Chinese Medical Journal 2009;122(20):2449-2454 2449 Original article

Therapeutic effect of human amniotic epithelial cell transplantation into the lateral ventricle of hemiparkinsonian rats

YANG Xin-xin, XUE Shou-ru, DONG Wan-li and Kong Yan

Keywords: Parkinson’s disease; human amniotic epithelial cell; transplantation; brain derived neurotrophic factor

Background Human amniotic epithelial cells (HAECs) are able to secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on . Previous study showed that when human amniotic epithelial cells were transplanted into the striatum of 6-hydroxydopamine (6-OHDA)-induced Parkinson disease rats, the cells could survive and exert functional effects. The purpose of this study was to investigate the survival and the differentiation of human amniotic epithelial cells after being transplanted into the lateral ventricle of Parkinson′s disease (PD) rats, and to investigate the effects of grafts on healing PD in models. Methods The Parkinson’s model was made with stereotactic microinjection of 6-hydroxydopamine (6-OHDA) into the striatum of a rat. The PD models were divided into two groups: the HAECs group and the normal saline (NS) group. Some untreated rats were taken as the control. The rotational asymmetry induced by apomorphine of the HAECs group and the NS group were measured post cell transplantation. The expression of nestin and vimentin in grafts were determined by immunohistology. Ten weeks after transplantation the density of tyrosine hydroxylase positive cells in the substantia nigra of the HAECs group, NS group and the untreated group was determined. The differentiation of grafts was determined by TH immunohistology. High performance liquid chromatography (HPLC) was used to determine monoamine levels in the striatum. Results The rotational asymmetry induced by apomorphine of the HAECs group was ameliorated significantly compared to the NS group two weeks after transplantation (P <0.01). The grafts expressed nestin and vimentin five weeks after transplantation. TH immunohistochemistry indicated that the TH positive cells in the substantia nigra of the HAECs group increased significantly compared to the NS group (P <0.01). Tyrosine hydroxylase (TH) positive cells in the substantia nigra of the HAEC group and the NS group were decreased compared to the untreated group (P <0.01). Dopamine and DOPAC levels in the striatum of the HAECs group increased significantly compared to the NS group (P <0.05). Homovanillic acid (HVA) levels in the striatum of the HAECs group increased significantly compared to the NS group (P <0.01). In addition dopamine, DOPAC, and HVA levels in the striatum and dopamine levels in the cerebrospinal fluid of the HAECs group and the NS group were decreased compared to the untreated group (P <0.05). Conclusions Human amniotic epithelial cells could be used to ameliorate the rotational asymmetry induced by apomorphine of the PD models. This could have been due to the increased content of dopamine and its metabolic products, DOPAC and HVA, in the striatum in the PD models. Chin Med J 2009;122(20):2449-2454

arkinson’s disease (PD) is a neurodegenerative Levodopa can not cure Parkinson’s disease completely. P disorder characterized by a progressive death of So we must find new treatments for PD. nigral dopamine neurons with a resultant loss of striatal dopamine levels.1 When the number of dopaminergic Some studies indicate that tissue transplantation is a neurons in the substantia nigra pars compact is reduced therapeutic modality that can supplement dopamine in the by over 70%, classical clinical symptoms, such as brain using a PD model in rats. The potential donor cells akinesia, rigidity, tremor and postural dysfunction, are for transplantation include neural stem cells, embryo stem 2 cell, mesenchymal stem cells, olfactory ensheathing cells, produced. The pathogenesis of PD is complicated and 7-9 the cause of idiopathic PD is obscure. The major risk retinal pigment epithelial cells, and Schwann cells. But factors for PD include aging, environmental neurotoxins these doners are limiting in source. Human amniotic such as paraquat,3 and genetic defects. Oxidative stress epithelial cells (HAECs) are formed from epilates on the and excitotoxicity are involved in the mechanisms of 8th day after fertilization, and constitute the inner layer of 4,5 PD. Levodopa has been long employed as a dopamine DOI: 10.3760/cma.j.issn.0366-6999.2009.20.010 replacement therapy for Parkinson’s disease, but the Department of Neurology, First Affiliated Hospital of SooChow efficacy of the drug in alleviating motor symptoms in PD University, Suzhou, Jiangsu 215006, China (Yang XX, Xue SR, is gradually attenuated with the progress of the disease. Dong WL and Kong Y) Correspondence to: XUE Shou-ru, Department of Neurology, First Long-term application of levodopa can result in severe Affiliated Hospital of SooChow University, Suzhou, Jiangsu toxicity or debilitating side effects, such as dyskinesias, 215006, China (Tel: 86-512-67780317. Fax: 86-512-65228072. 6 symptomatic fluctuation, and hallucinations. Moreover, Email: [email protected]). 2450 Chin Med J 2009;122(20):2449-2454 the amnion, opening the possibility that they might Switzerland) fitted with a steel cannula following the maintain the plasticity of pregastrulation embryo cells. below coordinate: AP –0.8, ML –1.3, D –4.8. We HAECs lack major histocompatibility complex antigens transplanted 8 µl HAECs for each rat, containing 106 cells. and have a low risk of tissue rejection when transplanted After the completion of the injections, the cannula was into other individuals.10 In addition, they are readily left in situ for 5 minutes before being slowly retracted. In available and may be a useful and noncontroversial the NS group, we injected NS into the lateral ventricle of source of stem cells for cell transplantation and recipient rats in the same manner. regenerative medicine. Behavior test METHODS Apomorphine induced rotational behavior13 was measured for 30 minutes following intraperitoneal Human amniotic epithelial cell cultures administration of 0.5 mg/kg D-amphetamine sulphate. Three placentas were involved, cultures of HAECs were The net rotational asymmetry was measured for 10 prepared as described previously.11 Briefly, human consecutive weeks post cell transplantation. amniotic membrane was mechanically peeled from the chorion of a placenta obtained from an uncomplicated Immunohistochemistry elective caesarean section with the informed consent of Pathological change in the substantia nigra of a PD each donor patient. The HAE cell layer was thoroughly model scraped out from the underlying tissues such as the The rat was deeply anesthetized by hydral and spongy and fibroblast layers. The HAECs layer was then transcardially perfused with physiological saline followed treated with 0.125% trypsin three times each for 20 by 4% paraformaldehyde. After 6 hours postfixation in minutes to obtain dissociated HAECs. The cells were the same fixative, the brain was immersed in 25% sucrose cultured in RPMI-1640 medium (Sigma, USA) until it sank. Sections were cut at 30 µm in a cryostat. containing 10% fetal calf serum at 37°C in a 95% air/5% After being blocked by 0.3% endogenous peroxidase for CO2 humidified atmosphere. The culture medium was 3 minutes, the sections were incubated in 1.5% normal changed every 3 days. goatserum (Vector, USA). Then the sections were incubated overnight at 4°C with rabbit anti goat-TH 6-OHDA lesions (1:1000, Chemicon, USA) antibody and 10% normal goat Female Sprague–Dawley rats weighing 180 g–220 g were serum. After several rinses in PBS, sections were included. After being anesthetized by hydral, incubated for 30 minutes in biotinylated donkey 6-hydroxydopamine was stereotaxically injected at four anti-rabbit IgG (1:1000, Sigma, USA), then for 30 sites. The coordinates which were calculated with minutes in avidin-biotin-peroxidase complex (1:200, reference to bregma for the anterioposterior (AP) and the Vector, USA). Subsequently the sections were treated mediolateral (ML) coordinates using the rat brain atlas12 with 3,4-diaminobenzidine (DAB, Sigma, USA) and were as follows: (1) AP +1.3, ML –2.6; (2) AP +0.4, ML hydroxygen peroxide, mounted on albumin-coated slides –3.2;(3) AP –0.4, ML –4.2; (4) AP –1.3, ML –4.5. The and embedded with a cover glass. dorsoventral position of all injections was 5.0 mm below the dura and the tooth bar set to 0.0. 6-OHDA (Sigma, Tracking of the transplanted cells by detecting human USA) was prepared freshly in the dark to avoid specific nestin and vimentin expression on HAECs autooxidation, and was administered using a 5 µl The rats were perfused, fixed and brains cut as described microinjector at a rate of 0.5 µl/min. The syringe was left above. The brain sections were preincubated in equine in place for 5 minutes before slowly retracting it to allow serum for one night. The sections were then incubated for for toxin diffusion and prevent toxin reflux. One week 1 hour at 4℃ with primary antibodies against nestin after the surgery, the effect of the 6-OHDA lesions was (1:200, Chemicon, USA) or vimentin (1:2000, Sigma, assessed by monitoring apomorphine induced rotational USA). The sections were then incubated for 1 hour at asymmetry over a period of 30 minutes to select animals room temperature with secondary donkey anti-rabbit IgG with profound dopamine-denervating lesions in the antibodies conjugated to rhodamine (1:200, Santacruz, striatum following an intraperitoneal injection of 0.5 USA). Sections were observed under the fluorescence mg/kg of D-amphetamine sulphate (Sigma, USA). The microscope. behavior test was assessed for consecutive 4 weeks. The rats that exhibited a stable net rotational asymmetry of at The density of TH-positive cells in the substantia nigra. least 7 full turns per minute away from the lesioned side Ten weeks after transplantation, the density of were selected for the next experiment. All involved rats TH-positive cells in the substantia nigra of rats receiving were divided into two groups: HAECs group and NS HAECs and PBS was determined by TH group. Some untreated rats were taken as the control. immunohistology described above and analyzed with a computerized analysis system (Olympus Sp-1000, Japan). Grafting surgery In the HAECs group, HAECs were stereotaxically Dopamine in the cerebrospinal fluid measured by transplanted into the lateral ventricle of the recipient rats HPLC using a 10 µl Hamilton microsyringe (Hamilton, The dopamine levels in the cerebrospinal fluid of the Chinese Medical Journal 2009;122(20):2449-2454 2451

Figure 1. The HE staining of cultured HAECs (original magnification×400). Figure 2. TH immunohistochemistry shows TH positive cells in the stritum of lesion side and untreated side of a PD model (original magnification×100).

HAECs group, NS group and the untreated group rats could reach half of the cell diameter (Figure 1). were determined by HPLC. All rats were killed by decapitation. The CSF samples, with a volume up to 40 µl, Morphology of a PD model were filled up with 10 µl 0.02 mol/L perchloric acid. And TH immunohistochemistry shows that the number of then the homogenates were centrifuged for 10 minutes TH-positive cells decreased significantly in the substantia (10 000 rpm, 4°C), HPLC system was used to determine nigra of the 6-OHDA lesioned side compared to the monoamine levels in the supernatant. The striatae were untreated hemisphere (Figure 2). dissected out and weighted. Each sample was sonicated in ice cold 1 ml of 0.1 mol/L perchloric acid until Transplanted HAECs attenuate 6-OHDA induced homogeneity was achieved. The samples were rotational behavior in rats centrifuged for 15 minutes (12 000 r/min, 4°C), and the The rotational asymmetry in the PD rat model is related supernatants were collected and transferred onto a 0.2 µm to the number of dopamine neurons, so the rotational nylon filter tubes (Corning). The samples were asymmetry of PD rats can be used as an indicator of the centrifuged again (6 000 r/min, 5 minutes, 4°C) and the damage to dopamine neurons. As shown in Figure 3 the filtrates were stored in −80°C until analyzed. An HPLC rotational asymmetry of the HAECs group was system was used to measure the filtrates. significantly ameliorated from 2 weeks post cell transplantation, and it did not rebound by the end of the Monoamine in the striatum measured by HPLC experiment. The rotational asymmetry of the NS group After transplantation for 10 weeks, monamine levels in showed no obvious change. the striatum of the three groups above were determine by HPLC. All rats were anesthetized by hydral and the striatum was quickly disserted and put on ice. After being weighted, each sample was sonicated in ice cold 1 ml of 0.1mol/L perchloric acid until homogeneity was achieved. The samples were centrifuged for 10 minutes (10 000 r/min, 4°C), and the supernatants were collected and transferred into a 0.2 µm nylon filter tubes (Corning, USA). The samples were centrifuged again (6 000 rpm, 5 minutes, 4°C). An HPLC system was employed to measure the filtrates.

Statistical analysis Data are expressed as the means ± standard error (SE). One-way analysis of variance (ANOVA) test and

Student’s two-way t test were used for statistical analysis. A probability value of less than 0.05 was considered Figure 3. Transplantation of HAECs attenuated 6-OHDA- significant. induced behavioral changes.

RESULTS Transplantation of HAECs resulted in a greater preservation of TH-positive cells in the 6-OHDA Cultured human amniotic epithelial cells lesioned substantia nigra HE staining showed that cultured human amniotic We found that HAECs demonstrated a protective effect epithelial cells were round or oval and they grew to on substantia nigra. As shown in Figures 4 and 5, the confluence. The cell nucleus was relatively big; some number of TH positive cells in the substantia of the 2452 Chin Med J 2009;122(20):2449-2454

The differentiation of survival HAECs in the lateral ventricle of a PD rat Being transplanted for 10 weeks some of the survived HAECs showed positive labeling by TH immuohistochemistry. But the number was relatively small (Figure 7).

Changes of dopamine levels in the cerebrospinal fluid Dopamine levels in the cerebrospinal fluid of the HAECs group increased significantly compared to the NS group (P <0.01) (Figure 8).

Cellular transplantation inhibited 6-OHDA-induced Figure 4. Treatment with HAECs salvaged TH-positive cells in dopamine depletion in the lesioned striatum the substantia nigra damaged by 6-OHDA. *P <0.01, compared ** HAECs were found to prevent the fall of striatal to the NS group. P <0.01, compared to untreated group. dopamine levels induced by 6-OHDA injection. As shown in Figure 9, dopamine and DOPAC levels in the HAECs HAECs group was about 37.7±2.7%, but the number of group increased significantly compared to the NS group cells in the NS group was only 24.4±2.8%. There is a (P <0.05). HVA levels in the HAECs group also increased statistically significant difference between the two groups. significantly compared to the NS group (P <0.01). Dopamine, DOPAC and HVA levels in the HAECs group Tracking of transplanted grafts by histology did not reach the levels of the untreated group (P <0.05). After transplantation for 5 weeks, the grafts showed positive immunoreactivity to human specific nestin and DISCUSSION vimentin antibody. The grafts grew around the lateral ventricle and were not overgrown. The same changes Currently, the most effective treatment for PD is were not found in the NS group of rats (Figure 6.) dopamine replacement therapy via oral supplementation

Figure 5. TH positive cells in the substantia nigra (original magnification×200). A: untreated group; B: NS group; C: HAECs group.

Figure 6. HAECs expressed nestin (A, C) and vimentin (B, D) in the lateral ventricle of a PD rat after transplantation for 5 weeks, and the same result was not detected in the brain of the NS group (original magnification×200). Chinese Medical Journal 2009;122(20):2449-2454 2453

The amnion is the inner of two membranes surrounding the fetus. It arises from embryonic epiblast cells prior to gastrulation, and the possibility exists that they might maintain the plasticity of pregastrulation embryo cells. Human amniotic epithelial cells express the pluripotent -specific transcription factors octamer-binding protein 4, nanog, SSEA-4, TRA 1-60, TRA 1-81, but do not express SSEA-1.14 Human amniotic epithelial cells are clonogenic and they have the potential to differentiate into all three germ layers in vitro; endoderm (liver, ), (cardiomyocyte), and (neural cells).15-17 In addition, studies show that HAECs can differentiate into neuronal, glial and ligodendrocyte cells.11,18 HAECs have the capacity to synthesize and release brain-derived neurotrophic factor, insulin-like Figure 7. The surviving grafts show positive to TH growth factors, neurotrophin-3, NGF and other factors. immuohistochemistry staining after being implanted for 10 weeks in the lateral ventricle and third ventricle (original Brain derived neurotrophic factor has been shown to exert magnification×200). A: TH-positive cells of grafts in the lateral trophic effects on dopaminergic neurons against ventricle; B: TH-positive cells of grafts in the third ventricle. neurotoxin. HPLC showed that cultured HAECs can also synthesize and release catecholamines19 and it is indicated that HAECs contain dopamine metabolizing enzymes.

In our study, similar to a previous study, we induced a moderate lesion of the dopaminergic terminals with 6-OHDA. HAECs were then transplanted into the lateral cerebral ventricle of PD rats at the 5th week after lesion induction, when the acute phase of degeneration of the nigral dopamine was complete. We found transplantation of HAECs was beneficial and partially attenuated amphetamine-induced rotation, striatal dopamine level reduction, as well as the loss of Figure 8. Transplantation of HAECs increased dopamine levels TH-immunoreactive cells. It could have been due to the * in the cerebrospinal fluid of PD rats. P <0.01, compared to the neurotrophic factors secreted by the human amniotic NS group, **P <0.01, compared to the untreated group. epithelial cells: That would be in accordance with what

was reported by Kakishita et al.13 Neurotrophic factors are a family of proteins that are essential for neuronal development and survival. Specific factors are essential for subpopulations of cells. Brain derived neurotrophic factor is an important example relevant to PD since it mediates dopaminergic neuronal survival and induces re-innervation of tissue deafferented following exposure to specific toxins.20 Insulin-like growth factor 1 has also been shown to be effective for the restorative effect on the dopaminergic fibers in animal models.21 HAECs conditioned media also rescues 6-OHDA-treated neuroblastoma cells.

The reasons that transplantation of human amniotic Figure 9. HAECs transplantation prevented the fall of striatal * † epithelial cells into the ventricle of PD model rats may dopamine levels induced by 6-OHDA. P <0.05, P <0.01, compared to the NS group. **P <0.01 compared to untreated ameliorate rotational asymmetry are complicated. We group. assume that the neurotrophic factors secreted by human amniotic epithelial cells may slow down the apoptosis of of levodopa, but it can not prevent the progressive dopamine neurons induced by 6-OHDA. The surviving degeneration of nigral dopamine neurons. New therapies dopamine neurons can secrete more dopamine for PD are required. Transplantation of cells, like embryo cells, is rats. used to treat PD but it too has defects; such as limited cell sources, tissue rejection, ethics, etc. So we are forced to In conclusion, it is effective to transplant HAECs for find new types of replacement cells for transplantation. treatment in a PD rat model. 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