Astrocytes derived from fetal neural progenitor cells as a novel source for therapeutic adenosine delivery.
ABSTRACT Intracerebral delivery of anti-epileptic compounds represents a novel strategy for the treatment of refractory epilepsy. Adenosine is a possible candidate for local delivery based on its proven anti-epileptic effects. Neural stem cells constitute an ideal cell source for intracerebral transplantation and long-term drug delivery. In order to develop a cell-based system for the long-term delivery of adenosine, we isolated neural progenitor cells from adenosine kinase deficient mice (Adk(-/-)) and compared their differentiation potential and adenosine release properties with corresponding wild-type cells.
Fetal neural progenitor cells were isolated from the brains of Adk(-/-) and C57BL/6 mice fetuses and expanded in vitro. Before and after neural differentiation, supernatants were collected and assayed for adenosine release using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Adk(-/-) cells secreted significantly more adenosine compared to wild-type cells at any time point of differentiation. Undifferentiated Adk(-/-) cells secreted 137+/-5 ng adenosine per 10(5) cells during 24 h in culture, compared to 11+/-1 ng released from corresponding wild-type cells. Adenosine release was maintained after differentiation as differentiated Adk(-/-) cells continued to release significantly more adenosine per 24 h (47+/-1 ng per 10(5) cells) compared to wild-type cells (3+/-0.2 ng per 10(5) cells).
Fetal neural progenitor cells isolated from Adk(-/-) mice--but not those from C57BL/6 mice--release amounts of adenosine considered to be of therapeutic relevance.
- [show abstract] [hide abstract]
ABSTRACT: Population-based epidemiological studies on epilepsy are available mainly from the UK and the Nordic, Baltic and western Mediterranean countries. No studies were identified from large areas of Europe, especially from the former eastern Europe (except the Baltic countries) and the eastern Mediterranean countries. Based on the prevalence of epilepsy in different studies and accounting for incomplete case identification the estimated number of children and adolescents in Europe with active epilepsy is 0.9 million (prevalence 4.5-5.0 per 1000), 1.9 million in ages 20-64 years (prevalence six per 1000) and 0.6 million in ages 65 years and older (prevalence seven per 1000). Approximately 20-30% of the epilepsy population have more than one seizure per month. Based on the age-specific incidence rates in European studies, the estimated number of new cases per year amongst European children and adolescents is 130,000 (incidence rate 70 per 100,000), 96,000 in adults 20-64 years (incidence rate 30 per 100,000) and 85,000 in the elderly 65 years and older (incidence 100 per 100,000). The proportion of both new and established cases with epilepsy in the young, adults and elderly in individual countries may differ substantially from total European distribution because of differences in age structure.European Journal of Neurology 05/2005; 12(4):245-53. · 4.16 Impact Factor
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ABSTRACT: The incidence of epilepsy and of all unprovoked seizures was determined for residents of Rochester, Minnesota U.S.A. from 1935 through 1984. Age-adjusted incidence of epilepsy was 44 per 100,000 person-years. Incidence in males was significantly higher than in females and was high in the first year of life but highest in persons aged > or = 75 years. Sixty percent of new cases had epilepsy manifested by partial seizures, and two thirds had no clearly identified antecedent. Cerebrovascular disease was the most commonly identified antecedent, accounting for 11% of cases. Neurologic deficits from birth, mental retardation and/or cerebral palsy, observed in 8% of cases, was the next most frequently identified preexisting condition. The cumulative incidence of epilepsy through age 74 years was 3.1%. The age-adjusted incidence of all unprovoked seizures was 61 per 100,000 person-years. Age- and gender-specific incidence trends were similar to those of epilepsy, but a higher proportion of cases was of unknown etiology and was characterized by generalized onset seizures. The cumulative incidence of all unprovoked seizures was 4.1% through age 74 years. With time, the incidence of epilepsy and of unprovoked seizures decreased in children and increased in the elderly.Epilepsia 01/1993; 34(3):453-68. · 3.91 Impact Factor
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ABSTRACT: More than 30 percent of patients with epilepsy have inadequate control of seizures with drug therapy, but why this happens and whether it can be predicted are unknown. We studied the response to antiepileptic drugs in patients with newly diagnosed epilepsy to identify factors associated with subsequent poor control of seizures. We prospectively studied 525 patients (age, 9 to 93 years) who were given a diagnosis, treated, and followed up at a single center between 1984 and 1997. Epilepsy was classified as idiopathic (with a presumed genetic basis), symptomatic (resulting from a structural abnormality), or cryptogenic (resulting from an unknown underlying cause). Patients were considered to be seizure-free if they had not had any seizures for at least one year. Among the 525 patients, 333 (63 percent) remained seizure-free during antiepileptic-drug treatment or after treatment was stopped. The prevalence of persistent seizures was higher in patients with symptomatic or cryptogenic epilepsy than in those with idiopathic epilepsy (40 percent vs. 26 percent, P=0.004) and in patients who had had more than 20 seizures before starting treatment than in those who had had fewer (51 percent vs. 29 percent, P<0.001). The seizure-free rate was similar in patients who were treated with a single established drug (67 percent) and patients who were treated with a single new drug (69 percent). Among 470 previously untreated patients, 222 (47 percent) became seizure-free during treatment with their first antiepileptic drug and 67 (14 percent) became seizure-free during treatment with a second or third drug. In 12 patients (3 percent) epilepsy was controlled by treatment with two drugs. Among patients who had no response to the first drug, the percentage who subsequently became seizure-free was smaller (11 percent) when treatment failure was due to lack of efficacy than when it was due to intolerable side effects (41 percent) or an idiosyncratic reaction (55 percent). Patients who have many seizures before therapy or who have an inadequate response to initial treatment with antiepileptic drugs are likely to have refractory epilepsy.New England Journal of Medicine 02/2000; 342(5):314-9. · 51.66 Impact Factor
Astrocytes derived from fetal neural progenitor cells as a novel source for
therapeutic adenosine delivery
Annelies Van Dyckea,*, Robrecht Raedta, Alain Verstraeteb, Panos Theofilasc, Wytse Wadmana,d,
Kristl Voncka, Detlev Boisonc, Paul Boona
aLaboratory for Clinical and Experimental Neurophysiology, Department of Neurology, Ghent University Hospital, 1K12, 185 De Pintelaan, 9000 Ghent, Belgium
bLaboratory of Clinical Biology, Ghent University Hospital, Ghent, Belgium
cRobert Stone Dow Neurobiology Laboratories, Legacy Research, Portland, OR, USA
dSwammerdam Institute of Life Sciences, Department of Neurobiology, University of Amsterdam, Amsterdam, The Netherlands
Epilepsy is a chronic neurological disorder with a prevalence
between 0.5 and 1%.1,2Despite the availability of several anti-
uncontrolled seizures or medication-related side effects.3Intrace-
rebral delivery of anti-epileptic compounds represents a novel
strategy for the treatment of refractory epilepsy.4,5Adenosine is a
possible candidate for local delivery because of its proven anti-
epileptic effects. Systemic application of adenosine is not possible
because of severe side effects such as decreased heart rate, blood
pressure and body temperature.6Adenosine is a ubiquitous
neuromodulator of the brain and acts via binding to G-protein
coupled adenosine receptors. The inhibitory effect of adenosine is
mainly due to binding to the high-affinity A1 receptor that is
expressed in cerebral cortex, hippocampus, thalamus, cerebellum,
brain stem and spinal cord.7,8Several in vivo studies have already
proven the feasibility and anti-seizure effects of local delivery of
adenosine in different animal models.9–13In those studies, a
synthetic adenosine-releasing polymer or encapsulated adeno-
sine-releasing cells from different sources were implanted into the
lateral ventricle of kindled rats. However, the anti-seizure effect of
adenosine decreased during the second week of treatment because
of expiration of adenosine release from the polymer or limited
long-term viability of the encapsulated cells. Further attempts to
extend the adenosine release resulted in the successful develop-
ment of a silk protein-based release system for adenosine.
Implantation of this system in the infrahippocampal fissure of
rats resulted in seizure protection and retardation of kindling
acquisition.14,15However, these therapeutic effects lasted for a
maximum of 10 days, corresponding to the duration of adenosine
release from this system. Since epilepsy is a chronic disorder and
lifelong treatment is required, development of an implantable
adenosine source that is able to produce a sufficient and stable
Seizure 19 (2010) 390–396
A R T I C L E I N F O
Received 31 March 2010
Received in revised form 8 May 2010
Accepted 21 May 2010
Neural stem cell
Neural progenitor cell
A B S T R A C T
Purpose: Intracerebral delivery of anti-epileptic compounds represents a novel strategy for the
treatment of refractory epilepsy. Adenosine is a possible candidate for local delivery based on its proven
anti-epileptic effects. Neural stem cells constitute an ideal cell source for intracerebral transplantation
and long-term drug delivery. In order to develop a cell-based system for the long-term delivery of
adenosine, we isolated neural progenitor cells from adenosine kinase deficient mice (Adk?/?) and
compared their differentiation potential and adenosine release properties with corresponding wild-type
Methods: Fetal neural progenitor cells were isolated from the brains of Adk?/?and C57BL/6 mice fetuses
and expanded in vitro. Before and after neural differentiation, supernatants were collected and assayed
for adenosine release using liquid chromatography–tandem mass spectrometry (LC–MS/MS).
Results: Adk?/?cellssecretedsignificantlymore adenosinecomparedtowild-type cells atanytimepoint
of differentiation. Undifferentiated Adk?/?cells secreted 137 ? 5 ng adenosine per 105cells during 24 h in
culture, compared to 11 ? 1 ng released from corresponding wild-type cells. Adenosine release was
maintained after differentiation as differentiated Adk?/?cells continued to release significantly more
adenosine per 24 h (47 ? 1 ng per 105cells) compared to wild-type cells (3 ? 0.2 ng per 105cells).
Conclusions: Fetal neural progenitor cells isolated from Adk?/?mice – but not those from C57BL/6 mice –
release amounts of adenosine considered to be of therapeutic relevance.
? 2010 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +32 9 332 6946; fax: +32 9 332 4971.
E-mail address: Annelies.VanDycke@UGent.be (A. Van Dycke).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yseiz
1059-1311/$ – see front matter ? 2010 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
dose without the need of future replacement is necessary. A
promising technique may be local delivery via implantation of
In brain, adenosine can be metabolized and removed by three
enzymes that control levels of ambient intracellular adenosine:
adenosine kinase (ADK), adenosine deaminase (ADA) and S-
adenosyl-homocysteine hydrolase (SAHH). Based on its low KM
for adenosine, the key enzyme for the control of adenosine levels is
ADK,19,20which, in adult brain, is almost exclusively expressed in
epileptic brain and associated with a deficiency of the endogenous
anticonvulsant adenosine and the expression of spontaneous
seizure activity.22–25Therefore, inhibition of ADK constitutes a
seizure control. To this end adenosine-releasing embryonic stem
the Adk-gene,26and successfully been used to suppress kindling
acquisition in rats27and the development of epilepsy in mice.24In
these experiments it was necessary to differentiate the embryonic
stem cells before implantation to avoid teratoma formation and
genetic alterations by undifferentiated embryonic stem cells.28,29
Several strategies are available to improve functional and safety
cells can be controlled by specific selection and culture procedures
or genetic modification. Genetic aberrations must be excluded by
early passage karyotyping and tumor formation can be excluded by
depletion of tumorigenic cells or enrichment of non-tumorigenic
cells.30Nevertheless it remains to be proven whether these
strategies are sufficient to guarantee long-term survival and
therapeutic action. Isolation of fetal or adult stem cells could avoid
these ethical and practical problems coupled to embryonic stem
addition, the transplantation of neural stem cells may have
additional benefits since they have the potential ability to integrate
in the neural network.31Combination with local delivery of an anti-
epileptic substance like adenosine may lead to a promising and
effective therapy. As a potential cell source, an adenosine kinase
knockout mouse (Adk?/?) has been developed.32Since Adk?/?mice
die within 14 days due to hepatic steatosis, the isolation of adult
neural Adk?/?stem cells is not a possibility.32In this paper we
describe the isolation and characterization of Adk?/?fetal neural
progenitor cells as a source for therapeutic adenosine delivery.
Neural differentiation in vitro and adenosine secretion is compared
with wild-type cells derived from C57BL/6 mice.
2. Materials and methods
Mice [Adk+/?– breeders in the C57BL-6 background – (Robert
Stone Dow Neurobiology Laboratories, Legacy Research, USA) and
matching wild-type C57BL/6 mice (Harlan, The Netherlands)] were
Committee (decree 86/609/EEC). The study protocol was approved
by the Animal Experimental Ethics Committee of Ghent University
controlled conditions (12 h normal light/dark cycles, 21–22 8C and
50% relative humidity) with food and water ad libitum.
2.2. Isolation of neural stem cells
Fetal neural stem cells were isolated from Adk?/?and wild-type
C57BL/6 mice fetuses at embryonic day 14 (E14). Four pregnant
mice from Adk+/?? Adk+/?matings (all mutants were maintained
in the C57BL/6 background) and two pregnant C57BL/6 mice were
sacrificed by cervical dislocation; the uteri were removed and
transferred to a dish with ice-cold phosphate buffered saline (PBS).
The uterine horns were opened and the fetuses removed. Their
brains were removed and placed in separate dishes with ice-cold
PBS. The cortex was isolated via microdissection and put into
culture medium (see below). The tissue was dissociated by
trituration, followed by centrifugation at 80 g for 10 min. The
pellet was resuspended in fresh medium and the cells were
counted. Their viability was checked with the trypan blue
exclusion method and cells were cultured in T25 flasks at a
density of 10,000/cm2.
(StemCell Technologies SARL, Grenoble, France) with an additional
2 mM L-glutamine (Cambrex, Verviers, Belgium), 3 mM D-glucose
(Sigma, Bornem, Belgium), 2% B27 (Invitrogen, Merelbeke, Bel-
gium), 1% N2 supplement (Invitrogen), 100 U/ml penicillin
(Cambrex), 100 U/ml streptomycin (Cambrex), 20 ng/ml of human
recombinant epidermal growth factor (EGF, Sigma) and 20 ng/ml
of recombinant human basic fibroblast growth factor (bFGF, R&D,
Abingdon, UK). Cells were grown at 37 8C in 5% CO2and 95% air
with saturated humidity. They were passaged once cell clusters,
with a diameter of about 100 mm, were formed, approximately 2
weeks after initial isolation. Subsequent passages were done every
were further cultured in T75 flasks at a density of 10,000 cells/cm2.
2.3. PCR of adenosine-releasing cells
Since the fetuses were derived from two heterozygote
Adk+/?? Adk+/?matings, cell colonies of three different genotypes
(Adk+/+, Adk+/?and Adk?/?) were isolated and genotyped by PCR.
DNA from expanded cell colonies was extracted using the GenElute
Mammalian GenomicDNAPurificationKit(Sigma)and subjectedto
PCR with allele-specific primer sets. PCR reactions were performed
under standard conditions using three primers simultaneously:
o107, 50-CTC ACT TAA GCT GTA TGG AGGTGACCG-30- (sense primer
GGT GAG-30- (antisense primer specific for wild-type Adk), and
o109, 50-ACT GGG TGC TCA GGT AGT GGT TGT CG-30- (antisense
primer specific for targeting construct).
2.4. Western Blot
lysates of undifferentiated and differentiated Adk?/?cells. Corre-
sponding wild-type cells served as control. GAPDH expression was
used to control for protein concentration.
Following harvesting and washing the cells with PBS, total cell
lysates were made by adding Laemmli buffer followed by
sonication. Protein concentration was measured and protein
extracts were applied to a 4–12% Bis–Tris gel (Invitrogen) for
electrophoresis by using MES/SDS running buffer (Invitrogen).
Then proteins were transferred to Protran Nitrocellulose Hybrid-
ization Transfer membrane (0.2 mm pore size; PerkinElmer,
Belgium) with transfer buffer (Invitrogen). After the blotting
process, the membranes were blocked for 1 h in Tris Buffered
Saline (TBS) containing 0.075% Tween 20 (Invitrogen) and 5%
nonfat milk, followed by addition of the primary antibodies: rb
anti-ADK 1:6000 (custom made at RS Dow Neurobiology
Laboratories, Legacy Research, Portland, USA) or ms anti-GAPDH
1:1000 (Santa Cruz Biotechnology, sc-47724). Primary antibodies
were incubated for 1 h. After rinsing, secondary antibodies anti-rb/
ms IgG Horseradish Peroxidase 1:3000 were added during 90 min
at room temperature (GE Healthcare, Buckinghamshire, UK,
NA934/NA931). After thorough rinsing, signals were visualized
using a commercial enhanced bioluminescence detection method
(ECL) kit (Invitrogen).
A. Van Dycke et al./Seizure 19 (2010) 390–396
To evaluate the expression of neural stem cell markers (nestin,
GFAP and Sox-2), Adk?/?cells and corresponding wild-type cells
were plated at a density of 20,000 cells/cm2on laminine coated
(3 mg/cm2, Roche Diagnostics, Vilvoorde, Belgium) chamber slides
(VWR International, Leuven, Belgium). The cells were plated in
neural stem cell growth medium without addition of the growth
factor EGF for 24 h. Then they were fixed with 4% paraformalde-
hyde (PFA) for 15 min and subjectedto immunocytochemistry (see
For neural differentiation, Adk?/?cells and corresponding wild-
type cells were plated for 24 h in neural stem cell growth medium
without the addition of EGF at a density of 50,000 cells/cm2on
laminine coated (3mg/cm2) chamber slides. Then the medium was
changed to neural stem cell growth medium without added growth
factors but with the addition of 3% Fetal Calf Serum (FCS, Serum
Supreme, Cambrex). On day 7 cells were fixed with 4% PFA and
subjected to immunocytochemistry for the three different neural
phenotypes: neurons (tau), glial cells (GFAP) and oligodendrocytes
(RIP). The fixed cells were rinsed with 50 mM NH4Cl for 10 min
(quenching), permeabilized and blocked in PBS containing 0.4% fish
followed by incubation overnight at 4 8C with primary antibodies
(ms, mouse monoclonal; rb, rabbit polyclonal): rb anti-Sox2 1:1000
(Chemicon, Brussels, Belgium, AB5603), rb anti-nestin 1:5000
1:2000 (Dako, Heverlee, Belgium; A0024), rb anti-GFAP 1:400
(Dako, Z0334), ms anti-RIP 1:5000 (Chemicon, MAB1580). After
rinsing, the cells were incubated with the secondary antibodies
Alexa Fluor 594 goat anti-rabbit IgG 1:1000 (Invitrogen, A11072) or
Alexa Fluor 594 goat anti-mouse IgG 1:1000 (Invitrogen, A11020)
diluted in PBS/FSG/TX100 for 2 h each. Chamber slides were then
rinsed and nuclei were stained with 0.3mM 40-6-diaminido-2-
phenylindole (DAPI, Invitrogen) for 1 min. After additional rinsing,
slides were mounted with Vectashield mounting medium (Labcon-
sult, Brussels, Belgium). To assess the fraction of cells expressing a
specific marker, immunopositive cells were counted in nine
randomly selected high power fields. The relative amount of cells
positive for a specific marker was obtained by dividing the total
number of immunopositive cells by the total number of nuclei
counted.The cells wereevaluated undera fluorescencemicroscope.
In control slides the primary antibody was omitted and no
immunostaining was detected in these controls.
2.6. In vitro adenosine release
The amount of adenosine released from the cells was assessed
from plated cells. Adenosine release from Adk?/?cells was
compared with adenosine release from corresponding wild-type
cells and this under different experimental conditions. First,
samples were collected from supernatants of non-differentiated
cells. Therefore cells were plated at densities of 10,000 cells/cm2
and 100,000 cells/cm2in a laminine coated (3 mg/cm2) 96-well
plate (200 ml medium/well) in neural stem cell growth medium
without EGF. After 24 h, medium was replaced with fresh medium
without growth factors but with addition of the adenosine
deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hy-
drochloride (EHNA, 50 mM, Sigma). The addition of EHNA was
necessary to prevent adenosine breakdown in the culture medium.
It has been shown that addition of EHNA to cultured neurons or
astrocytes is not toxic for the cells.33At different time points after
medium replacement (1 h, 2 h, 4 h, 8 h, 12 h and 24 h) samples of
200 ml supernatants were taken, each time from a different well.
Samples were subsequently frozenat ?20 8C untillateranalysis. At
each time point six samples were taken.
Second, a similar protocol was performed to collect samples
from differentiated cells. For this condition, cells were plated at a
density of 50,000 cells/cm2in a laminine coated (3 mg/cm2) 96-
well plate (200 ml medium/well) in neural stem cell growth
medium without EGF. After 24 h the medium was changed to
neural stem cell growth medium without growth factors but with
addition of 3% FCS. Seven days later medium was replaced with
fresh medium without growth factors or FCS but with the addition
of EHNA (50 mM) and samples of supernatants were collected (1 h,
2 h, 4 h, 8 h, 12 h and 24 h after medium replacement; six samples
per time point) and frozen for later analysis.
At each sample collection, cells were counted with the trypan
blue exclusion method and averaged. These data were used for
normalization and quantification of adenosine release per cell
2.7. Sample analysis
Samples were analyzed using liquid chromatography–tandem
mass spectrometry (LC–MS/MS) according to an earlier described
protocol.34Liquid chromatographic separation was performed
using an Agilent LC 1100 HPLC system (Agilent Technologies, Santa
Clara, US) equipped with a quaternary pump, a column oven and a
100 well-plate autosampler. The LC was coupled to an API 2000
Triple Quadrupole system (Applied Biosystems/MDS Sciex, Foster
City, US) equipped with an APCI interface. Chromatographic
separation was done using a reversed phase column, the XBridge
C8column 4.6 mm ? 75 mm, 3.5 mm (Waters, Zellik, Belgium) and
the temperature of the column oven was set at 40 8C. The solvent
system consisted of 2 mM ammonium acetate in water (A) and
2 mM ammonium acetate in methanol (B). An isocratic elution
minandaninjectionvolumeof5 ml.Thetotalruntimewas4.5 min
per analytical run. Mass spectrometer analysis was set up in
selected reaction monitoring (SRM) in positive polarity. Based on
the component dependent parameters the SRM transitions of m/z
268.2/136.1 and 302.2/170.0 were selected, respectively for
adenosine and IS.
Before analysis of the samples, 2-chloroadenosine (1000 ng/ml,
20 ml) was added as internal standard to each sample. At each
analytical run a new calibration curve was made consisting of a
dilution series of 10 concentrations of adenosine in medium with
addition of internal standard.
2.8. Data analysis and statistics
At each sample collection, six different replicates were taken
and the mean was calculated. Adenosine secretion from differen-
tiated and non-differentiated cells was normalized to secretion per
105cells. Statistical evaluation (SPSS 15.0) was performed using
parametric tests. All data were expressed as means and standard
errors of the mean. p < 0.05 indicates a significant difference.
Neurosphere-forming cells were isolated as described above
from the cortex of E14 fetal mice derived from Adk+/?? Adk+/?
matings and from respective wild-type dams. Approximately 2
to 150 mm – neurospheres could be passaged mechanically and
further passages were performedevery 4–5 days. At passage 7, PCR
analysis with allele-specificprimer sets (o107, o108 and o109)was
performed (Fig. 1A). The wild-type specific primer set (o107/o108)
gave rise to a 640 bp band, whereas the knockout specific primer
set (o107/o109) showed a 840 bp band. Based on PCR results one
cell clone homozygous for Adk?/?and one from the C57BL/6
A. Van Dycke et al./Seizure 19 (2010) 390–396
control were selected for all subsequent experiments. Additionally
protein extracts from both cell clones were investigated with
Western Blot analysis for ADK expression (Fig. 1B). The control
cells showed expression for ADK whereas no expression was found
in the Adk?/?cell clones, confirming the PCR results.
To analyze stem cell properties for both cell clones, we
performed immunohistochemical staining of the undifferentiated
isolated cells. Three neural stem cell markers (nestin, GFAP and
Sox-2) were evaluated and expression of all markers was found in
bothAdk?/?and wild-typecells(Fig. 2A–D). Forthe nuclear marker
Sox-2, we performed a quantitative analysis by randomly selecting
nine high power fields (200?) under a fluorescence microscope.
The amount of immunopositive cells was counted together with
361/570 (63%) of the Adk?/?cells and in 324/528 (61%) of the
corresponding wild-type cells.
Since Adk?/?cells were obtained by genetically manipulation,
their differentiation potential was evaluated and compared with
wild-type cells. Therefore cells were subjected to growth in
differentiation medium for 7 days followed by immunohistochem-
ical staining for the three different neural phenotypes: neurons
(tau), glial cells (GFAP) and oligodendrocytes (RIP). Both Adk?/?
cells and wild-type cells expressed tau and GFAP (Fig. 3A–D).
Quantitative analysis – as described above for the Sox-2 marker –
demonstrated expression of tau in 6% (42/694) and GFAP in 28%
(178/640) of the Adk?/?cells. Similar ratios were obtained in wild-
Fig. 1. PCR and Western Blot. (A) PCR: results of isolated cells from Adk+/?? Adk+/?matings. Based on these results, an Adk?/?clone needed for the experiment could be
detected. The genomic DNA was amplified using the three allele-specific primers o107, o108 and o109. DNA from Adk+/?cells gives a combination of the wild-type specific
640 bp band (primers o107/o108) and the knockout-specific 840 bp band (primers o107/o109), while DNA from Adk?/?cells gives rise to only a 840 bp band (primers o107/
o109), demonstrating the bi-allelic genetic disruption of the Adk locus. (B) Western Blot: above the GAPDH immune reactivity for protein control is shown (38 kDa). Beneath
ADKimmunereactivityis shown(44–46 kDa). Aclearreactivityisfoundinwild-type cells,whereas noreactivityis foundinnon-differentiated anddifferentiatedAdk?/?cells
(CO = control wild-type; KO 1d = Adk?/?after 24 h; KO 1w = Adk?/?after 1 week).
Fig. 2. Expression of neural stem cell markers. Left: Immunostaining with nestin (red) and GFAP (green) in Adk?/?cells (A) and wild-type cells (C). Scale bar = 100 mm. Right:
Immunostaining with Sox2 (red) in Adk?/?cells (B) and wild-type cells (D). Scale bar = 50 mm. Nuclei are stained with DAPI (blue).
A. Van Dycke et al./Seizure 19 (2010) 390–396
type cells: 7% (29/441) tau+and 29% (200/679) GFAP+. The
the cells expressed the oligodendrocyte marker RIP. The isolated
fetal cells therefore have a bipotential differentiation capacity
consistent for neural progenitor cells.
different densities and cultured in undifferentiated (10,000 and
100,000 cells/cm2) and differentiated (50,000 cells/cm2) condi-
tions. After sample collection, cells from different wells per
condition were counted and averaged. These data were used for
normalization of adenosine release per cell number. Already 1 h
after medium replacement adenosine was measured in samples
from undifferentiated(3.40 ? 0.3 ngper105cells)anddifferentiated
(3 ? 0.2 ng per 105cells) Adk?/?cells, whereas no adenosine
was found in samples from wild-type cells. Comparison of adenosine
release at the different time points after medium replacement
showed an accumulation of adenosine over time in (un)differentiated
Adk?/?cells, whereas corresponding wild-type cells did not release
release in samples taken 24 h after medium replacement. A
significant difference is seen between adenosine release from
cells compared to wild-type, both in undifferentiated
(Fig. 4A) and differentiated(Fig. 4B) conditions: 137 ? 5 ng compared
to 11 ? 1 ng adenosine per 105cells (non-differentiated cells,
p < 0.05) and 47 ? 1 ng compared to 3 ? 0.2 ng adenosine per 105
cells (differentiated cells, p < 0.05). In undifferentiated conditions,
results of Adk?/?cells plated at 10,000/cm2showed a release of
152 ? 4 ng per 105cells after 24 h, indicating that cell density has no
effect on the amount of adenosine secretion. These results indicate
that Adk?/?cells release significantly more adenosine compared to
wild-type cells. Furthermore the higher amount of adenosine release
is sustained in differentiated cells, meaning that they can be used as a
therapeutic source for adenosine release.
In this experiment we successfully isolated a neural novel line
of Adk?/?neural progenitor cells. We demonstrated their ability to
secrete amounts of adenosine – in contrast to matching wild-type
cells – which are considered to be of therapeutic relevance. Sample
analysis revealed adenosine release from 47 ? 1 ng (differentiated
Adk?/?cells) to 137 ? 5 ng (undifferentiated Adk?/?cells) per 105
cells per 24 h. These results are comparable with adenosine secretion
from Adk?/?embryonic stem cell derived glial cells.26In those former
Fig. 3. Glial and neural differentiation of neural progenitor cells in vitro. Left: Immunostaining with GFAP (red) in Adk?/?cells (A) and wild-type cells (C). Right:
Immunostaining with tau (red) in Adk?/?cells (B) and wild-type cells (D). Nuclei are stained with DAPI (blue). Scale bar = 100 mm.
Adenosine release in non-differentiated and differentiated conditions from Adk?/?cells and corresponding wild-type cells. After medium replacement six samples per time
point per condition were collected and themean was calculated. An accumulationof adenosine releaseis seen over timein Adk?/?cells in both conditions, whereas wild-type
cells do not release significant amount of adenosine. Adenosine release is expressed in ng per 105cells.
Time after medium replacementNon-differentiated cells Differentiated cells
9 ? 1
A. Van Dycke et al./Seizure 19 (2010) 390–396
studiesnon-differentiatedembryonicstemcellssecreted2.6 ? 0.4 ng
adenosine per 105cells per hour (i.e. around 62 ng adenosine per 105
cells per 24 h), whereas non-differentiated glial precursor cells
secreted 2.7 ? 0.8 ng to 11.7 ? 1.7 ng per 105cells per hour (i.e.
around 65–280 ng adenosine per 105cells per 24 h) depending on the
The lower adenosine secretion of differentiated Adk?/?cells
compared to undifferentiated Adk?/?cells during the first 24 h of
samplecollectionwas a remarkablefinding.Sincestemcellshavea
higher metabolic rate compared to differentiated cells, differences
in adenosine release during the first 24 h might be due to
differences in their overall metabolic rate. However, adenosine
release from our differentiated cells is considered to be of
therapeutic relevance since previous studies with local adenosine
delivery in the lateral brain ventricle of rats revealed that a
continuous release of relatively low (20–50 ng per day in vitro)
adenosine concentrations lead to seizure suppression in the rat
kindling model.9,11–13Subsequent studies with release of 1000 ng
adenosine per day using a silk-based delivery system implanted
into the infrahippocampal cleft showed sustained suppression of
evoked seizures in the kindling model.14,15These studies demon-
strate the therapeutic potential of focal adenosine augmentation
therapies in refractory epilepsy.
Genetically engineered neural stem cells have been investigat-
ed as a tool for different neurological diseases, including refractory
epilepsy.31,35Compared to other cell sources, such as fetal brain
tissue, survival capacities of neural stem or progenitor cells are
higher and more stable.18Therefore transplantation of neural
stem/progenitor cells combined with local delivery of an anti-
epileptic substance may be a successful alternative treatment
option for refractory epilepsy.
Since Adk?/?cells are genetically manipulated, it was necessary
and wild-type neural progenitor cells to in vitro differentiation,
and compared their differentiation potential towards neurons and
astrocytes. We previously demonstrated the ability of neural
progenitor cells to survive in a sclerotic hippocampus in the rat
kainic acid induced status epilepticus model.36We found that the
majority of surviving cells differentiated towards astrocytes in
vivo. Likewise, in our current experiment we found that neural
progenitor cells differentiated towards astrocytes in vitro. This
differentiation towards astrocytes may be a major advantage in
transplantation strategies for epilepsy since astrocytes are key
regulators of adenosine.22The ultimate rationale for using this cell
source is transplantation of astrocytes derived from the Adk?/?
progenitor cells after predifferentiation in vitro. These cells might
be superior to integrate into the astrogliotic environment of an
epileptic hippocampus and interact with the epileptogenic process
by releasing adenosine. We recently demonstrated that local
delivery of adenosine directly into the epileptic hippocampus has
an antiseizure effect in rats with spontaneous seizures.37Together
with the results of our former transplantation experiment36the
new cells described here indicate that Adk?/?cells offer the
possibility to stably integrate after transplantation into the brain,
and to become a long-term source for continuous local adenosine
Compared with data from previous studies,9,11–15we conclude
that the amount of secreted adenosine – both from non-
differentiated and differentiated Adk?/?cells – is sufficient to
obtain a therapeutic effect in the treatment of refractory epilepsy.
Since astrocytes play a major role in the regulation of adenosine in
the brain, astrocytes derived from Adk?/?neural progenitor cells
seem a promising source for local delivery of adenosine in an
epileptic brain. Further in vivo studies in relevant animal models
with spontaneous seizures should focus on effect on seizure
Conflicts of interest
None of the authors has any conflict of interest to disclose.
Annelies Van Dycke is supported by a junior researcher
(‘Aspirant’) grant from the Fund for Scientific Research-Flanders.
Professor Kristl Vonck is supported by a grant from the BOF
Fund of Ghent University Hospital.
Detlev Boison is Director of the Epilepsy Program at Legacy
Researchand supported by
NS058780, NS057538 and MH083973 from the National Institutes
of Health (NIH).
Professor Paul Boon is a Senior Clinical Investigator of the Fund
for Scientific Research-Flanders and is supported by grants from
the Fund for Scientific Research-Flanders and Ghent University
Research Fund and by the Clinical Epilepsy Grant from Ghent
‘We confirm that we have read the Journal’s position on issues
involved in ethical publication and affirm that this report is
consistent with those guidelines’.
Fig. 4. Adenosine releases from Adk?/?cells (blue) and corresponding wild-type
cells (red) in 24 h. (A) Adenosine released from 100,000 non-differentiated cells:
adenosine release from Adk?/?cells is significantly higher compared to wild-type
cells. (B) Adenosine released from 100,000 differentiated cells: adenosine release
from Adk?/?cells is significantly higher compared to wild-type cells. Standard
errors are marked with black bars. Significance is marked with an asterisk (*):
p < 0.05.
A. Van Dycke et al./Seizure 19 (2010) 390–396
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