Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
Iranian Institute of Cell and Gene therapy, Tehran, Iran
Bioviva Science USA, Seattle, USA
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Neuroscience Lab, Department of Anatomy and Cell Biology, School of Medicine, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
Shefa Neuroscience Research Center, Khatam-al-Anbia Hospital, Tehran, Iran
Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
P.O. BOX: 14155/6451
Department of Toxicology and Pharmacology
Faculty of Pharmacy
Tehran University of Medical Sciences
Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Babaei Abraki Shahnaz,
Ghahremani Mohammad Hossein.
Stable Knockdown of Adenosine Kinase by Lentiviral Anti-ADK
miR-shRNAs in Wharton’s Jelly Stem Cells.
In this study, we describe an efficient approach for stable knockdown of adenosine kinase (ADK) using lentiviral
system, in an astrocytoma cell line and in human Wharton’s jelly mesenchymal stem cells (hWJMSCs). These sources of stem
cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited
numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy.
Materials and Methods
In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding
sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA (shRNAs). First, these cassettes with
scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain
reaction (qRT-PCR) was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and
measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing
mesenchymal specific marker (CD44+) and lack of expression of hematopoietic lineage marker (CD45-). Then, the lentiviral
vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs.
Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We
successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miR-
shRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral
system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miR-
shRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by
western blot analysis at the protein level.
Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with
genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors (AAV) might
be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy.
Previous molecular studies indicated that up-regulation of
adenosine kinase (ADK), a key enzyme in the metabolism
of adenosine, is one of the most important processes
involved in astrogliosis (1, 2). RNA interference (RNAi)
or post-transcriptional gene silencing (PTGS) is an
interesting molecular tool for gene knockdown. Knock
down of ADK increases intracellular adenine and results
in extracellular adenosine augmentation. Adenosine has
known protective effects on the central nervous system (3,
4). ADK gene could be targeted by RNAi in human cells which
is an effective way to produce adenosine-releasing cells (5,
6). Adenosine augmentation exhibits a paracrine therapeutic
effect and has potential for therapeutic applications in
neurological diseases like refractory epilepsy (7).
Among children, the highest incidence of epilepsy is seen at
ages less than five years old. Therefore, finding a new source
of cells with therapeutic applications is highly required (8).
Wharton’s jelly stem cells (WJMSCs) are an alternative
for bone marrow mesenchymal stem cell (BMSCs).
They are multipotent cells which are easily isolated in
unlimited numbers with long-term ex vivo proliferation and
immunomodulatory properties (9). WJMSCs are obtained
from discarded human umbilical cord, with no ethical concern
(10). These cells express specific MSCs markers like CD44
and are negative for CD45 hematopoietic lineage marker (11,
12). Being easily accessible, makes WJMSCs an alternative
and attractable source for cell-based gene therapy.
In the present study, we used anti-ADK microRNA (miR)
in a shRNA lentiviral systems (miR-shRNA) for ex vivo gene
therapy in U-251 MG cell line. We screened eight cassettes
of miR-shRNAs that target human ADK gene. In order to
screen and select the most efficient anti-ADK miR-shRNA,
astrocytoma cell line was employed. Human U-251 MG
cell line highly expresses ADK gene. Pseudo lentiviruses of
eight anti-ADK miR-shRNAs were used for transducing of
astrocytoma cell lines. The most efficient anti-ADK miRshRNA
for knockdown of ADK was selected by quantitative
real time-polymerase chain reaction (qRT-PCR) analysis
of established cells. Furthermore, human WJMSCs were
isolated and cultured after characterizing with flow cytometry
for specific mesenchymal markers. Knockdown of ADK gene
in WJMSCs was confirmed by western blot analysis as well
as qRT-PCR after transduction using the most efficient anti-
ADK miR-shRNA lentiviral vector.
Materials and Methods
In this experimental study, human U-251 MG cell line
(Sigma-Aldrich, USA) was cultured with Dulbecco’s
Modified Eagle’s Medium (DMEM, Gibco-BRL, Japan)
and 10% fetal bovine serum (FBS, Gibco, USA). This cell
line highly expresses ADK gene. The third passage of these
cells was used for screening the anti-ADK miR-shRNAs to
knockdown ADK gene. All the experiments including animal
works were approved by TUMS Ethics committee No. 9301-
87-25045-109011 and were performed based on the
Lentiviral constructs for the expression of anti-ADK
The eight different pre-miRNAsequences and a randomizedscrambled control (SC) sequence were purchased (GE
Healthcare). All miR-shRNA cassettes were cloned into
the pGIPZ lentiviral vector, which contained a TurboGFPgreen fluorescence protein (tGFP) as a reporter gene, internalribosome entry site (IRES) and a puromycin resistance gene;
thus, it allowed co-expression of the respective miR-shRNAwith tGFP and selection of stably transduced cells withpuromycin. Expression of tGFP, puromycin and miR-shRNAs
were under Cytomegalovirus (CMV) constant promoter. All
genes were expressed as a single mRNA. At first, mRNA wasprocessed in nuclear for producing premature miR-shRNAand bicistronic GFP-puromycin mRNA. pGIPZ lentiviralexpression vector harbored internal long terminal repeats(LTRs) zeocin selection marker for selection of correct intact
vector during bacterial propagation.
Production of recombinant pseudo lentiviruses
Recombinant lentiviruses were produced according to the
Prof. Trono lab protocol with some modifications (13, 14).
Briefly for all 8 miR-shRNAs and the positive control vector,
1×106 HEK 293T cells (Invitrogen, USA) were cultured
in a 10-cm2 plate in DMEM medium supplemented with
10% FBS one day prior to transfection. Two hours before
transfection, the medium was replaced with fresh medium.
The transfection mixture contained 21 µg of pGIPZ-miR-
sh/SC, 15 µg of pCMV-dR8.2, 10.5 µg of pMD2, 33 µl of
TE 1X, 105 µl of 2.5 M CaCl2, and 1064 µl of 2X HEPES-
buffered saline (HeBS) finally reaching a volume of 2100 µl
using buffered water.
All the components were mixed, and HeBS 2X was addedwhen the solution was being vortexed vigorously. The
final volume of transfection mixture used for each 10-cm2
plate, was 2100 µl. HEK 293T cells were incubated withtransfection solution in 37°C for 14 hours. The transfection
medium was replaced with fresh medium 14 hours aftertransfection and cells were assayed for GFP expression usinga fluorescent microscope (LaboMed, USA). GFP expressionindicated transfection efficiency. To estimate the transfectionrate, five fields were randomly observed under the fluorescentmicroscope. Supernatant of cells containing recombinantviruses was collected at three time points (24, 48 and 72 hoursafter transfection) after 14 hours post-transfection. Next,
collected supernatants were centrifuged at 180 g and filteredthrough a 0.25 µm filter before concentration and titration.
For concentration of the supernatants, we used PEG 6000 and9000 g centrifuge. The lentiviral titers from the 9 different miR-shRNA constructs due to tGFP gene in our vectors weredetermined using flow cytometry method. The determinationof the lentiviral titer allowed us to estimate the multiplicity ofinfection (MOI) and thus to reduce the infectious activity of
the viral stocks.
Determination of lentivirus titration
Since the stocks of vector carry GFP transgene that can beeasily monitored by flow cytometer, we used this method fortitration of lentivectors. We used Prof. Trono lab protocol forlentivirus titration (15). For this purpose, 293T cells werecultured in DMEM medium supplemented with 10% FBS in a12-well plate with 1×105 cells in each well. Then, the medium
was removed and cells were transduced in 500 µl of fresh
DMEM with serial dilutions of the vector that correspondedto the final amount of 1, 10-1, 10-2, 10-3, 10-4 and 10-5 µl of the
vector. After 24 hours, the medium was removed and one ml
of fresh medium was added to each well. Then, 72 hours after
transduction, cells were processed for
Fluorescence ActivatedCell Sorting (FACS) analysis.
The following formula wasused for calculating titer
by flow cytometry:
"Titer HEK 293T transducing U/ml=[Number of target
cells(counted on day 1)×(% of GFP-positive cells/100)]/volume
of supernatant (ml)"
Ideally, the cells should present 2-20% GFP+ to confirm
that cells that received multiple copies of the virus, were
not counted. The flow cytometer apparatus did not have
enough sensitivity for determination of GFP+ <1%.
Genetic engineering of U-251 MG cell line
After three passages, U-251 MG was cultured in six-wellcell culture plates at a density of 2×105 cell /well /2 ml. Beforeadding fresh recombinant viruses, U-251 MG cell line waswashed with PBS. Recombinant viruses in culture medium
with an MOI of 5 were used for transduction of cells in each
well. To increase the rate of transduction, the spinfectionmethod was used. After infection, the plates were incubated at 37°C and the medium was changed 24 hours later. Transducedcells were assayed for GFP expression with a fluorescentmicroscope (LaboMed, USA), 72 hours after transduction.
GFP expression indirectly indicated the expression of miRshRNA
and transduction efficiency. To select stably transducedcells, from day 4, the medium was replaced with fresh mediumcontaining 2 µg/ml puromycin for 5-7 days.
Quantitative analysis of ADK knockdown by real-time
analysis in U-251 MG cells
A QuantiFast SYBR Green RT-PCR Kit (Qiagen, USA)
was employed to determine and monitor ADK expression afterknockdown of this gene by recombinant lentiviral constructsthat express anti-ADK miR-shRNAs. After puromycinselection, 2×106 cells from stable U-251 MG cell line was
used for RNA extraction and cDNA synthesis (Qiagen kit,
USA). According to the manufacturer protocols, cDNA wasused for standard real-time PCR. Specific primer pairs wereused for ADK and TBP (TATA Sequence-Binding Protein).
The expression of ADK mRNA was evaluated in the lentiviralengineered
U-251 MG cells. For improving the reliability ofrelative RT-PCR, TBP was used as a reference gene.
Isolation and expansion of human Wharton’s jelly
mesenchymal stem cells
Human Wharton’s jelly tissues were obtained from
newborn umbilical cord in accordance with bioethics
agreement following obtaining the consent from its mother
at Taleghani Hospital, Tehran, Iran. Mucoid connective tissueor Wharton’s jelly were separated from blood vessels (twoarteries and one vein of the umbilical cord) then, washedtwice with PBS containing penicillin, streptomycin andamphotericin. The cord was rinsed with PBS and isolatedfrom amniotic membrane. After separation of the matrixfrom cord vessels, the jelly matrix was cut into small piecesand transferred into culture dishes supplemented with DME/
F12 medium, 10% FBS and antibiotics. Two weeks later,
the tissues were discarded and the isolated growing cells(WJMSCs) were fed with the same medium. The cells weregrown to 60% confluence and passaged by trypsinization.
The third passage of WJMSCs was used for characterization.
A number of 1×105 cultured WJMSCs were washed, fixed
and incubated for 15 minutes at 4°C with a 1:9 dilution of
normal goat serum in phosphate buffered saline (PBS, Sigma-
Aldrich, USA). Then, cells were incubated for 1 hour withFITC-conjugated antibodies (CD44 and CD45) for labelling.
Cells washed with 2% FBS in PBS were used for analyzingwith FACSCalibur apparatus (Becton Dickenson, USA). Thecontrol population was stained with isotype-matched antibodies(FITC-conjugated and PE-conjugated mouse IgG monoclonalisotype standards) and confirmed by positive fluorescence ofthe limbal samples. For each sample, at least 1×104 events were
recorded and analyzed by WinMDI software (USA).
Quantitative analysis of ADK knockdown by selected
anti-ADK miR-shRNA in Wharton’s jelly mesenchymal
A QuantiFast SYBR Green RT-PCR Kit (Qiagen, Germany) was employed to monitor knockdown of ADK after
transduction of WJMSCs by the most efficient recombinant
lentiviral construct that expressed anti-ADK miR-shRNA.
After puromycin selection with 2 µg/ml for 5-7 days, 1×106
cells from WJMSCs were used for RNAextraction and cDNA
synthesis (Qiagen, Gemany). In the second step, cDNA was
used for standard real-time PCR. Specific primer pairs were
used for ADK and TBP. The expression of ADK mRNA was
evaluated in the lentiviral-engineered WJMSCs cells.
Preparation of total protein extracts
Stable transduced umbilical cord mesenchymal stem cells
(WJMSCs) were lysed using ReadyPrep™ mammalian
cell lysis reagent (Bio-Rad) comprising complete protease
inhibitor cocktail. Cellular proteins were prepared according
to manufacturer’s protein extraction protocol. The samples
were stored at -80°C until western blot analysis. The protein
content of sample lysate was measured using Bradford’s
Western blot analysis
An equal amount of proteins was loaded on 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE)
and transferred to polyvinylidene fluoride membranes.
Blots were blocked using non-fat dry milk for 1 hour. After
blocking, membranes were probed with ADK primary
antibody (Santa Cruz, 1/1000). Then anti-rabbit IgG antibody
conjugated with horseradish peroxidase (Santa Cruz, 1/8000)
was used for 1 hour at room temperature. Subsequently, the
blots were reprobed with a ß-actin antibody (1:2000, Abcam,
ab8227, USA). Then, the blots were treated with Electro
Chemi Luminescence reagents (Amersham Biosciences,
UK). For quantification of protein intensities, western blot
bands were visualized on radiograph film and evaluated by
Image J software. Human umbilical cord tissue was used in
accordance with the Declarations of Tehran University of
Medical Science and Stem Cell Research Center Committee
after obtaining written informed consent from the mother.
Transfection efficiency of lentiviral anti-ADK miRshRNAs
vectors in packaging 293T cell line
All of nine transfer vectors (eight anti-ADK miRshRNAs
and scrambled control) with helper vectors
(pCMV-dR8.2 and pMD2) were separately co-transfected
into HEK293T cell line using CaCl2 protocol. The
transfection efficiency, as evaluated based on GFP marker
under the fluorescent microscope, was more than 90-95%
for eight anti-ADK miR-shRNAs (Fig .1,) and scrambled
Titration of lentivirus by FACS
The titer of viral particles was approximately 1.5-2×107
U/ml. Serial dilutions of the vector that corresponded to
the final amount of 10-3-10-4 µl of the vector, were used
for calculating the titration because the GFP+ cells in other serial dilutions were more than 20% or under 1%.
Transduction of U-251 MG cell line
As U-251 MG cell line highly expresses ADK gene,
we chose it for screening anti-ADK miR-shRNAs
(Fig .2,). Nine recombinant pseudo lentiviruses (eight
anti-ADK miR-shRNAs and scrambled control) were separately used for transduction of U-251 MG
cell line. GFP reporter gene showed high efficiency
of transduction and expression of anti-ADK miRshRNAs
and scrambled pseudo lentiviruses in U-251
MG cells (Fig .3,). Selection of GFP+ cells, 72 hours
after transduction, was done by using puromycin (2
µg/ml) (Fig .4,).
- Transfection efficiency of lentiviral anti-ADK miR-shRNAs vectors in packaging 293T cell line. A-H. HEK 293T cells were transfected with anti-ADK miRshRNAs
and I. Scrambled control and with high efficiency (90-95%). The rate of GFP expression evaluated undera fluorescent, microscope confirmed the
high efficiency of transfection and expression of anti-ADK miR-shRNAs.
- Morphological characteristics of U-251 MG cells. A. These cells are shown under a phase-contrast microscope with low confluence and B. High
- Lentiviral Transduction efficiency in U-251 MG cell line. High rate of tGFP expression was seen under a fluorescent microscope 72 hours after
transduction with anti-ADK miR-shRNAs lentiviral vectors. A-H. Transduction with sh1-sh8 and I. Transduction of cells with scrambled control.
- Antibiotic selection of transduced U-251 MG cell line. The stable cell line was produced by puromycin selection after 7 days. A, B. Depict the second
and third day after using puromycin (2 µg/ml), C and D. Show the 5th and 7th day of selection.
Screening of anti-ADK miR-shRNAs in U-251 MG
cells by semi-quantitative real-time polymerase chain
For selection of the most efficient anti-ADK miRshRNAs
for ADK knockdown, quantitative real-time
PCR was employed. Expression of ADK gene in each of
target groups (U-251 MG cell line transduced with 8 anti-ADK miR-shRNAs) was measured in proportion to control
group (U-251 MG cell line transduced with scrambled
control). For improving the reliability of relative RT-PCR,
TBP was used as a reference gene.Analysis by quantitative
real-time PCR was done by REST2009 software (Fig .5A,).
Data showed that anti-ADK miR-shRNAs, except for sh8,
could knockdown ADK more than 60% and sh4 and sh7
were the most efficient anti-ADK miR-shRNAs with 86
and 95% knockdown of ADK, respectively (Fig .5B,).
Morphological characteristics and mesenchymal stem
cell markers expression of human Wharton’s jelly cells
Umbilical cord matrix tissue was cultured by an explant
method. Afew days later, WJMSCs migrated away from tissues.
After two weeks, fibroblast-like cells appeared in culture dishes.
The cells grew fast and rapidly covered the surface. Flow
cytometric analyses indicated that the cultured WJMSCs do not
express hematopoietic marker CD45; however, they expressed
mesenchymal stem cell marker CD44 (Fig .6A,).
Genetic engineering of WJMSCs by anti-ADK miR-sh7
Analysis of real-time data showed that Anti-ADK miRsh7
is the most efficient (up 95%) anti-ADK miR-shRNA
for knockdown of this gene. For this reason, we used
pseudo lentiviruses of this miR-shRNA for transduction
of WJMSCs. The high efficiency of transduction was
seen under the fluorescent microscope. After selection
with puromycin (1.5 µg/ml) in the culture medium
(Fig .6B,), semi-quantitative real-time PCR was employed
for measuring ADK expression at mRNA level. Data also
showed knockdown of ADK gene in WJMSCs up to 95%
as well as down-regulation in the U-251 MG cell line.
Confirm of ADK knockdown in WJMSCs by western
After selection of transduced WJMSCs using anti-
ADK miR-sh7 lentiviral vector, cell lysates were used for
performing western blot. Cell lysates from transduced
WJMSCs with scrambled control viruses, cell lysates from
WJMSCs and HepG2 as a control were also employed.
To quantify and normalizing of ADK immunoreactivity,
ß-actin antibody were used. Analysis of data did not show
any reduction of ADK in WJMSCs, WJMSCs-miR-shSC,
and HepG2; however, down-regulation of ADK was
observed after transducing cells with the human specific
anti-ADK miR-sh7 (Fig .6C,).
- Screening of anti-ADK miR-shRNAs in U-251 MG cells by semiquantitative real-time polymerase chain reaction. A. The expression of ADK mRNA was
evaluated in the lentiviral-engineered U-251 MG cells by REST 2009 software. For improving the reliability of real-time polymerase chain reaction (RT-PCR),
TBP was used as a reference gene and B. Percent of ADK knockdown obtained by anti-ADK miR-shRNAs. Data showed that anti-ADK miR-shRNAs (except
for sh8) could knock down ADK more than 60% and sh4 and sh7 were the most efficient with 86 and 95% ADK knockdown, respectively.
- Knock down of ADK in human Wharton’s jelly cells. A. Morphological characteristics and mesenchymal stem cell markers expression of human
Wharton’s jelly cells. WJMSCs showed fibroblast-like phenotype under a phase-contrast microscope (a, b). Flow cytometric analyses indicated that the
cultured WJMSCs significantly express mesenchymal stem cell marker CD44 (c). These cells were almost negative for hematopoietic marker CD45 (d),
B. Genetic engineering of WJMSCs by anti-ADK miR-sh7. WJMSCs observed under phase-contrast microscope (b). Stable transduced WJMSCs with anti-
ADK miR-sh7 under fluorescent microscope (a). Puromycinselected WJMSCs transduced with anti-ADK miR-shSC (1.5 µg/ml) (c), and C. Western blot
analysis confirmed ADK knockdown in WJMSCs. Western blot analysis was performed on cell lysates from WJMSCs, WJMSCmiR-sh7, WJMSC-miR-shSC,
and HepG2. ADK staining (top) and ß-actin staining (bottom). WJMSCmiR-sh7 showed the most marked reduction of ADK expression at protein level.
In adult brain, ADK is a key enzyme in astrocytes
that regulates adenosine level by converting
adenosine to 5'-adenosine monophosphate (AMP)
and subsequently generates ATP (17). When trauma
or epilepsy happens, adenosine, as a modulator of
inflammation increases and induces proliferation of
astrocytes via A2ARs, leading to astrocyte activation
and ADK overexpression. ADK expression results in
adenosine decline. In an acute injury or intractable
epilepsy, proliferation of astrocytes (i.e. astrogliosis),
is the pathological hallmark of the disease and at the
molecular level, adenosine deficiency is the consequence
of ADK overexpression induced by over-activation of
astrocytes (18). So, ADK down-regulation and increasing
adenosine release are among the strategies used for the
treatment of some neurological disorders like epilepsy.
Although inhibition of ADK with chemical and small
molecule drugs can suppress seizures but systemic long
term use of this therapeutic approach may increase the
risk of side effects like brain hemorrhage (19). Post
transcriptional gene silencing is a molecular tool that
has opened new windows in recent years. Knockdown
of ADK by viral vectors that express anti-ADK miRNAs
can increase adenosine and results in suppressing seizures
via a paracrine effect (20). Epilepsy is a chronic disease
so permanent release of adenosine is needed. In this
therapeutic approach, the combination of cell therapy
with gene therapy is the key to the riddle.
Cell therapy provides long-lasting focal delivery of
antiepileptic molecules like adenosine and prevents
systemic pharmacological side effects. Cell therapy also
has the potential to restore cells that are destroyed by
neurological conditions, especially in refractory epilepsy.
In cell therapy approaches, safer and more controllable
gene delivery of inhibitory neurotransmitters or other
therapeutic compounds is possible. Chemical mutations
for engineering cells to release therapeutic agents like
adenosine, were induced in 2001. In that study, fibroblasts
that release adenosine were generated by induction a
deficiency in ADK and adenosine deaminase (ADA) gene
(21). Transplantation of these cells implicated nearly
complete protection against seizures up to 24 days but
lost their antiepileptic effects after that as the viability of
grafting fibroblast cells decreased. Stem cells are more
attractive sources to be used for solving this problem.
Stem cells in addition to having higher viability, can
be differentiated into multi-lineage cells such as neuroprogenitor
cells to integrate into damaged neural networks
that are created during epilepsy or other neurological
disorders, they can slow seizure development and they
have anti-epileptogenesis effects by releasing inhibitory
Different sources of stem cells (e.g. adult stem cells,
fetal stem cells, embryonic stem cells and iPS cells)
have been used to evaluate treatment of animal model of
epilepsy (22-25). Therapeutic application of ADK gene
knockout in mouse embryonic stem cells and ADK gene
knockdown in bone marrow mesenchymal stem cells
have been investigated by Li et al. (26). These studies
implicated reduction of seizures in a mouse model after
transplantation of hMSCs and the results were promising
to be used for treatment of epilepsy. However, ethical
concerns about embryonic stem cells and technical issues
related to bone marrow mesenchymal stem cells, have
made WJMSCs as an attractive source for cell therapy
and gene delivery systems.
WJMSCs could be obtained from the human umbilical
cord that is normally discarded after the birth. These cells
are actually a by-product of childbirth. WJMSCs have the
potential to differentiate into ectodermal and mesodermal
derived cells (27, 28). In addition to the easy access to
WJMSCs and their potential of differentiation, these
cells have anti-inflammatory and immunomodulatory
properties. Some studies have shown that xenograft
transplantation of WJMSCs are not rejected in an animal
model of human disease without immune-suppression
(29). Studies suggested that production of cytokines
and growth factors by WJMSCs, results in their anti-
inflammatory and neuro-protective activities (30). In
the present study, we improved therapeutic benefit of
WJMSCs by knocking down ADK gene and producing
adenosine releasing WJMSCs to suppress seizures in
families with status epilepticus and we stored these
cells for possible future applications. WJMSCs are an
attractive alternative source for cell-based gene therapy
as they are i. Easily accessible stem cell sources without
ethical and technical concerns, ii. Accessible in unlimited
numbers and, iii. Amenable to genetic modification, and
iv. They possess therapeutic potential. Based on these
considerations, we selected these cells for engineering
with lentiviral anti-ADK miR-shRNA expressing systems.
miRNA-mediated downregulation of ADK was
previously reported by Li et al. (26). They used five
anti-ADK miRNA expression cassettes and showed
downregulation (up to 85%) of ADK in human MSCs
(hMSCs). In the present study, we considered human
miR 30 for designing stem-loop and flanking sequences
of anti-ADK miR-shRNAs to increase the efficiency of
RNAi-based gene therapy. We used eight anti-ADK miRshRNAs
that target ADK at different sense and antisense
sequences but at common stem-loop and flanking
By developing this system, we succeeded to
diminish ADK expression up to 95% in astrocytoma cell
line and in WJMSCs. ADK mRNA has 4 variants (isoform
a, b, c and d). These eight anti-ADK miR-shRNAs can
target all transcript variants of ADK. Antisense of sh6 and
sh8 target ADK mRNA at 3´ outside of coding sequence
(CDs) while other shRNAs (sh1, sh2, sh3, sh4, sh5 and
sh7) target ADK mRNA at 3´ including CDs. Using
human miR30 for designing shRNAs caused 60-95%
knockdown of ADK (except for sh8 which resulted in
23% downregulation). Anti-ADK miR-sh4 and Anti-
ADK miR-sh7 were the most efficient cassettes with up
to 86 and 95% downregulation of ADK, respectively.
These results show that the usage of miR-based shRNAs
is an efficient method in knocking down ADK. Based on
these results, in our future research, we work on using
engineered WJMSC for the survey of the therapeutic
potential of ADK down-regulation and adenosine delivery
in an animal model.
Results show that lentiviral system expressing anti-
ADK miR-shRNAs that was used in this study is a
promising tool for ADK knockdown in all transcript
variants. Manipulated WJMSCs by Adeno, AAV, non-
integrated lentiviral, CRISPR/Cas9 and RNA transfer
instead of lentivirus, might be a suitable source in cell-
based and ex vivo gene therapy of epilepsy and could be
evaluated for ADK knockdown, in vivo.
This study was performed as a part of a thesis for
obtaining Ph.D. in Molecular Medicine at School of
Advanced Medical Technologies, Tehran university of
Medical Sciences (Tehran, Iran) and supported by a grant (NO. 25045) from Tehran University. In addition,
we would like to thank Stem Cell Technology Research
Center (Tehran, Iran), Shefa Neuroscience Research
Center (Tehran, Iran) and Iranian Institute of Cell and
Gene Therapy (Tehran, Iran) for their support. None of
the authors has any conflict of interest to disclose.
M.H.Gh.; Supervised the project and conceived the
study and were in charge of overall direction and planning.
A.F.; Contributed to conception, design and revising the
manuscript. H.E.; Worked out almost all of the technical
details and wrote the manuscript. M.S.; Helped in the
primary design of the experiments. A.A.; Helped in
animal surgeries and stem cell isolation. F.K.; Analysed
the data of EEG. Sh.B.A.; Contributed to the western blot
experiment. All authors read and approved the final study.
Astrogliosis in epilepsy leads to overexpression of adenosine kinase, resulting in seizure aggravation.
Theo las P,
Adenosine kinase as a target for therapeutic antisense strategies in epilepsy.
Endogenously released adenosine regulates excitability in the in vitro hippocampus.
Adenosine dysfunction in epilepsy.
Suppression of kindled seizures by paracrine adenosine release from stem cell-derived brain implants.
Engineered adenosine-releasing cells for epilepsy therapy: human mesenchymal stem cells and human embryonic stem cells.
Progress and prospects in stem cell therapy.
Acta Pharmacol Sin.
Overview of drugs used for epilepsy and seizures: etiology, diagnosis, and treatment.
Wharton’s jelly-derived mesenchymal stem cells: phenotypic characterization and optimizing their therapeutic potential for clinical applications.
Int J Mol Sci.
Discarded Wharton jelly of the human umbilical cord: a viable source for mesenchymal stromal cells.
Mesenchymal stem cells derived from Wharton’s Jelly of the umbilical cord: biological properties and emerging clinical applications.
Curr Stem Cell Res Ther.
Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord.
Evaluation of AD-MSC (adipose-derived mesenchymal stem cells) as a vehicle for IFN-β delivery in experimental autoimmune encephalomyelitis.
A new approach in gene therapy of glioblastoma multiforme: human olfactory ensheathing cells as a novel carrier for suicide gene delivery.
Trono D. Production and titration of lentiviral vectors. :
Curr Protoc Neurosci;
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Adenosine kinase is a target for the prediction and prevention of epileptogenesis in mice.
J Clin Invest.
How does adenosine control neuronal dysfunction and neurodegeneration?.
Analgesic and anti-in ammatory effects of A-286501, a novel orally active adenosine kinase inhibitor.
Lentiviral RNAi-induced downregulation of adenosine kinase in human mesenchymal stem cell grafts: a novel perspective for seizure control.
Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy.
Proc Natl Acad Sci USA.
Adenosine and epilepsy: from therapeutic rationale to new therapeutic strategies.
Genetically engineered GABA-producing cells demonstrate anticonvulsant effects and long-term transgene expression when transplanted into the central piriform cortex of rats.
Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy.
Human mesenchymal stem cell grafts engineered to release adenosine reduce chronic seizures in a mouse model of CA3-selective epileptogenesis.
Identi cation of cord blood-derived mesenchymal stem/ stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression pro les.
Stem Cells Dev.
Optimization and scale-up of Wharton’s jelly-derived mesenchymal stem cells for clinical applications.
Stem Cell Res.
Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells: role of B7-H1 and IDO.
Immunol Cell Biol.
Verga Falzacappa L,
Wharton’s jelly derived mesenchymal stromal cells: Biological properties, induction of neuronal phenotype and current applications in neurodegeneration research.