Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive death of nigral dopamine neurons with a resultant loss of striatal dopamine levels.1 When the number of dopaminergic neurons in the substantia nigra pars compact is reduced by over 70%, classical clinical symptoms, such as akinesia, rigidity, tremor and postural dysfunction, are produced.2 The pathogenesis of PD is complicated and the cause of idiopathic PD is obscure. The major risk factors for PD include aging, environmental neurotoxins such as paraquat,3 and genetic defects. Oxidative stress and excitotoxicity are involved in the mechanisms of PD.4,5 Levodopa has been long employed as a dopamine replacement therapy for Parkinson's disease, but the efficacy of the drug in alleviating motor symptoms in PD is gradually attenuated with the progress of the disease. Long-term application of levodopa can result in severe toxicity or debilitating side effects, such as dyskinesias, symptomatic fluctuation, and hallucinations.6 Moreover, Levodopa can not cure Parkinson's disease completely. So we must find new treatments for PD.
Some studies indicate that tissue transplantation is a therapeutic modality that can supplement dopamine in the brain using a PD model in rats. The potential donor cells for transplantation include neural stem cells, embryo stem cell, mesenchymal stem cells, olfactory ensheathing cells, retinal pigment epithelial cells, and Schwann cells.7–9 But these doners are limiting in source. Human amniotic epithelial cells (HAECs) are formed from epilates on the 8th day after fertilization, and constitute the inner layer of the amnion, opening the possibility that they might maintain the plasticity of pregastrulation embryo cells. HAECs lack major histocompatibility complex antigens and have a low risk of tissue rejection when transplanted into other individuals.10 In addition, they are readily available and may be a useful and noncontroversial source of stem cells for cell transplantation and regenerative medicine.
Human amniotic epithelial cell cultures
Three placentas were involved, cultures of HAECs were prepared as described previously.11 Briefly, human amniotic membrane was mechanically peeled from the chorion of a placenta obtained from an uncomplicated elective caesarean section with the informed consent of each donor patient. The HAE cell layer was thoroughly scraped out from the underlying tissues such as the spongy and fibroblast layers. The HAECs layer was then treated with 0.125% trypsin three times each for 20 minutes to obtain dissociated HAECs. The cells were cultured in RPMI-1640 medium (Sigma, USA) containing 10% fetal calf serum at 37°C in a 95% air/5% CO2 humidified atmosphere. The culture medium was changed every 3 days.
Female Sprague-Dawley rats weighing 180 g-220 g were included. After being anesthetized by hydral, 6-hydroxydopamine was stereotaxically injected at four sites. The coordinates which were calculated with reference to bregma for the anterioposterior (AP) and the mediolateral (ML) coordinates using the rat brain atlas12 were as follows: (1) AP +1.3, ML -2.6; (2) AP +0.4, ML -3.2;(3) AP -0.4, ML -4.2; (4) AP -1.3, ML -4.5. The dorsoventral position of all injections was 5.0 mm below the dura and the tooth bar set to 0.0. 6-OHDA (Sigma, USA) was prepared freshly in the dark to avoid autooxidation, and was administered using a 5 μl microinjector at a rate of 0.5 μl/min. The syringe was left in place for 5 minutes before slowly retracting it to allow for toxin diffusion and prevent toxin reflux. One week after the surgery, the effect of the 6-OHDA lesions was assessed by monitoring apomorphine induced rotational asymmetry over a period of 30 minutes to select animals with profound dopamine-denervating lesions in the striatum following an intraperitoneal injection of 0.5 mg/kg of D-amphetamine sulphate (Sigma, USA). The behavior test was assessed for consecutive 4 weeks. The rats that exhibited a stable net rotational asymmetry of at least 7 full turns per minute away from the lesioned side were selected for the next experiment. All involved rats were divided into two groups: HAECs group and NS group. Some untreated rats were taken as the control.
In the HAECs group, HAECs were stereotaxically transplanted into the lateral ventricle of the recipient rats using a 10 μl Hamilton microsyringe (Hamilton, Switzerland) fitted with a steel cannula following the below coordinate: AP -0.8, ML -1.3, D -4.8. We transplanted 8 μl HAECs for each rat, containing 106 cells. After the completion of the injections, the cannula was left in situ for 5 minutes before being slowly retracted. In the NS group, we injected NS into the lateral ventricle of recipient rats in the same manner.
Apomorphine induced rotational behavior13 was measured for 30 minutes following intraperitoneal administration of 0.5 mg/kg D-amphetamine sulphate. The net rotational asymmetry was measured for 10 consecutive weeks post cell transplantation.
Pathological change in the substantia nigra of a PD model
The rat was deeply anesthetized by hydral and transcardially perfused with physiological saline followed by 4% paraformaldehyde. After 6 hours postfixation in the same fixative, the brain was immersed in 25% sucrose until it sank. Sections were cut at 30 μm in a cryostat. After being blocked by 0.3% endogenous peroxidase for 3 minutes, the sections were incubated in 1.5% normal goatserum (Vector, USA). Then the sections were incubated overnight at 4°C with rabbit anti goat-TH (1:1000, Chemicon, USA) antibody and 10% normal goat serum. After several rinses in PBS, sections were incubated for 30 minutes in biotinylated donkey anti-rabbit IgG (1:1000, Sigma, USA), then for 30 minutes in avidin-biotin-peroxidase complex (1:200, Vector, USA). Subsequently the sections were treated with 3,4-diaminobenzidine (DAB, Sigma, USA) and hydroxygen peroxide, mounted on albumin-coated slides and embedded with a cover glass.
Tracking of the transplanted cells by detecting human specific nestin and vimentin expression on HAECs
The rats were perfused, fixed and brains cut as described above. The brain sections were preincubated in equine serum for one night. The sections were then incubated for 1 hour at 4 with primary antibodies against nestin (1:200, Chemicon, USA) or vimentin (1:2000, Sigma, USA). The sections were then incubated for 1 hour at room temperature with secondary donkey anti-rabbit IgG antibodies conjugated to rhodamine (1:200, Santacruz, USA). Sections were observed under the fluorescence microscope.
The density of TH-positive cells in the substantia nigra.
Ten weeks after transplantation, the density of TH-positive cells in the substantia nigra of rats receiving HAECs and PBS was determined by TH immunohistology described above and analyzed with a computerized analysis system (Olympus Sp-1000, Japan).
Dopamine in the cerebrospinal fluid measured by HPLC
The dopamine levels in the cerebrospinal fluid of the HAECs group, NS group and the untreated group rats were determined by HPLC. All rats were killed by decapitation. The CSF samples, with a volume up to 40 μl, were filled up with 10 μl 0.02 mol/L perchloric acid. And then the homogenates were centrifuged for 10 minutes (10 000 rpm, 4°C), HPLC system was used to determine monoamine levels in the supernatant. The striatae were dissected out and weighted. Each sample was sonicated in ice cold 1 ml of 0.1 mol/L perchloric acid until homogeneity was achieved. The samples were centrifuged for 15 minutes (12 000 r/min, 4°C), and the supernatants were collected and transferred onto a 0.2 μm nylon filter tubes (Corning). The samples were centrifuged again (6 000 r/min, 5 minutes, 4°C) and the filtrates were stored in −80°C until analyzed. An HPLC system was used to measure the filtrates.
Monoamine in the striatum measured by HPLC
After transplantation for 10 weeks, monamine levels in 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.
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 significant.
Cultured human amniotic epithelial cells
HE staining showed that cultured human amniotic epithelial cells were round or oval and they grew to confluence. The cell nucleus was relatively big; some could reach half of the cell diameter (Figure 1).
Morphology of a PD model
TH immunohistochemistry shows that the number of TH-positive cells decreased significantly in the substantia nigra of the 6-OHDA lesioned side compared to the untreated hemisphere (Figure 2).
Transplanted HAECs attenuate 6-OHDA induced rotational behavior in rats
The rotational asymmetry in the PD rat model is related to the number of dopamine neurons, so the rotational asymmetry of PD rats can be used as an indicator of the damage to dopamine neurons. As shown in Figure 3 the rotational asymmetry of the HAECs group was significantly ameliorated from 2 weeks post cell transplantation, and it did not rebound by the end of the experiment. The rotational asymmetry of the NS group showed no obvious change.
Transplantation of HAECs resulted in a greater preservation of TH-positive cells in the 6-OHDA lesioned substantia nigra
We found that HAECs demonstrated a protective effect on substantia nigra. As shown in Figures 4 and 5, the number of TH positive cells in the substantia of the HAECs group was about 37.7±2.7%, but the number of cells in the NS group was only 24.4±2.8%. There is a statistically significant difference between the two groups.
Tracking of transplanted grafts by histology
After transplantation for 5 weeks, the grafts showed positive immunoreactivity to human specific nestin and vimentin antibody. The grafts grew around the lateral ventricle and were not overgrown. The same changes were not found in the NS group of rats (Figure 6.)
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 dopamine depletion in the lesioned striatum
HAECs were found to prevent the fall of striatal dopamine levels induced by 6-OHDA injection. As shown in Figure 9, dopamine and DOPAC levels in the HAECs group increased significantly compared to the NS group (P <0.05). HVA levels in the HAECs group also increased significantly compared to the NS group (P <0.01). Dopamine, DOPAC and HVA levels in the HAECs group did not reach the levels of the untreated group (P <0.05).
Currently, the most effective treatment for PD is dopamine replacement therapy via oral supplementation of levodopa, but it can not prevent the progressive degeneration of nigral dopamine neurons. New therapies are required. Transplantation of cells, like embryo cells, is used to treat PD but it too has defects; such as limited cell sources, tissue rejection, ethics, etc. So we are forced to find new types of replacement cells for transplantation. 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 stem cell-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, pancreas), mesoderm (cardiomyocyte), and ectoderm (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 growth factors, neurotrophin-3, NGF and other factors. Brain derived neurotrophic factor has been shown to exert trophic effects on dopaminergic neurons against 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 neuron 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 TH-immunoreactive cells. It could have been due to the neurotrophic factors secreted by the human amniotic 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 epithelial cells into the ventricle of PD model rats may ameliorate rotational asymmetry are complicated. We assume that the neurotrophic factors secreted by human amniotic epithelial cells may slow down the apoptosis of dopamine neurons induced by 6-OHDA. The surviving dopamine neurons can secrete more dopamine for PD rats.
In conclusion, it is effective to transplant HAECs for treatment in a PD rat model. The cells are from the discarded placenta. They are in unlimited supply and easily available, and their use is not encumbered by ethical arguments.22 HAECs have a great advantage for treatment of Parkinson's Disease in the future.
1. Sherer TB, Betarbet R, Greenamyre JT. Pathogenesis of Parkinson's disease
. Curr Opin Investig Drugs 2001; 2: 657-662.
2. Le Couteur DG, Muller M, Yang MC, Mellick GD, McLean AJ. Age-environment and gene-environment interactions in the pathogenesis of Parkinson's disease
. Rev Environ Health.2002; 17: 51-64.
3. Li X, Yin J, Cheng CM, Sun JL, Li Z, Wu YL. Paraquat induces selective dopaminergic nigrostriatal degeneration in aging C57BL/6 mice. Chin Med J 2005; 118: 1357-1361.
4. Lee FJ, Liu F. Genetic factors involved in the pathogenesis of Parkinson's disease
. Brain Res Rev 2008; 58: 354-364.
5. Yamashita H, Matsumoto M. Molecular pathogenesis, experimental models and new therapeutic strategies for Parkinson's disease
. Regen Med 2007; 2: 447-455.
6. Jankovic J. Parkinson's disease
therapy: treatment of early and late disease. Chin Med J 2001; 114: 227-234.
7. Ming M, LE WD. Retinal pigment epithelial cells: biological property and application in Parkinson's disease
. Chin Med J 2007; 120: 416-420.
8. Jiang CC, Xia Y, Ding ZL. Effects of co-engraftment of Schwann cells with neural stem cells into rats with Parkinson disease. Chin Med J 2006; 119(12): 1030-1033.
9. Bouchez G, Sensebé L, Vourc'h P, Garreau L, Bodard S, Rico A, et al. Partial recovery of dopaminergic pathway after graft of adult mesenchymal stem cells in a rat model of Parkinson's disease
. 2008; 52: 1332-1342.
10. Adinolfi M, Akle CA, McColl I, Fensom AH, Tansley L, Connolly P, et al. Expression of HLA antigens, beta 2-microglobulin and enzymes by human amniotic epithelial cells. Nature 1982; 295: 325-327.
11. Sakuragawa N, Thangavel R, Mizuguchi M, Hirasawa M, Kamo I. Expression of markers for both neuronal and glial cells in human amniotic epithelial cells. Neurosci Lett 1996; 209: 9-12.
12. Carlsson T, Winkler C, Burger C, Muzyczka N, Mandel RJ, Cenci A, et al. Reversal of dyskinesias in an animal model of Parkinson's disease
by continuous L-DOPA delivery using rAAV vectors. Brain 2005; 128(Pt 3): 559-569.
13. Kakishita K, Elwan MA, Nakao N, Itakura T, Sakuragawa N. Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson's disease
: a potential source of donor for transplantation
therapy. Exp Neurol 2000; 165: 27-34.
14. Miki T, Lehmann T, Cai H, Stolz DB, Strom SC. Stem cell characteristics of amniotic epithelial cells.Stem Cells 2005; 23: 1549-1559.
15. Tamagawa T, Oi S, Ishiwata I, Ishikawa H, Nakamura Y. Differentiation of mesenchymal cells derived from human amniotic membranes into hepatocyte-like cells in vitro
. Hum Cell 2007; 20: 77-84.
16. Enosawa S, Sakuragawa N, Suzuki S. Possible use of amniotic cells for regenerative medicine. Nippon Rinsho 2003; 61: 396-400.
17. Ilancheran S, Michalska A, Peh G, Wallace EM, Pera M, Manuelpillai U. Stem cells derived from human fetal membranes display multilineage differentiation potential.Biol Reprod 2007; 77: 577-588.
18. Ishii T, Ohsugi K, Nakamura S, Sato K, Hashimoto M, Mikoshiba K,et al. Gene expression of oligodendrocyte markers in human amniotic epithelial cells using neural cell-type-specific expression system. Neurosci Lett 1999; 268: 131-134.
19. Elwan MA, Sakuragawa N. Evidence for synthesis and release of catecholamines by human amniotic epithelial cells. Neuroreport 1997; 8: 3435-3438.
20. Sadan O, Bahat-Stromza M, Barhum Y, Levy YS, Pisnevsky A, Peretz H, Y, et al. Protective effects of neurotrophic factors secreting cells in a 6-OHDA rat model of Parkinson disease. Stem Cell Dev 2009. Epub ahead of print.
21. Guan J, Krishnamurthi R, Waldvogel HJ, Faull RL, Clark R, Gluckman P. N-terminal tripeptide of IGF-1 (GPE) prevents the loss of TH positive neurons after 6-OHDA induced nigral lesion in rats. Brain Res 2000; 859: 286-292
22. Fan W, Yang Z, Deng L. Basic study on the development of amniotic membrane and its application. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2006; 20: 65-68.
Keywords:© 2009 Chinese Medical Association
Parkinson's disease; human amniotic epithelial cell; transplantation; brain derived neurotrophic factor