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Transplantation of retinoic acid treated murine embryonic stem cells & behavioural deficit in Parkinsonian rats.

Parkinson's disease (PD) affects more than 2 per cent of the elderly population over the age of 65 (1). The disease is characterized by a progressive degeneration of the dopaminergic neurons in the substantia nigra pars compacta, which results in dopamine deficiency of the striatal region (2). Many causes have been suggested for the degeneration of the dopaminergic (DA) neurons, including oxidative stress, mitochondrial dysfunction, excitotoxicity and genetic factors (1,3). An interaction between predisposing genetic factors and environmental factors, such as pesticides and viruses, has also been suggested as a cause for the disease (4).

One of the approaches for treating PD is cell replacement using human embryonic tissue grafts (3), such as foetal-derived dopamin-producing neurons (3). For this purpose, neurons from 6 to 10 foetuses, aged between 6-10 wk, need to be harvested to treat a single patient (1,3). Other cell types have been used with variable results (3). Recently, pluripotent embryonic stem cells (ESCs) have been tried for cell replacement therapy. ESCs can adapt themselves into the host environment following their transplantation (7,8). Pluripotent human embryonic stem cells (hESCs) (3,4), which potentially proliferate indefinitely in culture, may supply unlimited numbers of DA neurons for transplantation. The potential of mouse ES cells to generate functional DA neurons and to correct behavioural deficits after engraftment into Parkinsonian rats has been demonstrated (7,8). When low numbers of undifferentiated mouse ES cells were implanted into the rat dopamin-depleted striatum, the cells proliferated and differentiated into functional DA neurons that reduced Parkinsonism. However, lethal teratomas developed in 20 per cent of the animals (7). In an alternative approach, highly enriched populations of midbrain neural precursors were developed in vitro from mouse ES cells and then implanted into Parkinsonian rats. The engrafted cells led to the recovery from Parkinsonism, and teratoma tumour formation was not observed (9).

Evidence for an in vitro differentiation of ES cells into the neuronal lineage has been reported (10,11). Bain et al (12) showed that retinoic acid (RA) induces the differentiation of ES cells into neuronal-like cells with a high efficiency. Strubing et al (13) reported that ES cells differentiate in vitro into different subsets of neurons forming functional networks of synaptically coupled cells. These cells revealed electrophysiological and biochemical properties almost indistinguishable from primary cultures of embryonic central nervous system (CNS) neurons (13). Ion channel expression in BLC 6-derived neurons followed a similar temporal pattern as found in certain embryonal neurons (14). Many laboratories have reported that embryonic stem (ES) cells can efficiently differentiate into neural progenitor (NP) cells and then into dopaminergic neurons via optimal culture conditions and/or genetic manipulation (15-18). Thus, multipotent NP cells can be efficiently derived from ES cells, because ES cell-derived NPs (ES NP) do not go through the same maturation process during in vitro expansion as foetal brain-derived NPs do (19). Chung et al (20) hypothesized that ES NPs maintain their potential to differentiate to the neuronal and/or DA fate during mitogenic expansion. They could differentiate ES cells into neural progenitor or nestin positive cells by retinoic acid. They reported that ES NPs maintain the potential to generate Tuj1neurons as well as functional DA neurons after extensive in vitro expansion (20).

The purpose of this study was to evaluate the therapeutic benefits of using murine ES NPs for cell therapy in Parkinsonian rats by evaluating their ability to improve impaired movement.

Material & Methods

The animal model and experimental groups: Adult female Sprague-Dawley rats (n=16), weighing between 250-300 g, obtained from the Pasteur Institute of Tehran, Iran, and transferred to animal facility of Tarbiat Modarres University, Tehran. They were maintained for 15 days, 3 to 4 per cage, in a temperature controlled colony room under standard light-dark cycle with free access to food and water. The study protocol was approved by Research Council of Tarbiat Modares University (Tehran, Iran). The animals were divided into two groups: a control group (5 animals) grafted with culture medium, and an experimental group (11 animals) grafted with ES cells treated with RA. In experimental group, six animals were used for ultrastructural and histological evaluations (these animal were behaviourally tested before transplantation to make sure of validity of the generated model) and five animals were used for behavioural testing (before and 8 wk after transplantation) same as of control group (Fig.1). The ESCs in experimental group were cultured without leukaemia inhibitory factor (LIF), for the formation of embryoid bodies (EBs). The EBs were exposed to retinoic acid, trypsinized and transplanted into left striatum of animals in experimental group.

All animals received unilateral stereotaxic injections of 6-hydroxy dopamine (6-OHDA) (Sigma, USA) into the corpus striatum. For this purpose, they were anaesthetized by intraperitoneal injection of 60 mg/kg ketamine and 3 mg/kg xylazine mixture. The anaesthetized animals were injected unilaterally in the intrastriatal region with 6-OHDA using a 10 (J Hamilton microsyringe (21). The injections were carried out using stereotaxic apparatus (Stoelting, Wood Dale, USA). The co-ordinates of the injection site were L -3 mm, AP +9.2 mm, V +4.5 mm from the center of the interaural line (Paxinos and Watson (22)). Upon completion of the injection, the needle was left in place for an additional 5 min and then withdrawn at a rate of 1 mm/min. Each animal received a single injection of 5 [micro]l 0.9 per cent saline that contained 2.5 mg/ml of 6-OHDA and 0.2 per cent (w/v) ascorbic acid (Sigma, USA) at a rate of 1 [micro]l/min. The flow rate was controlled by a microsyringe pump (EICOM EP-60, Japan). The animals were housed one per cage under conditions mentioned above until the end of experiment.


Behavioural test: The behavioural test was done according to the protocol by Zheng et al (23). The rotational behaviour of the animals in both groups was evaluated twice: the first time following an injection of 2.5 mg/ kg (ip) apomorphine hydrochloride (Sigma, USA), two wk after surgery and before cell transplantation. The second evaluation was performed at eighth week after cell transplantation. In this test, the animals were allowed to habituate for 10 min in a cylindrical container with 33 cm diameter and 35 cm height. After the injection of the apomorphine, full contralateral rotations were counted for 60 min in a quiet isolated room (22,23).

Propagation and preparation of ES cells for transplantation: The undifferentiated murine ESCs were maintained on gelatin-coated dishes and cultured in Dulbecco modified essential medium (DMEM, Gibco, Paisley, UK) supplemented with: 2 mM glutamine (Gibco, Paisley, UK), 0.001 per cent [beta]-mercaptoethanol (Sigma Aldrich, St. Louis, USA), 1X non-essential amino acids (Gibco, Paisley, UK), 15 per cent foetal bovine serum (Gibco, Paisley, UK), and a leukaemia inhibitory factor (10 ng/ml) (Sigma Aldrich, USA), and incubated under 5 per cent C[O.sub.2] (24) differentiation of mESCs into nestin positive cultures was accomplished by the modified protocol of Bain et al (12). The CCE cells were trypsinized (0.25% trypsin0.04% EDTA, Gibco, UK), resuspended and seeded at 1x[10.sup.6] cells/ml in 100-mm bacteriological petri dishes. The DMEM media were supplemented with 10 per cent FBS, and no addition of LIF and [beta]-mercaptoethanol. The cultures were kept for 48 h till the formation of EBs. RA (12) (Sigma Aldrich, USA) with the concentration of 5 x [10.sup.-7] M was added for another 2 days, and then the aggregated cells were transferred to a 15 ml sterile culture tube. After settling, the cells were centrifuged at 200 g for 5 min, collected and rinsed once in calcium and magnesium-free Dulbecco's PBS (Gibco, Paisley, UK). After rinsing, the PBS was removed and 1.5 ml of trypsin-EDTA solution was added. The cells were incubated for 5 min at 37[degrees]C and then triturated with fire-polished Pasteur pipettes with decreasing aperture size in order to fully dissociate the cells. Upon completion of trituration, the cells were centrifuged at 200 g for 5 min. The trypsin-containing supernatant was replaced with 200 [micro]l of culture medium. The viability and concentration of the ESCs were determined by using a haemocytometer after staining with trypan blue.

Transplantationprocedures: 6-OHDA-lesionedanimals were anaesthetized with ketamine-xylazine (60 mg/kg and 3 mg/kg, respectively, im) and were placed on a stereotaxic frame (Stoelting, USA). Two weeks after surgery the apomorphine test was performed and then each experimental model received an injection of 5 [micro]l of ES cell suspension (Experimental group) or medium (Control group) into the left striatum (L-3 mm, AP +9.2 mm, V +4.5 mm from the center of the interaural line, as described elswhere (22), using a 10 [micro]l Hamilton microsyringe. A 5-min waiting period allowed the ES cells to settle before the needle being removed. Animals received 100000 ES cells per 5 [micro]l. Eleven rats received ES cell injections, and five rats received sham surgery by injection of medium. The medium was free of RA, LIF, [beta]-mercaptoethanol and FBS.

Bromodeoxyuridine (BrdU) labelling and detection: The ESCs prepared for replacement therapy were labelled with bromodeoxyuridine (BrdU) (Sigma, UK) for 14 h by adding 10 [micro]/ml BrdU to the culture medium. The incorporation of BrdU in a sample of labelled cells was evaluated by immunocytochemistry. Briefly, the cells were cultured on coverslips and fixed with 70 per cent ethanol at 2-8[degrees]C for at least 30 min. The nuclear DNA was denatured using 2 N HCl added to the culture for 60 min at 37[degrees]C. The cultures were neutralized by immersing the slides in 0.1 M borate buffer, pH 8.5 twice for 10 min. The slides were then washed three times with PBS, with each wash lasting 10 min. The cells were immunolabelled with 100 [micro]l antibody against anti-bromodeoxyuridine (Chemicon,Temecula, CA, USA) in a humidified chamber for 120 min at 37 [degrees]C. The slides were then washed three times with PBS solution, with each wash lasting 10 min. Peroxidase activity in the cells was detected by incubating the slides for 30 min in a solution that contained 1 mg/ ml diaminobenzidine tetrahydrochloride DAB (Dako, Carpinteria, CA, USA) and 0.02 per cent [H.sub.2][O.sub.2] in PBS, pH 7.4. The reaction was stopped by washing the slides in PBS, pH 7.4. The cells were then dehydrated in ethanol and cleared in xylene and mounted.

For detecting BrdU-labelled cells in vivo, the animals were anaesthetized and perfused intracardially with 100 ml of heparin saline (0.1% heparin in 0.9% saline), and then with 200 ml of a fixative solution containing 4 per cent paraformaldehyde (Sigma-Aldrich, USA) in 0.1 M phosphate buffer, pH 7.4. The brains were then removed and fixed in 4 per cent paraformaldehyde in phosphate buffer for 2 h, after which they were placed in 20 per cent sucrose until they sank. Coronal sections through the entire transplantation site were cut at 30 (m using a cryostat (Leica CM1850, Germany) and stored in phosphate-buffered saline pH 7.4. For the immunocytochemical detection of BrdU-labelled nuclei, cellular DNA was first pretreated in 50 per cent formamide-2xSSC buffer that contained 0.3 M NaCl, 0.03 M sodium citrate at 65 [degrees]C for 2 h and then incubated in 2 N HCl for 30 min at 37[degrees]C. Upon completion of the incubation, the sections were rinsed for 10 min in 0.1 M borate buffer (pH 8.5) in order to neutralize HCl and then immuonostained with BrdU antibody, as described above. The reaction was terminated by transferring the sections to PBS then mounted on glass slides, dehydrated, and coverslipped.

Immunosuppression: Transplant rejection was prevented by using the immunosupression method recommended by Bjorklund et al (7). The animals were injected subcutaneously with 15 mg/kg cyclosporine A (Sandimmune Neoral, Novartis Pharmaceuticals, UK) each day, starting with a 30 mg/kg injection one day before surgery. Immunosuppression was continued throughout the 8 wk after transplantation.

Histological and immunohistochemical procedures: Four treated animals were sacrificed either 5 or 15 days following surgery for detection of SSEA1 antigen or tyrosine hydroxylase (TH). The animals were anaesthetized and perfused intracardially with 100 ml of heparin saline (0.1% heparin in 0.9% saline) followed by immersion in 200 ml of fixative solution that contained 4 per cent paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed immediately and kept for 24 h in the same solution, then equilibrated in sucrose (20% in PBS at 4[degrees]C) and sectioned at 20-30 (m using cryostat. For the animals killed, 5 and 15 days following cell transplantation, prepared sections were used for immunostaining with monoclonal antibody against tyrosine hydroxylase (Chemicon, Temecula, CA, USA) at 1:200 dilution, detecting SSEA1 antigen using a monoclonal SSEA1 antibody (Developmental Studies Hybridoma Bank, Iowa city at 1:100 dilution) while the alternative sections were stained with 0.1 per cent cresyl violet (Sigma Aldrich, St Louis, MO, USA). For the immunohistochemical study, the sections were rehydrated, blocked using 10 per cent goat serum (Gibco, Paisley, UK), labelled with antibody solution that contained 0.1 per cent BSA-PBS for 24 h at 4[degrees]C, incubated in HRP conjugated goat-antimouse IgG (Serotec, UK Ltd, Cambridgeshire, UK) (at a dilution of 1:100 in 0.1% BSA-PBS for 2 h in DAB, 5 mg/5 ml of PBS and 3% [H.sub.2][O.sub.2]), counterstained with haematoxylin solution (Sigma Aldrich, USA), dehydrated, cleared, and cover slipped. The specificity of the immunolabeling was verified by preparing brain sections in which the step using the primary antibody was omitted. The specificity of the antibody was checked using an undifferentiated CCE cell line.

Ultrastructural Study: In day 15 after transplantation two treated animals were anaesthetized and perfused intracardially with 100 ml of heparin saline (0.1% heparin in 0.9% saline) followed by immersion in 200 ml of fixative solution that contained 4 per cent paraformaldehyde with 0.5 per cent glutaraldehyde (Taab Lab Equipment, Reading, UK), and 0.1 M phosphate buffer at pH 7.4. The brains were removed immediately and immersed in 2.5 per cent glutaraldehyde for 24 h. The brains were then fixed with 1 per cent osmium tetroxide (Taab Lab Equipment, Reading, UK) in 0.1 M phosphate buffer, washed in phosphate buffer, and embedded in resin (Taab Lab Equipment, Reading, UK). Thin 30-50 nm sections of the brains were cut and then stained with uranyl acetate and lead citrate for ultramicroscopy examination, using a transition electron microscopy (EM 900, Ziess microscope, Germany).

Statistical analysis: The data were analyzed statistically using SPSS software (Version 8.0 for windows XP) using Wilcoxon signed rank test.


Cultures of CCE cell line, with a density of 1x [10.sup.5] cells/ml, were trypsinized, dispersed and plated on bacteriological grade petri dishes, and allowed to aggregate in the culture medium containing 10 per cent FBS and without LIF. The cultures were kept for 48 h, and the number of formed EBs was counted. RA (5 x [10.sup.-7] M) was then added for another 2 days, and the aggregated cells were transferred to tissue culture grade plastic dishes (Nunc) coated with 1 per cent gelatin in DMEM supplemented with 10 per cent FBS. Nestinpositive cells were detected in day 5 of experiment in vitro (data not shown). Fig. 2 shows a tissue section, stained with cresyl violet, from the striatal region of a Parkinsonian rat transplanted with RA-treated ESCs in day 15. As it is evident from the morphology of the engrafted cells, some of them have undergone differentiation into neuron-like cells.

To trace the fate of engrafted cells, the cells were labelled with BrdU. Fig. 3A shows the results of immunostaining of the cells in culture with BrdU antibody. In the tissue sections from the striatal region of the animals, transplanted cells labelled with BrdU were seen Fig. 3B. Fig. 4 shows a tissue section from an animal in which the ESCs cells treated with RA were injected and stained immunohistochemically for the presence of SSEA1 and TH positive cells in days 5 and 15 of experiment. There was positive reaction for SSEA1 in day 5 (Fig. 4A) and no SSEA1 immunoreactivity in day 15 (Fig. 4C). There was absence of TH-positive cells in day 5 (Fig. 4B), while the RA-treated ESC cells transplanted into striatal region showed high immunoreactivity to tyrosine hydroxylase staining in day 15 (Fig. 4D). The ultrastructure of the transplanted cells revealed the morphology of neuron-like cells after 15 days of the lesion. Moreover, some of the cells showed an oligodendrocyte-like morphology (Fig. 5A and 5B, respectively).


The rotational response to the apomorphine injection was evaluated eight weeks following injection of the ESCs (Fig. 6). The Parkinsonian rats injected with RA-treated ES cells showed recovery over time (248 [+ or -] 17vs. 208 [+ or -] 15 rotations, P<0.005). On the other hand, there was no significant difference in the behaviour of untreated (control, injected with the medium alone) animals (258 [+ or -] 13 vs. 261 [+ or -] 15 rotations).



Parkinson's disease (PD) is an ideal model to test cell therapy, since the biochemistry of the disease is well documented, and a treatment, like supplementation of dopamine into striatum, is feasible. Dopaminergic neurons, derived from ES cells are a suitable choice for this purpose. Our results showed that RA-treated ES cells can be differentiated into TH-positive cells in transplantation site and relieve apomorphine-induced asymmetric motor behaviour. Interestingly, in the experimental group, some SSEA1 positive cells were detected in day 5, but no positive cells on day 15. This is in accordance with an earlier study (7) in which implanted undifferentiated ES cells were shown to gradually lose expression of early embryonic markers, such as the SSEA1 antigen, but start to express neuron markers such as TH. Similar results were reported by Deacon et al (8) where the number of transplanted mouse ES cells was (1x[10.sup.5] cells). However, no behavioural test was performed by this group to validate the functioning of the transplanted cells (8).


We found neural- and oligodendrocyte-like cells in day 15 after transplantation, where these probably interact to produce and maintain stable myelinated axons. Recent evidence proposes a role for oligodendrocytes in providing trophic support for neurons during development as well as in the mature nervous system. The study of Wilkins et al (25) provided evidence that oligodendrocyte progenitor cells and differentiated oligodendrocytes support neuronal survival by both contact-mediated and soluble mechanisms, and that IGF-1 significantly contributes to this effect. Takeshima et al (26) have shown that oligodendrocyte progenitor cells increase the survival of mesencephalic dopaminergic neurons grown under serum deprivation. When E14, mesencephalic, dopaminergic neurons were co-cultured with established O-2A progenitor cells in a serum-free growth medium, the survival of tyrosine hydroxylase-positive ([TH.sup.+]) neurons increased 23-fold and 668-fold at the 5th and 10th days, respectively, compared with the control cultures plated on poly-L-lysine. Conditioned medium from the O-2A progenitor cultures also decreased the death of [TH.sup.+] neurons. The mitotic inhibitor, cytosine arabinoside (1.0 [micro]M), did not block the protective effect of the O-2A progenitor cells. O-2A progenitor cells produce a potent, soluble factor that mediates the increased survival of dopaminergic neurons in vitro (26).


Cell-based therapy has been widely proposed for the treatment of neurodegenerative diseases (27). Lindvall and Hagell (28) emphasized that one of the major problems in cell replacement therapy is the lack of sufficient amounts of tissue for transplantation. They further, indicated that the ESCs could be useful as an unlimited source of DA neurons. Levy et al (29) mentioned that ESCs are a real hope for replacement therapy in a wide range of diseases, particularly Parkinson's disease, because of their ability to self-renew and to differentiate into DA neurons. Sonntag et al (30) added the advantage that there is an unlimited source of these cells compared with foetal tissues. However, the ethical factor concerning the use of stem cells as well as low efficiency and variability in the clinical outcome remain major concerns for many scientists.


A possible disadvantage of this mode of treatment in clinical practice is that if the degenerative agent persists in the environment, the degeneration may continue to exert its effect on newly differentiated dopaminergic neurons (30). The undifferentiated ES cells that were transplanted in the mouse study gave rise to teratomas in a high percentage of the animals (7). Our cultures of differentiated transplanted cells did not include undifferentiated mESCs, as suggested by the lack of expression of SSEA1 on day 15. In addition, serial haematoxylin & eosin-stained sections covering the entire brain did not reveal teratomas or any other tumour formation in transplanted rats.

Coyne et al (31) transplanted marrow stromal cells (MSCs) double-labelled with BrdU and bis- benzamide (BBZ) into the adult hippocampus or striatum noticed that BrdU labelling was colocalized to host phagocytes, astrocytes, and neurons in both regions. Based on the appearance and morphology of the Brdu-labelled cells under the microscope, we concluded that the positive cells are indeed RA-treated ESCs and not the host cells at the site or margin of implants. However, this conclusion needs further validation to rule out the possible diffusion of BrdU and cross-labelling of the host cells. Using other reporters such as green fluorescent protein (GFP) could provide more precise data in this regards.

In conclusion, while the study outcomes encouraging, additional and more extensive long-term studies are required to determine the safety of ESC-derived neural progeny transplantation and to rule out potential hazards such as tumour formation or the development of unwanted non-neural cells.

Received August 24, 2007


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Reprint requests: Dr Fardin Fathi, Department of Anatomy, KDRC, School of Medical Sciences, Kurdistan University of Medical Sciences P.O. Box: 66177-13446, Sanandaj, Iran e-mail:

Fardin Fathi, Taki Altiraihi *, Seyed Javad Mowla ** & Mansoreh Movahedin *

Kurdistan Center for Cellular & Molecular Research, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, * Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University Tehran & ** Department of Genetics, Faculty of Basic Sciences, Tarbiat Modarres University, Tehran, Iran
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Author:Fathi, Fardin; Altiraihi, Taki; Mowla, Seyed Javad; Movahedin, Mansoreh
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:7IRAN
Date:Apr 1, 2010
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