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Transplantation of human chorionic mesenchymal stem cells-derived dopaminergic neurons into a Parkinsonian rat model ameliorates behavioral and structural impairments

Abstract

Background: Parkinson’s disease (PD) is known as a neurodegenerative disorder related to motor coordination. The main cause of the disease is related to the loss of dopaminergic neurons in the substantia nigra (SN). Human chorionic mesenchymal stem cells (HCMSCs) are a good choice for neural differentiation and cell therapy in neurodegenerative models.

Objectives: This study was performed with the purpose of evaluate the striatum alterations following injection of the 6-hydroxydopamine (6-OHDA). In addition, the effects of transplantation of HCMSCs-derived dopaminergic neurons into the striatum were evaluated using stereological methods.

Methods: Eighteen rats were randomly divided into three groups. Group І (Control), Group ІІ (6-OHDA/vehicle), and Group ІІІ (6-OHDA/cells). HCMSCs were isolated, characterized and then treated with neural and dopaminergic differentiation factors for 2 weeks. Next, the immunoreactivity for dopaminergic specific marker, tyrosine hydroxylase (TH) was evaluated. Behavior status of animals was assessed by the open-field and Apomorphine tests. Moreover, volume of striatum, number of neurons and glial cells and length of dendrites were measured using quantitative stereological methods.

Results: Our results showed a significant reduction in the volume of striatum, number of neurons and length of dendrite in the striatum of treatment group, whereas number of glial cells increased in the striatum. The results also showed a significant increase in the total distance travelled and velocity in the dopaminergic neuron-treated rats.

Conclusion: The current study revealed that treatment of the 6-OHDA rats with HCMSCs-derived dopaminergic neurons prevents structural and behavioral changes in the striatum after 14 weeks.

Keywords: Human chorionic mesenchymal stem cells, Dopaminergic neurons, Parkinson’s disease, Stereology, Histology, Cell culture, Differentiation

Introduction

Parkinson’s disease (PD) is known as a neurodegenerative syndrome related to motor coordination impairment with tremor, rigidity, changes in gait and bradykinesia. The main cause of the disease is related to the loss of approximately 70% dopamine (DA) producing neurons in the midbrain (1, 2).One of the major problems in dealing with the treatment of neurodegenerative disease, especially PD is significant variability in the appearance and progression of its symptoms. On the other hand, there is no well-known definite cure for PD and current treatments including medications and some types of surgical procedures can only relieve the part of unfriendly effects of disease (3, 4). Nowadays, cell therapy approach for PD has entered a new point and recent exciting advances in the clinic phase give reason for hopefulness. Several types of potential sources such as neural stem cells, embryonic stem (ES), and induced pluripotent stem (iPS) cells, and somatic cells which are differentiated to dopaminergic neurons have been recommended for animal models of PD. The experimental and clinical evidences showed that an appropriate clinical candidate cell must have the properties of substantia nigra (SN) neurons to be capable to induce optimum neural function recovery (5, 6).This means that differentiated DA-producing neurons from human candidate source can be able to express typical factors of dopaminergic neurons of the brain and also have excitation and neurotransmitter release ability. Thus, these sources will be valuable to induce substantial symptomatic relief (7). Accordingly, human placental tissue, an available rich source of pluripotential cells called human chorionic mesenchymal stem cells (HCMSCs), is a brilliant choice for neural differentiation and cell therapy in neurodegenerative models (8). HCMSCs show common properties of other mesenchymal stem cells such as adipose tissue or bone marrow and have exclusive immunoregulating properties. HCMSCs also represent the ability to express pluripotency-associated factors for example SOX2, c-MYC, KLF4, NANOG, SSEA4 and SSEA3. HCMSCs usually do not express various types of HLA including HLA-ABC or HLA-DR. Therefore, there is minimal risk and low ethical issues in xenograft of HCMSCs and specific neurons differentiated from these cells into animal models of neurodegenerative disorders (9, 10). Based on previous studies, we supposed that in vivo transplantation of HCMSCs-derived dopaminergic neurons into the striatum of rat models of PD is a confident approach for initiating symptomatic relief and improving neural function subsequently. With the goal of better understanding the effects of 6-OHDA and cell transplantation on behavioral changes and histological structure, we used stereological methods to determine the changes of striatum volume, number of neuron and glial cells as well as nerve fiber length in a Parkinsonian rat model.

Materials and Methods

Animals

In this study, 18 adult male Sprague–Dawley rats (250±20 g) were obtained from the laboratory animal house of Shahid Beheshti University of Medical Sciences, Tehran, Iran. The Ethics Committee of the University approved the animal experiment (IR.SBMU. MSP. REC.1395.320). Male rats were randomly categorized into three groups. Each group included 6 animals that were kept under standard conditions including room temperature (22–24°C), 12-12 h light-dark cycle, and free access to water and food.

Isolation and culture of HCMSCs

Chorionic villous stem cells were isolated from third trimester (34-40 weeks GA) human placental tissue after cesarean sections of healthy donor mothers. All our experiments on the cells were approved by informed consent and the Ethical Committee of Shahid Beheshti University of Medical Sciences. The chorionic tissue was washed in phosphate-buffered saline (PBS) (Gibco, Germany) and then, it was cut into small slices and digested with 0.1% type II collagenase solution (Gibco, Germany) at 37°C for 30 minutes. The cells were filtrated through 100-μm filter, the enzyme was deactivated using FBS, and the cell suspension was centrifuged at 1200 rpm for 3 min. Finally, almost 3-3.5×103 cells/cm² was cultured in T75 tissue culture flask for cell attachment (TC treated) (TPP, Switzerland) that contained growth and expansion medium composed of DMEM/F12, complemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco, Germany). Afterward, the cell cultures were placed into a humidified chamber at 37°C and 5% CO₂ conditions. Cultured HCMSCs at passage 3 (P#3) were used for following experiments. The medium was changed every 3 days. At 70–80% cell confluence, the cells were replaced into new freshly prepared flasks with media.

Characterization of HCMSCs (flowcytometric Analysis for surface antigens)

For flowcytometric analysis, HCMSCs were detached with 0.05% trypsin after P#3, the samples were centrifuged, the number of 5×105 cell pellets resuspended in 10% FBS, and then reserved on ice packs for 10 minutes. The cells were incubated with 10% serum at 4°C for 1 hour. Then the serum was removed and the cells were incubated with fluoresceinisotiocyanate (FITC) or phycoerythrin (PE)-labeled monoclonal antibodies against human surface markers at 4°C for 1 hour. CD44, and CD73 (all from BD Bioscience, USA) were used against mesenchymal stem cell markers. CD34 and CD45 (both from BD Bioscience, USA) were used against hematopoietic markers and HLA-DR (MHC class II, BD bioscience, USA) was used against immunologic marker as well. Isotype antibodies also were used as control samples. Flowcytometric analysis was carried out by a PartecCyFlow Space cytometer using FloMax software.

In vitro differentiation of HCMSCs (mesodermal differentiation)

Differentiation potential into a variety of mesodermal cell lineages such as adipocytes and osteocytes is one of the most prominent criteria to identify HCMSCs.

Adipogenic differentiation

Medium of 70–80% confluent culture of the cells was substituted with adipogenic medium composed of DMED/F12 supplemented with 0.5 mM 3- isobutyl-1-methylxanthine (IBMX), 50 μM indomethacin (Sigma-Aldrich) and 10 nM dexamethasone (Sigma-Aldrich). The cultures were returned back in humidified chamber at 37°C and 5% CO₂. The medium was changed every 3 days. After 15 days of incubation, the cells were fixed in 4% paraformaldehyde and then prepared for staining with 0.5% oil red O solution for 30 min. Formation of multiple lipid droplets inside the mesodermal stem cells were photographed by phase contrast microscope.

Osteogenic differentiation

At 70–80% confluence, the medium was changed with osteogenic medium composed of DMEM/F12 complemented with 10% FBS, 50 μg/ml ascorbic 2-phosphate, 10 nM dexamethasone and 10 mM β-glycerol phosphate (all from Sigma-Aldrich). The culture plate was incubated again in humidified chamber at 37°C and 5% CO₂. After approximately 21 days, the cells were fixed in 4% paraformaldehyde and then stained with 2% Alizarin Red solution for 20 min to detect the formation of mineralized crystals in extracellular matrix.

Pre- differentiation of HCMSCs to neural lineage

Pre-induction of HCMSCs into the neural differentiation at P#3 with 70–80% confluence, the cells were prepared for pre-differentiation into neural lineage. To do that, expansion primary medium was removed. The cells were detached with 0.05% trypsin, enzyme was deactivated using FBS. The number of 5000 cell/cm² was plated in 24-well plates that contained DMEM/F12 supplemented with 10% FBS, antibiotic/antimycotic and pre-induction medium enriched by neural factors such as 2% B27, 20 ng/mL FGF2, and 200 Μm Butylated hydroxyanisole (BHA). Afterwards, the cells were incubated in humidified chamber at 37°C and 5% CO₂ for 3 days.

Final neural differentiation (dopaminergic differentiation of HCMSCs)

After 3 days, neural pre-induction medium was removed and then replaced with final dopaminergic medium composed of DMEM/F12 complemented with 10% FBS, antibiotic/antimycotic, 100 ng/mL FGF8 and 100 ng/mL sonic hedgehog (Shh). Culture plates immediately were incubated again in humidified chamber at 37°C and 5% CO₂ conditions for 14 days.

Immunocytochemistry

Cells were washed with phosphate buffered saline (PBS), fixed by paraformaldehyde 4% and then washed with PBS (3 times) and air dried. Slides were blocked with normal blocking serum (%10) in PBS for 40 minutes and washed by PBS, and incubated by primary antibody including anti tyrosine hydroxylase (TH) (Abcam, USA) in 1.5% normal blocking serum in PBS overnight. Then slides were washed 3 times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Abcam, USA) for 60 minutes. Fluorescence microscopy was used for visualizing immunoreactive cells.

Experimental model of PD

Induction of PD in animals was performed by a single dose (4µg of 6-hydroxydopamine (6-OHDA) in 2µl of physiological saline containing 0.02% ascorbic acid) unilateral stereotaxic injection by a Hamilton microsyringe into the right substantia nigra (11). Coordinates were set according to the Atlas of Paxinos and Watson (12) as follows: antero-posterior (AP): -4.3 mm, lateral (L): 1.6 mm, and Dorso-ventral (DV):8.2 mm.

Apomorphine turning behavior

To test apomorphine-induced turning behavior the animals received a subcutaneous injection of 0.05 mg/kg apomorphine hydrochloride (Sigma-Aldrich) dissolved in 1% ascorbic acid 0.9% NaCl in the neck, and placed on metal testing bowls for 30 minutes. The number of contralateral rotations was digitally recorded.

Behavioral and locomotor measurements (open-field test)

Behavior status of animals was assessed by the open-field test. The test was conducted at 7 days after 6-OHDA stereotaxic surgery and post-cell transplantation by introducing the animals into a 30 cm (L) × 30 cm (W) × 50 cm (H) arena. Total distance travelled and velocity was recorded automatically by an automated monitoring system. At the end of 5-min observation period, animals were returned to their cages and the arena was cleaned and then dried. All experiments were performed from 8 a.m. to 1 p.m. in a silent room illuminated by an overhead fluorescent light. The videos were analyzed by EthoVision XT software (Noldus).

Experimental design and transplantation procedures

All animals were randomly arranged into three groups as follows: control group (n=6), 6-OHDA group (n=6), and 6-OHDA/Cells (treatment) group (n=6).  Animals were anesthetized with ketamine and xylazine cocktail (1 ml/kg IP, contains 90 mg/kg and 9 mg/kg, respectively).Then, animals were placed in stereotaxic frame. Treatment group animals received injection of approximately 3×10⁵ HCMSCs-derived dopaminergic cells suspension into right striatum (antero-posterior (AP):0.24 mm, lateral (L): 3.2, and Dorso-ventral (DV): 5.2) using a Hamilton microsyringe. Coordinates were set according to the Atlas of Paxinos and Watson (12). HCMSCs-derived dopaminergic neurons were labeled with Höchst. After implantation, HCMSCs-derived dopaminergic neurons were found in the striatum zone of rat brains. At the second and fourth weeks after transplantation, the Höchst-positive cells were detected in the striatum of HCMSCs-derived dopaminergic neurons -injected animals Parkinsonian models.

Tissue preparation

Animals were deeply anesthetized and decapitated. Then, their brains were exposed by an incision along the midline of the skull. A small amount of fixative was poured on the exposed brain immediately. The brains were removed and immersed in 10% formalin for one week and then embedded in paraffin blocks. Then, complete serial sections (10 μm and 60 μm) using a microtome were prepared.  Every 10 section was sampled starting with a random number between 1 and 10.  Finally, about 8–10 sections in each animal were selected in a systematic random manner. The sections were stained with Haematoxylin and Eosin (H&E) (0.1% in distilled water) and also rapid Golgi method.

Estimation of the striatum volume

Using a projecting microscope, the live image of each brain section was evaluated according to the rat brain atlas (Atlas of Paxinos and Watson) (12). Using the stereological software designed at Histomorphometry and Stereology center (Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences), a grid of points was superimposed on the images (Figure 1A). The volume of the striatum was estimated by the following formula:

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Legends for the figures/images:

Figure 1. (A) Point counting method (Cavalieri’s method). (B) Optical dissector method. The cells whose nuclei came into focus during scanning the dissector height and did not touch the left and lower borders of the frame were counted (arrow head).

Figure 2. Micrograph of the striatum stained with Golgi. Four cycloids were located at a rectangle. The length of each cycloid was equal to twice the length of its minor axis (r). The area associated with the cycloids was calculated by multiplying X by Y and dividing by the length of the four cycloids to achieve the area per length. When the sections were scanned, the number of cell bodies of the neurons was counted using the optical dissector method and unbiased counting frame. The total number of intersections between the dendrite axes and the cycloid was counted (arrow head).

Figure 3. (A) Morphology, proliferation and colony formation of HCMSCs. (B) neural dopaminergic differentiation of HCMSCs.

Figure 4. Characterization of HCMSCs. Flowcytometric analysis of passage 3 MSC culture for CD45, CD34, CD44, and CD73 cells.

Figure 5. HCMSCs potential for multilineage differentiation. (A) Osteogenic lineage forming deposits of mineralized calcium under osteogenic induction medium (Alizarin Red). (B) Adipogenic lineage forming lipid droplets under adipogenic induction medium (Oil Red).

Figure 6. Expression of TH (tyrosine hydroxylase) protein in human chorion-derived cells. Immunocytochemical analyses revealed that a subpopulation of TH positive dopaminergic neurons differentiated from HCMSC samples.

Figure 7. Apomorphine-induced turning behavior revealed of 6-OHDA lesioned animals with intense turning behavior in comparison with control animals that showed no turning behavior (p < 0.05).

Figure 8. Open field test revealed the total distance travelled (A) and velocity (B) in the different groups. The total distance travelled and velocity of 6-OHDA group are significantly different in comparison to control and 6-OHDA/cells groups (* p<0.05).

Figure 9. The total volume of the striatum in the 6-OHDA group (6-OHDA; n=6), HCMSCs-derived dopaminergic neurons treated animals (6-OHDA/cells; n=6) and Control (n=6) were compared. 6-OHDA/cells animals showed significant difference as compared to the 6-OHDA after 10 days. * p<0.05, ** p <0.01, and ## p <0.01.

Figure 10.The total number of neuron and glial cells in the 6-OHDA rats (6-OHDA; n=6), HCMSCs -derived dopaminergic neurons treated rat (6-OHDA/cells; n=6) and Control (n=6) were compared. 6-OHDA/cells rats significantly difference with compared to the 6-OHDA after 10 days. *** p<0.001 and ### p <0.001.

Figure 11.The total dendritic length in the 6-OHDA rats (6-OHDA; n=6), HCMSCs -derived dopaminergic neurons treated rat (6-OHDA/cells; n=6) and Control (n=6) were compared. 6-OHDA/cells rats showed significant difference as compared to the 6-OHDA after 10 days. *** p<0.001 and ### p <0.001.