Selegiline induced differentiation of rat bone marrow mesenchymal stem cells to dopaminergic neurons in vitro

Today, the use of mesenchymal stem cells (MSCs) for treating human diseases has attracted wide attention. The aim of this study is the expression of dopaminergic genes such as Nestin, patched Tumor Suppressor (PTCH), Sonic Hedgehog (SHH), Tyrosine Hydroxylase (TH) and Nuclear receptor-related factor 1 (NURR1) in MSCs after induction with selegiline. Rat bone marrow mesenchymal stem cells (rBMSCs) were extracted from femur and tibia bones and incubated with alpha Minimum Essential Medium (α-MEM) and 10% Fetal bovine serum (FBS). The stemness of cells at passage 4 was determined by the positive response to CD71 and CD90 markers and their differentiation into adipocytes and osteoblasts. The expression of SHH, PTCH, TH, NURR1 and Nestin genes in the cells after induction by 10-8 M selegiline for 48 hours was investigated by Reverse transcription polymerase chain reaction (RT-PCR) and Real Time-PCR methods. Isolated rBMSCs expressed CD71 and CD90 markers in culture conditions and could differentiate into adipocytes and osteoblasts. Induced cells showed neuronal morphology, positive response to Nestin and TH immunostaining. There was a significant increase of dopaminergic genes TH and NURR1 compared to the untreated cells. The results showed that selegiline with a dose of 10-8 M for 48 hours can lead to dopaminergic differentiation in rBMSCs.


Introduction
Mesenchymal stem cells (MSCs) are non-hematopoietic cells discovered about 40 years ago from bone marrow by Friedenstein (Charbord et al. 2010).Many studies have shown that stem cells are in different tissues and successfully harvested from other organs such as the brain, liver, kidney, muscle, thymus, pancreas, skin, adipose tissue, bone marrow, lymph nodes, spleen, thymus, and um-bilical cord.Also, MSCs are known as multipotent cells, which can differentiate into adipocytes, myocytes, osteocytes, and chondrocytes.Studies indicate that MSCs can differentiate into non-mesodermal cells such as intestinal epithelial cells, skin, hepatocytes, pneumocytes, and neurons (Lim et al. 2021).In 2006, the International Society of Cell Therapy proposed at least three characteristics for the human MSCs: 1-positive expression of CD105, CD90, and CD73 markers.2-the negative expression of CD45, CD34, CD14, CD11b, CD79α, CD19 markers, and HLA-DR surface molecules, and 3-the ability to adhere to the cell culture dish and to differentiate into osteocytes, chondrocytes, and adipocytes (Dominici et al. 2006).The stem cells can be divided into three main groups: embryonic, germline, and somatic (Hong et al. 2012).These pluripotent cells originate from the embryo's inner cell mass or blastocysts and have an unlimited life span related to telomerase activity.However, using ESCs in scientific studies and clinical trials is limited due to ethical considerations (Warmflash et al. 2012).Induced pluripotent stem cells (iPSCs) can be replaced with ESCs.iPSCs from a patient's somatic cells could be valuable for drug discovery and cell transplantation therapies.The differentiation ability of iPSCs is similar to ESCs.Still, these cells do not have the ethical or immunogenic limitations of ESCs, so for this reason, these cells may be used more clinically than ESCs (Worku 2021).Germinal stem cells originate from the embryo's primary germ layers; these cells can differentiate into progenitor cells to produce specific cells of the body tissues.Somatic or adult stem cells (SSCs) are precursor cells with less differentiation ability than embryonic stem cells.SSCs are in mature tissues such as hematopoietic, nervous, digestive, and mesenchymal tissues.The most common adult stem cells include: bone marrow stem cells, hematopoietic stem cells, and MSCs.BMSCs can differentiate into mesodermal cells such as cartilage, bone, fat, muscle, connective tissue, and tendons in vitro.In addition to the capacity for self-renewal and differentiation potential, these cells can suppress the immune system.Also, these cells have anti-tumor and migration properties.Stem cells express growth factors and cytokines that regulate an innate immune response.Stem cells can secrete factors such as MCP-1/CCL2 and physically interact with tumor cells.The evidence indicates that the types of chemokine and growth factor receptors expressed by MSCs may participate in the implantation of tumors.CXCR4/SDF1 factor plays a prominent role in migrating different stem cells (Hong et al. 2012;Prentice 2019).
Stem cells are used as autograft transplantation in neurological diseases such as stroke, spinal cord injuries, Parkinson's, Alzheimer's, Huntington's, and Autism.Differentiation of MSCs into neural-like cells has been reported both in vitro and in vivo after migration to the brain and spinal cord (Kousha et al. 2022;Pradhan et al. 2022).
Recent studies indicate that MSCs can differentiate into different types of nerve cells after treatment with growth factors and chemical inducers (Iglesias-García et al. 2013).So, under proper conditions, these cells can differentiate into particular neurons, including dopaminergic neurons, and can be used to treat Parkinson's disease.
Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by a progressive and extensive loss of neurons in the substantia nigra pars compacta (SNpc) and their terminals in the striatum, which results in debilitating movement disorders.This devastating disease affects over 1 million individuals in the United States and is increasing in incidence worldwide.Available pharmacological and surgical therapies ameliorate clinical symptoms in the early stages of the disease, but they cannot stop or reverse the degeneration of dopaminergic neurons.Stem cell therapies have come to the forefront of the PD research field as promising regenerative therapies (Obeso et al. 2010;Parmar et al. 2020).
Selegiline is known to increase the survival of cultured nigral DAergic neurons, protecting them from oxidative stress.It has been reported that selegiline can protect hippocampal neurons from excitotoxic damage, most likely by induction of NGF protein (Semkova et al. 1996).The trophic effects of selegiline may play a significant role in treating neurodegenerative diseases (Vezina et al. 1992).
Chemical induction is simple and cost-effective compared to other methods.Selegiline is a selective, irreversible monoamine oxidase B (MAO-B) inhibitor at the conventional dose (10-8 M).MAO-B is an enzyme in the body that breaks down several chemicals in the brain, including dopamine, and slows the progression of PD (Ghorbanian et al. 2010;Haji Ghasem Kashani et al. 2013).
There have been reports that selegiline induced the differentiation of BMSCs into dopaminergic neurons (Beiki et al. 2022).Some researchers believe that chemical inducers have a toxic effect on cells and cause 50% of cell apoptosis after treatment for 24 hours.These cells expressed neuronal markers in the early hours after induction, but this phenomenon is reversible (Hokari et al. 2008).Many studies show that ascorbate, selegiline, vitamin E, and DMSO as essential antioxidants that protect against neurotoxic damage of dopaminergic neurons in the substantia nigra.Selegiline has been shown to prevent dopamine neuron death in Parkinson's disease (Hao et al. 1995;Kontkanen et al. 1999).This drug can improve the symptoms of Parkinson's disease by several mechanisms, which include: 1-it has trophic effects on dopaminergic neurons (Koutsiliere et al. 1994).Selegiline increased neuronal survival and the average neurite length and created branching in neurons and synapses (trophic effects) between neurons of dopaminergic neurons in rats treated with MPP+ (1-methyl-4-phenylpyridinium).2-Selegiline, as a MAO-B inhibitor, prevents the production of peroxides resulting from dopamine oxidation and prevents disease progression.So, a neuroprotective role for this drug is considered (Mytilineou et al. 1997).3-Selegiline reduces the apoptosis of neurons or stops it (Tatton et al. 1997).Selegiline at a concentration of 10-8 M had an anti-apoptotic effect.In addition, its concentrations of 10-7 to 10-9 M reduce oxidative damage of nerve cells in the culture condition (Suuronen et al. 2000).This drug can slow down the progress, weakness, and disability of the disease (Mytilineou et al. 1997).Selegiline reduces the neurodegeneration of PD by astrogliosis.Therefore, by releasing astroglial-derived neurotrophic factors, astrocytes cause the survival and protection of damaged neurons in PD (Hao et al. 1995).This study aimed to investigate the quantitative expression of dopaminergic genes in the cells treated by selegiline.

Isolation and culture of rat bone marrow mesenchymal stem cells (rBMSCs)
In this experimental study, 15 adult male Wistar rats (weighing about 200-250 g) were purchased from the Pasteur Institute of Iran and housed at 20-24 °C under a 12-h light/ dark cycle with free access to water and food for one week.The animals were sacrificed by chloroform, and the femur and tibia bones were separated.Under the hood, the epiphyses were cut, and bone marrow was flushed with alpha Minimum Essential Medium (α-MEM), 10% fetal bovine serum (FBS), and 1% penicillin/ streptomycin into the culture flask.After 72 hours of incubation, the cell culture medium was replaced with a fresh medium.The Cells were passaged at 70%-80% confluency (Locke et al. 2009)

Differentiation potential of rBMSCs
rBMSCs (passage 3) were cultured in 12-well plates and treated with adipogenic medium (StemPro Adipogenesis Differentiation Kit, A10070-01, Invitrogen) or osteogenic medium (StemPro Osteogenesis Differentiation Kit, A10072-01, Invitrogen).The medium was changed every three days.After 21 days, the cells were fixed with 4% formaldehyde for 1 hour at 4 °C, washed with 70% alcohol, and stained with oil-red for 10-15 min or alizarin red for 2 min.The cells were washed with PBS and observed by an inverted microscope (E600-Eclipse Nikon) equipped with a digital camera (DXM 1200 Camera Nikon Digital) (Locke et al. 2005).

Experimental groups
The cells at passage four were cultured in α-MEM containing 10% FBS as control.The selegiline group was induced cells with 10 -8 M selegiline for 24 h and transferred to serum-free medium for 48 h (Suuronen et al. 2000).

Identification of neural cells
In this study, the expression of neural markers such as βIII-tubulin, nestin, and Tyrosine Hydroxylase (TH) was investigated by immunocytochemistry.The term Sonic Hedgehog (SHH), patched Tumor Suppressor (PTCH), TH, NES-TIN, and Nuclear receptor-related factor 1 (NURR1) genes was analyzed by RT-PCR.In addition, Real-time PCR was used to detect the expression levels of TH and NURR1 genes.

Immunocytochemistry
The differentiated cells were cultured on sterile coverslips coated with gelatin and fixed with 4% paraformaldehyde for half an hour at room temperature.After washing with PBS for 5 minutes, the samples were incubated with blocking serum.The samples were incubated overnight at 4 °C with monoclonal antibody to βIII-tubulin and HRP secondary antibody for 2 h.After incubation with DAB for 20 minutes, it was examined with a light microscope (Prentice 2019).

Extraction of total RNA
Total RNA was extracted from experimental groups using RNX-Plus (Sinaclon, Iran).The quality of the RNA was confirmed by agarose gel electrophoresis.

Reverse transcription polymerase chain reaction (RT-PCR)
cDNA synthesis was carried out using 0.5 µg of total RNA according to the manufacturer's protocol (k1622; Fermentase).Expressions of SHH, PTCH, Nestin, TH and NURR1 genes were measured by semiquantitative RT-PCR using a master cycler (Eppendorf, Germany).The PCR mix consisted of 5 µg of synthesized cDNA, 1× PCR buffer, 50 mM MgCl2, 10 mM dNTPs, 10 pmol forward and reverse primers, and 0.25 µl Taq DNA polymerase in a final volume of 25 µl.The PCR protocol comprised 2 min at 94 °C; 34 cycles of 30 sec at 94 °C, 30 sec at 55 °C, and 30 sec at 72 °C; and 5 min of a terminal extension at 72 °C.The primer sequences and product sizes were shown in Тable 1. β2 microglobulin  (β2M) was used as the housekeeping gene (internal control).The PCR products were analyzed by 1.5% agarose gel electrophoresis, then visualized and photographed on a UV transilluminator (UVIdoc, EU).The intensity of gene bands was checked by the Image J software (Freeman et al. 1999).

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR)
Real-time PCR was done by Rotor-Gene 6000 PCR system, using RealQ Plus Master Mix Green (Amplicon, Denmark).The final volume of the reaction solution was 10μl, and the program was set as denaturation at 95 °C for 15 min, followed by 50 cycles at 95 °C for 15s and annealing/extension for 45s at 60 °C.Primer sequences were designed by AlleleID software version 7.5 (Premierbiosoft, USA), as shown in Table 2.The relative expression of target genes was calculated through the 2 -ΔΔCt formula.

Statistical analysis
Data analysis was performed using SPSS software version 16.After ensuring the normal distribution of data, the Kolmogorov test and the Independent-sample T-Test investigated a significant difference between the groups.P<0.05 was considered a significant level.

Results rBMSCs morphology in culture condition
Colony formation and expansion during the second subculture were maximal when the cells were cultured in α-MEM containing 10% FBS (Fig. 1A, B).Cells with small rounded, fibroblast-like, and large flattened morphologies were observed in the 4 th passage (Fig. 1C).Gradu-ally, the fibroblast-like cells increased the heterogeneous cells decreased at high passage.The fibroblast-like cells (MSC) gradually increased, and the heterogeneous cells decreased in the culture.Rat BMSCs demonstrated a strong capacity for differentiation into adipogenic and osteogenic lineages.Control cells at passage 4 were shown in Fig. 2A, and Alizarin Red stained the cells of the control, and only the deposition of colored substances was observed in Fig. 2B.Osteogenic differentiation was confirmed by the production of calcium phosphate and mineralized extracellular matrix in   induced cells stained with Alizarin Red (Fig. 2C, D).Adipogenic differentiation was confirmed by the appearance of small lipid vesicles formed after treatment and stained with oil red (Fig. 2E, F).

Selegiline-induced cells morphology
Within 4 hours after selegiline induction, the cells showed a triangular appearance with long processes.The cells showed a neuron-like morphology, at 24 h after induction (Fig. 3).

PCR results
There was a significant increase of NURR1 and TH genes expression in selegiline-induced cells compared to the control.There was no significant difference between experimental groups in SHH, PTCH, and Nestin expression (Figs 7,8).

Real Time RT-PCR results
The expression of the TH and NURR1 genes in the cells treated with selegiline was significantly increased compared to the control (Fig. 9).

Discussion
Many reports indicate that MSCs, due to their ability to differentiate into dopaminergic neurons, are a great therapeutic cell source to treat Parkinson's disease.(Liu 2022).This study aimed to investigate the effect of selegiline on the neural differentiation of rBMSCs.MSCs derived from bone marrow are a heterogeneous population of cells that expand quickly in vitro.These cells expressed CD71 and CD90 (Friedenstein et al. 1970).There are also reported that rBMSCs differentiate into non-mesodermal cells, including neurons, under appropriate conditions (Xiang et al. 2008).In this study, a homogenous population of cells was obtained after the third passage.The cells in passage 3 were spindle-shaped but became flat upon cell aging.Positive response (more than 95%) to CD71 and CD90 markers showed stemness.
In addition, a positive response to nestin antigen confirmed the neural progenitor cells of selegiline-induced group.But its expression level differed in experimental groups, so it was expressed about 70% in the control group and more than 90% in the selegiline-treated group.The cells of the selegiline-treated group responded positively to TH and βІІІ -tubulin by immunocytochemical staining.The analysis of RT-PCR data showed no significant difference in dopaminergic genes such as SHH, PTCH, and nestin between the two experimental groups.Still, a significant increase of TH and NURR1 genes in the selegiline-treated cells was observed compared to the control.In addition, the Real Time-PCR data confirmed the mentioned results.Some researchers believed that the expression of neural factors occurs during the first hours after induction, and this feature was reversibly lost (Hokari et al. 2008).However, no such result was observed in this study.A dose 10-8 M of selegiline has recently been shown to exert anti-apoptotic and neuroprotective effects.Produced dopaminergic neurons expressing TH and NURR1 genes.The selegiline-induced cells showed a significant increase in these genes, as compared to the control.These results are under Barzilay and colleagues, who generated dopaminergic neurons from MSCs by 12 different induction protocols.They showed that a two-step protocol can produce dopaminergic neurons from bone marrow MSCs.In the first stage, the cells were cultured in the serum-free medium containing EGF and FGF-2 for 48 to 72 hours, then transferred to the induction medium containing cAMP, IBMX, and rich in various neurotrophic factors for another 48 to 72 hours (Barzilay et al. 2008).The results showed that a basic level of TH is expressed in all induction protocols.A significant increase of TH mRNA was found, using BDNF alone or combined with GDNF, TGF-β3.They showed that using various neurotrophic factors, about 30% of MSCs express a detectable level of TH.Dopamine secretion was also observed in the mentioned study (Ahmed et al. 1995;Herzog et al. 2003).In 2021, Kashani et al. reported that the exposure of hASCs cells to 10-7 M selegiline for 24 hours led to the induction of neural-like cells, as well as a significant increase in the expression of NTF3 and BDNF genes compared to cells cultured in serum-free medium.This treatment did not affect the expression of NGF and GDNF genes (Amiri et al. 2021).

Conclusion
This research showed that selegiline-induced cells expressed TH antigen and relatively some neurotrophic factors that play a role in different stages of survival and differentiation of dopaminergic neurons.Selegiline at a dose of 10-8 M causes the expression of specific genes of dopaminergic neurons, such as TH and NURR1.

Figure 7 .
Figure 7. Agarose gel images of PCR product of dopaminergic genes in experimental groups.(A) Control; (B) Selegiline-induced cells.

Figure 8 .
Figure 8.Comparison of SHH, PTCH, TH, NESTIN, and NURR1 gene expression levels in experimental groups.Data are represented as mean ± SD, n = 3. * Significant difference versus control.

Figure 9 .
Figure 9.Comparison of the expression level of TH and NURR1 genes in experimental.Data are represented as mean ± SD, n = 3. * Significant difference versus control.
. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 23-80, revised 1996) and were authorized by the Islamic Azad University of Damghan's research ethical standards (IR.IAU.DAMGHAN.REC.1400.028).

Table 1 .
The primers used for RT-PCR.

Table 2 .
The primers used for Real time RT-PCR.
Chen et al. have reported that MSCs can proliferate and differentiate into neurons and maintain survival in the endogenous neurogenic niche of the Alzheimer's disease model.They also observed that transplantation of MSCs in rodents' Alzheimer's models led to neurogenesis and improved spatial learning (Andrzejewska et al. 2021; Chen et al. 2021).Liu et al. reported that MSCs can stimulate astrocyte proliferation and maintain neurons' survival