RT-PCR
in pluripotent mESC colonies as well as in EBs derived from 2-day
mESCs in non-adherent dishes (Jiang et al., 2007, Figure 2). In
addition, this work provides evidence that endocannabinoids are
present at significant levels in mESCs colonies and EBs. Those
authors claimed that activation of CB1 and CB2 receptors
modulate the survival of mESCs (Jiang et al., 2007). This potential
role for CB1 and CB2 receptors was based on the morphological
observation that mESC colonies appeared more viable when
incubated with cannabinoid receptor antagonists, an effect that
was not quantified. The actual effect, if any, of CB1 and CB2
receptors on the survival of mESCs has not been appropriately
determined. The authors also suggested that cannabinoid
treatment favoured differentiation of mESCs into a haematopoietic
cell line, measured by the number of cells in a chemotaxis assay.
Moreover, it showed an increase in the number of haematopoie-
tic-differentiated EBs obtained from mESCs. Nevertheless, since
the effects of cannabinoids on survival and proliferation were not
fully determined, it was not possible to know what contribution
would these effects have in the increased number of differentiated
cells.
Our results showed that activation of the CB1 and CB2
receptors by cannabinoid agonists in differentiating EBs resulted
in reduced cell death measured by diminished number of TUNEL-
stained cells (Figures 3–5). Considering that activation of canna-
binoid receptors in neural stem cells resulted in an increase in cell
proliferation according to several authors (Aguado et al., 2005;
Palazuelos et al., 2006; Aguado et al., 2007; Molina-Holgado et al.,
2007), one might suggest that CB1 and CB2 could also increase
cell proliferation. However, the increase in size and cell number in
EBs following cannabinoid treatment could be sufficiently
explained by the reduction in cell death. In our study, no increase
in proliferation measured by two independent methods (percent-
age of histone H3-positive cells and thymidine incorporation) were
observed after treating differentiating EBs with a cannabinoid
agonist (Figure 6 and Supplementary Figure S2). Phospho H3
staining is a fairly common methodology to determine cell
proliferation (Kingsbury et al., 2003) and so is [
3
H]thymidine
incorporation (Sun et al., 2007). However, we cannot completely
rule out a contribution of changes in cell proliferation at this point,
since measurements of cell proliferation were made at the end of
cannabinoid treatment. Corroborating our findings, however, is
another work showing that cannabinoids can, in fact, diminish cell
proliferation, rather than increase it (Turco et al., 2008). Our
findings strongly suggest that activation of cannabinoid receptors
in differentiating EBs did not represent an increase in cell
proliferation, but rather a significant reduction in the percentage
of cells undergoing cell death.
As demonstrated, WIN was only able to prevent mESC death
at lower concentrations (peaked at 10 nM), whereas higher
concentrations did not influence the viability of cells (Figure 3). It
has been already reported by others that cannabinoids might
present unusual concentration-dependent effects in some sys-
tems. For instance, low doses of cannabinoids can be anxiolytic,
whereas high doses can be anxiogenic (reviewed by Viveros et al.,
2005). It has been shown that low doses of a CB1 receptor agonist
(WIN) elicit potent antidepressant-like behaviour and enhance 5-
HT (serotonin) neurotransmission, mediated by CB1 receptor
activation (Bambico et al., 2007). In contrast, high doses of this
agonist decrease 5-HT neurotransmission, through a non-CB1
mechanism. One explanation for the absence of cannabinoid
effects at higher doses/concentrations could be the desensitiza-
tion of the receptor and/or induction of its internalization. Indeed, a
reduction in CB1 responses due to desensitization and/or
internalization can occur via GRK3 and b-arrestin phosphorylation
of specific intracellular C-terminal residues (Jin et al., 1999).
Another possibility is that, at higher concentrations, there is
activity through an unknown receptor. Many authors have shown
Table 1 Activation of cannabinoid receptors does not interfere with
chromosome stability
Relative frequency of chromosome ploidy number from EBs treated with vehicle or 10
nM WIN.
Frequency (%)
Culture passage
Metaphases
counted Karyotype Control WIN
#13 >48 ,40 28.06 26.13
40 62.28 68.69
.40 9.65 5.17
Cell Biol. Int. (2010) 34, 399–408
EThe Author(s) Journal compilation E2010 Portland Press Limited Volume 34 (4) Npages 399–408 Nwww.cellbiolint.org 405
pharmacological evidences of a third cannabinoid receptor,
already coined CB3, even though its molecular identity remains
to be established (Ja
´rai et al., 1999; Di Marzo et al., 2000;
Breivogel et al., 2001). Interestingly, these authors reported that
WIN has a lower affinity for this putative receptor compared with
CB1. Therefore, it is feasible that higher WIN concentrations could
stimulate this other receptor, which could have opposing effects
on cell survival compared with CB1 and CB2.
Owing to a more permissive mitotic spindle checkpoint,
cultured mESCs carrying structural chromosome abnormalities
have better chances of survival than somatic cells carrying similar
abnormalities. Therefore, mESCs have a relatively high level of
aneuploidy when compared with somatic cells (Mantel et al.,
2007; Rebuzzini et al., 2008). Although aneuploidy is a character-
istic trait of some somatic cell populations, such as neurons
(Rehen et al., 2001; Kingsbury et al., 2005; Rehen et al., 2005), it is
also a trademark of cancer cells (Rajagopalan and Lengauer,
2004; Kops et al., 2005; Weaver and Cleveland, 2006). Therefore,
the occurrence of aneuploidy in cultured ESCs would be an
obstacle to their use, given that ESC-derived aneuploid cells
might be more prone to suffer transformation upon implantation.
The ability to maintain chromosomal stability, regardless of the
manipulations these cells go through, is an essential requirement
for their effective use in cellular therapy. Our results showed that
activation of cannabinoid receptors did not interfere with the level
of cell stability observed in EBs treated with 10 nM WIN, as no
differences in euploidy rates was observed between treated and
untreated controls (Table 1).
The activation of cannabinoid receptors is known to have many
roles in the physiology of the adult body. The data presented here
indicate that cannabinoid signalling is also functionally implicated
in the biology of ESCs. We suggest that activation of both CB1
and CB2 receptors reduces cell death. Although reduction of cell
death mediated by cannabinoid signalling has been demonstrated
before, our results represent the first description of cell death
reduction mediated by cannabinoid receptors in differentiating
EBs.
Our data suggest that both cannabinoid receptors, CB1 and
CB2, are involved in survival of differentiating embryonic stem
cells. This raises the possibility that there might be an in vivo
coordinated signalling mechanism that activates either one or the
other over the time course of development. Understanding how
these two receptors coordinate their actions during development
may shed light on their specific roles and on using cannabinoid
agonists to increase production of differentiated embryonic stem
cells for therapy as well as on the role of the endocannabinoid
system in early development.
Author contribution
Jader Nones, Marilia Guimara˜es and Stevens Rehen conceived
and designed the experiments. Jader Nones, Tania Spohr,
Rafaela Sartore and Bruna Paulsen performed the experiments.
Jader Nones and Marilia Guimara˜es analysed the data. Jader
Nones, Daniel Furtado, Marilia Guimara˜es and Stevens Rehen
wrote the paper.
Acknowledgements
We thank Ismael Gomes for technical assistance and Renata de
Moraes Maciel dos Santos for helping with the RT-PCRs.
Funding
This work was supported by grants and scholarships from the
Brazilian Research Funding Agencies Fundac¸a˜o Carlos Chagas
Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro
(FAPERJ), Ministe´rio da Sau´de, Conselho Nacional para o
Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o
Ary Frauzino para Pesquisa e Controle do Caˆncer (FAF),
Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de Nı´vel Superior
(CAPES), the Academy of Sciences of the Developing World
(TWAS) and Pew Latin American Fellows Program in Biomedical
Sciences (M.Z.P.G. and S.K.R.)
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Published as Immediate Publication 22 October 2009, doi 10.1042/CBI20090036
Cannabinoids in embryoid bodies
408 www.cellbiolint.org NVolume 34 (4) Npages 399–408 EThe Author(s) Journal compilation E2010 Portland Press Limited
... mES cells, named as USP1, were kindly provided by Dr. Lygia Pereira (University of Sã o Paulo-USP) and Dr. Irina Kerkis (Federal University of Sã o Paulo-Unifesp). mES cells and miPS cells were grown in ES medium as previously described [13,14]. Briefly, cell colonies were grown on mouse embryonic fibroblast (MEF), mitotic inactivated with mitomycin C (Sigma), in 15% knockout serum replacement (KSR; Gibco)–DMEM/F12 (Gibco) supplemented with 1% nonessential amino acid solution (Gibco), 2 mM l-glutamine (Gibco), 0.1 mM b-mercaptoethanol (Gibco), 40 mg/mL gentamicin sulfate (Scheing-Plough), and 0.2% of conditioned medium of CHO (Chinese hamster ovary) cells producing leukemia inhibitory factor (LIF). ...
... In all experiments , 0.1% vehicle (DMSO) group was determined as control. To allow neuronal migration in some experiments, at d10, EBs were plated onto 1 mg/mL laminin (Invitrogen) and 1 mg/mL fibronectin (Invitrogen)-coated dishes and cultured in basal medium DMEM/F12 with N2 supplement and 20 ng/mL of fibroblast growth factor-2 for additional 4 days [13]. ...
... After washing, cells were incubated for at least 1 hour with the secondary antibodies: goat anti-mouse IgG conjugated to Alexa Fluor 546 (Molecular Probe; 1:400) or goat antirabbit IgG conjugated to Alexa Fluor 488 (Molecular Probe; 1712 PAULSEN ET AL. 1:400). The samples were washed with PBS and nuclear staining was performed with 1.0 mg/mL solution of 4¢,6- diamidino-2-phenylindole (DAPI) [13]. Images of cells were accessed with optic fluorescence microscopy (Nikon T300). ...
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... Murine ESCs (cell line USP1) were kindly provided by Dr. Lygia Pereira (University of São Paulo-USP) and by Dr. Irina Kerkis (Federal University of São Paulo). Murine ESCs were grown as previously described (Marinho et al., 2010;Nones et al., 2010). Briefly, cell colonies were grown on a mouse embryonic fibroblast (MEF) feeder layer previously arrested with mitomycin C (Sigma), in 15% knockout serum replacement (KSR; Gibco) -DMEM/F12 (Gibco) supplemented with 1% nonessential amino acid solution (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 40 mg/mL gentamicin sulfate (Schering-Plough), and 0.2% of conditioned medium from Chinese hamster ovary (CHO) cells producing leukemia inhibitory factor (LIF). ...
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... To allow growth of nestin-positive cells, EB were plated onto tissue culture dishes coated with 1 lg/ml LN (Invitrogen) and 1 lg/ml fibronectin (FN; Invitrogen) for 24 hr in DMEM/F12 containing 1% N-2 supplements (Invitrogen) and 20 ng/ml fibroblast growth factor 2 (FGF-2; R&D Systems, Minneapolis, MN; Lee et al., 2000). Neuronal migration in EB was achieved after they had been plated onto dishes coated with 5 lg/ml LN (Invitrogen) and 1 lg/ml FN (Invitrogen) in DMEM/F12 with N-2 supplement and 20 ng/ml fibroblast growth factor-2 (R&D Systems) for an additional 6, 15, or 30 days (Nones et al., 2010). ...
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This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.
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