Conditioned Medium Protects Dopaminergic Neurons in
Adipose derived stem cells (ASCs) secrete numerous neurotrophic factors and cytokines in conditioned medium (CM), which protect neurons by its antioxidative and trophic effects. This research assesses the neuroprotective effect of ASC- CM on neurotrophins genes expressions and tyrosine hydroxylase positive (TH+) cell density in male Wistar rats lesioned by 6-hydroxydopamine (6-OHDA).
Materials and Methods
In this experimental study, the groups consisted of lesioned and sham rats with unilateral injections of 20 µg of 6-OHDA neurotoxin and phosphate buffered saline (PBS) into the striatum, respectively. Another groups received intravenous injections of 3×106 cells (ASCs group), 500 µl of CM (ASC-CM group) or medium [α-minimal essential medium (α-MEM) group)]. All rats underwent evaluations with the rotarod and apomorphine-induced rotation tests at 2, 4, 6, and 8 weeks post-injection. At 8 weeks we sacrificed some of the animals for real-time polymerase chain reaction (PCR) analysis, and evaluation of TH+cell counts.
We observed a significant decrease in contralateral turns to the lesions in the ASCs and ASC-CM groups
compared to the neurotoxin lesioned or α-MEM groups at 8 weeks post transplantation. Cell and CM- injected rats
showed a significant increase of staying on the rotarod compared to the lesion or α-MEM groups. Cell and CM-treated
rats showed significant increases in the
The results suggested that ASCs and ASC-CM protected dopaminergic neurons through the expressions of neurotrophin genes.
Motor disorders of parkinson’s disease (PD) are caused by dopamine loss of corpus striatum as the result of nigrostriatal pathway degeneration (1, 2). Adult stem cells have been used to treat neurodegenerative diseases such as PD over the past few years. Transplanted cells have the capability to differentiate into neural cells or secret neurotrophic factors and create an appropriate microenvironment to protect residual dopaminergic neurons of the substantia nigra (SN) pars compacta.
Adipose derived stem cells (ASCs) are a population of mesenchymal stem cells in the stromal or nonadipocyte compartment of adipose tissues. Intrastriatal transplantation of ASCs has been shown to protect against 6-hydroxydopamine (6-OHDA)-induced experimental PD in mice (3). Secreted neurotrophins, which modulate oxidative stress in the injured SN after cell therapy, are more effective than neural differentiation of transplanted cells to repair the nigrostriatal pathway (3, 4). The survival of transplanted cells increased when accompanied with nerve growth factor (NGF) injection. NGF played an antioxidative role to protect neurons (5).
Human ASC transplantation stimulated angiogenesis and neurogenesis by secreting vascular endothelial growth factor (VEGF) and transforming growth factor- beta (TGF-ß) (6). According to low survival and tumorigenesis of transplanted cells, another therapeutic application of stem cell is the use of cultured ASCs conditioned medium (ASC-CM) to protect surviving neurons or stimulate renewal of axonal sprouting. The secretory factors of cultured stem cells are called the secretome, microvesicles, or exosome; the medium is CM (7). Numerous studies showed that stem cells secreted various growth factors into the CM, which had therapeutic effects on various diseases (6-14).
The neuroprotective effect of ASC-CM has been
reported in an
Materials and Methods
In this experimental study, adult male Wistar rats that weighed 220-280 g were purchased from Pasteur Institute of Iran. They were kept in standard cages in a temperature- and climate-controlled room under a 12/12 hour light/dark cycle and had ad libitum access to water and food. The Research and Ethics Committee of Damghan University approved this experimental protocol. Animals were deeply anesthetized by an intramuscular injection of a mixture of ketamine hydrochloride and xylazine, and then placed in a stereotaxic frame. A total of 20 µg of 6-OHDA hydrobromide (Sigma-Aldrich, USA) in 4 µl of sterile saline that contained 0.2% ascorbic acid was injected into the right striatum by a 26-gauge Hamilton syringe (Hamilton, France) at a flow rate of 1 µl/minute. Stereotaxic coordinates from the bregma were: anteroposterior (AP)=-1.2 mm, mediolateral (ML)=-3.9 mm, and dorsoventral (DV)=-5 mm (17). The syringe was left in place for 5 minutes after the injection and then removed slowly to optimize toxin diffusion.
Preparation and culture of rat adipose derived stem cells
Fat tissues from the backs of the rats were cut under sterile conditions. The tissues were digested mechanically and enzymatically with 0.2% collagenase (Gibco, USA) (18). ASCs were extracted by adherence to the plastic flasks. We cultured the isolated cells with 10% fetal bovine serum (FBS, Gibco, USA) that contained a-minimal essential medium (α-MEM, Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). The cells were incubated at 37°C in air with 5% CO2. The culture medium was changed after the first 48 hours and every 3-4 days to remove any floating cells. When the culture reached 80% confluency (usually within a week), the cells were harvested by incubation with 0.25% trypsin and 0.02% EDTA (Merck) at 37°C for 3-4 minutes. Once harvested, the cells were sub-cultured (19).
Collection of adipose derived stem cell-conditioned medium
ASCs were cultured in α-MEM that contained 10% FBS. After four passages, 5×105 plastic-adherent cells were washed three times with PBS, and cultured in serum-free medium for 72 hours to allow secretion of neurotrophic factors. ASC-CM was then collected, centrifuged at 2000 rpm for 5 minutes, filtered through a 0.22 mm syringe filter, and stored in a -80°C refrigerator (4, 16, 20).
Treatment with adipose derived stem cells, ASC- conditioned medium and a-minimal essential medium
At one week after the 6-OHDA lesion (18), the rats were anesthetized with a mixture of ketamine hydrochloride and xylazine. The ASCs (3×106 cells, n=7) (21), ASC-CM (500 µl in four stages over a 2-month period, n=7) (22, 23), or α-MEM (500 µl in four stages over a 2-month period, n=7) were injected into the tail veins of the PD rats.
Apomorphine-induced rotation test
We used the apomorphine-induced rotational test to determine the extent of the retrograde nigrostriatal lesion. The animals received intraperitoneal injection of 0.5 mg/kg apomorphine hydrochloride (Sigma- Aldrich, Germany) dissolved in 1% ascorbic acid, and 0.9% NaCl. The animals were placed on a cylinder (diameter: 28 cm) to monitor rotational asymmetry for 5 minutes. The net rotation asymmetry score was calculated by subtracting the total number of contralateral turns to the lesion from the total number of ipsilateral turns to the lesion prior to transplantation (1 week after the 6-OHDA injection) as well as at 2, 4, 6, and 8 weeks after transplantation (or equivalent times in the other groups). We chose only rats that exhibited at least 4 net rotations/minute (24, 25).
Motor performance was evaluated on a Rotarod equipment (Hugo Basil, Biological Research Apparatus, Italy) with an accelerating protocol (26). The first 3 days of testing served as the training period. The animals underwent a 4 trial test under an accelerating protocol that went from 4 rpm to 40 rpm in 5 minutes, with a rest period for at least 20 minutes between trials. On the fourth day, using the same protocol, we recorded the latency to fall (24, 27).
After 8 weeks, all animals underwent perfusion through the ascending aorta with 150 ml of 0.9% NaCl, followed by 500 ml of 4% paraformaldehyde in 100 mM phosphate buffer. The animals’ brains were extracted, post-fixed, and paraffinized. Next, they were cut at a thickness of 7 µm, starting at 12.3-13.7 mm and 7.9-9.3 mm from the anterior pole of the brain for the SN and striatum, respectively. A total number of six coronal sections per rat were obtained. Sections were deparaffinized and incubated in 0.1% Triton X-100 (Merck, Germany) for 10 minutes followed by 5% goat serum for 30 minutes at room temperature.
The sections were then incubated with the primary antibody anti-TH (1:200, Millipore-AB152, USA) for 24 hours in a wet box at 4°C and then for 1 hour with goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Germany) as the secondary antibody. The sections were washed twice with phosphate buffered saline (PBS) for 10 minutes after each step. When the staining reaction was completed, the tissue sections were sealed after washing and dehydration. The density of TH+ neurons of SN was measured with ImageJ software (28). All data were represented as mean ± SEM values with statistical significance set at P<0.05.
Real-time polymerase chain reaction
After 8 weeks, all animals were killed and we
removed their brains. The ipsilateral and contralateral
striata (with respect to the lesion) were isolated
The mean of the three experiments was used as the
relative quantification value. Relative gene expression
was analyzed using the comparative Ct method, 2-ΔΔCt. All
samples were normalized to the level of
We used SPSS software version 16, for data analysis (SPSS Inc., Chicago). Differences between groups were assessed by one-way ANOVA followed by the Tukey and LSD, least significant difference tests. P<0.05 was considered statistically significant. All values were expressed as mean ± SEM.
Passage-4 of adipose derived stem cells with spindle- shaped morphology
Analysis of the cultured cells by inverted microscope showed fibroblast and spindle-like shaped passage-4 ASCs. In addition, we observed colonies of proliferative cells.
Intravenous administration of adipose derived stem cells and ASC-conditioned medium reduced rotational behavior of parkinson’s disease rats
We did not detect any changes in the numbers of contralateral rotations between groups before, and 2 and 4 weeks after transplantation. At 6 weeks after transplantation, only the ASC-CM group showed a significant decrease in rotations compared to the α-MEM and lesion groups (P=0.01). In contrast, there was a significant lower number of net rotations in the ASC and ASC-CM groups compared to both the lesion (P=0.02) and α-MEM (P=0.01) groups at 8 weeks post-transplantation (Fig .1,).
|Gene||Primer sequence (5ˊ-3ˊ)||Primer size||Amplicon length (bp)||Reference|
Intravenous administration of adipose derived stem cells and ASC-conditioned medium significantiy improved motor coordination on the rotarod test
There was a significant decrease in time spent on the spinning rods of the rotarod in the lesion and α-MEM groups compared to the sham group (P=0.000). The ASCs and ASC-CM groups showed significant increases in time spent on the spinning rod compared to the lesion (P=0.001) and α-MEM (P=0.01) groups. The ASCs and ASC-CM groups showed no significant difference compared to the sham group at 8 weeks post-transplantation (Fig .2,).
Rats with adipose derived stem cells and ASC-conditioned medium transplantation showed better preservation of TH+ neuron density in the substantia nigra
Immunohistochemical images of TH immunopositive neurons were shown (Fig .3A-E,). There was a significant decrease in TH+ neuron density in the SN of the lesion and α-MEM groups compared to the sham group. We observed no significant difference between the treated and sham groups. The density of TH+ neurons in the ASCs and ASC-CM groups was significantly higher than the lesion and α-MEM groups (Fig .3F,).
Neurotrophin gene expressions of the striatum
All groups showed a significant decrease in BDNF geneexpression in the striatum compared to the sham group. ASCs and ASC-CM groups showed a significant increase in geneexpression compared to the lesion (P=0.05) and α-MEM(P=0.02) groups. There was no significant difference betweenthe ASCs and ASC-CM groups. There was a significantincrease in expressions of the NGF and NT3 genes in the ASCs and ASC-CM groups compared to the lesion group (Fig .4,).
In this study, we observed that intravenous administration of ASCs and ASC-CM of benefit and reduced apomorphine-induced rotations, as well as preserved TH-immunoreactive neurons. McCoy et al. (18) reported that the neuroprotective property of ASCs following transplantation was not related to its in vivo differentiation into neurons; instead, infused cells caused high amounts of neurotrophic factors (BDNF, GDNF, and NGF) mRNAs at the lesioned site. These factors have trophic and neuroprotective effects on nigral dopaminergic neurons (30, 31). Gu et al. (16) demonstrated that mesencephalic and cerebellar granule neurons could be protected against 6-OHDA-induced neurotoxicity by ASC-CM. This effect might be related to the neurotrophic factors of CM secreted by ASCs. The use of CM has several advantages compared to stem cells. CM can be manufactured, freeze-dried, packaged, and transported more easily. CM contains no cells; therefore, there is no need to match the donor and the recipient to avoid rejection problems. CM contains various growth factors and tissue regenerative agents, which are secreted by stem cells. However, intravenous injection of cells results in poor cell viability when passing through a thin syringe into the tail vein.
In the mature nervous system, neurotrophic factors play a major role in neuronal protection and the maintenance of cellular homeostasis; therefore, any change in their expression can be associated with neurodegeneration (32). Neurotrophic factors have been shown to activate receptor tyrosine kinases. Within neural precursors and neurons, the pathways regulated by tyrosine kinases include proliferation and survival, axonal and dendritic growth and remodeling, assembly of the cytoskeleton, membrane trafficking and fusion, and synapse formation and function. Recently, many studies on the neurotrophic factors have shown that they regulate each of these functions (33).
BDNF is a neurotrophic factor for dopaminergic
neurons of the SN, the region affected by PD (30).
Reduced expression of BDNF within the SN has been
shown to cause the loss of dopaminergic neurons in PD.
Indeed, postmortem studies of PD patients showed that
a reduction in BDNF accompanied PD and BDNF was
required to preserve neurons of the SN pars compacta (34).
In this study, we assessed
The expressions of
Possibly transplanted ASCs need adequate time to migrate from the peripheral vasculature into the damaged area to protect and restore destroyed dopaminergic neurons. Salinas reported that in PD, NGF like an antioxidant reduced ROS induced cell death due to 6-OHDA (35). It has been revealed that high sensitivity of dopaminergic cells to toxins or free radicals related to glutathione reduction, which was known as an intracellular antioxidant (36, 37).
a result, we observed motor improvement. This
treatment slows neurodegeneration progression. These
reports have suggested that soluble factors of CM activate
endogenous restorative and preserve the level of
The present data provided evidence that neuroprotection
by ASC-CM was associated with stimulation of
This study was financially supported by the Biology School, Damghan University. The authors declare no conflicts of interest.
M.N., M.H.G.K.; Cell culture, preparation of conditioned medium and parkinsonian rats, cell and conditioned medium injection, immunohistochemical staining and article writing, editing and designing. A.R.; Real-time PCR study and article wirting and editing. I.G.; Behavioral study and statistical analysis and article wirting and editing. All authors read and approved the final manuscript.