Research Article |
Corresponding author: Maryam Haji Ghasem Kashani ( kashani@du.ac.ir ) Academic editor: Georgi Momekov
© 2023 Dhiya Altememy, Maryam Haji Ghasem Kashani, Pegah Khosravian.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Altememy D, Haji Ghasem Kashani M, Khosravian P (2023) Selegiline induced differentiation of rat bone marrow mesenchymal stem cells to dopaminergic neurons in vitro. Pharmacia 70(4): 959-965. https://doi.org/10.3897/pharmacia.70.e107909
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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.
Selegiline, dopaminergic genes, in vitro culture
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 umbilical 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 (
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 (
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 (
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 (
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 (
There have been reports that selegiline induced the differentiation of BMSCs into dopaminergic neurons (
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 (
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).
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 (
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, NESTIN, 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.
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 (
Total RNA was extracted from experimental groups using RNX-Plus (Sinaclon, Iran). The quality of the RNA was confirmed by agarose gel electrophoresis.
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 (
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
Accession number | Primer sequence | Product size (bp) | Gene |
---|---|---|---|
NM_012604 | F: 5’-TTA AAT GCC TTG GCC ATC TC-3’ | 173 bp | SHH |
R: 5’-CGA GCC AGC ATG CCA TAC TT-3’ | |||
NM_012842 | F: 5’-CCT CCT TTA CGG TGG ACA AA-3’ | 269 bp | PTCH |
R:5’-ATC AAC TCC TGC CCA TG-3’ | |||
NM_012512 | F: 5’-CCG TGA TCT TTC TGG TGC TT-3’ | 318 bp | Β2M |
R: 5’-TTT TGG GCT TCA GAG TG-3’ | |||
U_72345 | F: 5׳-TCC CGG AGG AAC TGC ACT TCG-3׳ | 683 bp | NURR1 |
R: 5׳-GTG TCT TCC TCT GCT CGA TCA-3׳ | |||
NM_012740 | F: 5׳-TGT CAC GTC CCC AAG GTT CAT -3׳ | 276 bp | TH |
R: 5׳ –CGT GGG ACC AAT GTC TTC AGT G-3׳ | |||
NM_012987 | F: 5׳- CAG- GCT –TCT- CTT- GGC- TTT-CTG-3׳ | 431 bp | Nestin |
R: 5׳- TGG- TGA- GGG-TTG -AGG -TTT- 3׳ |
Primer | Sequence | Product size |
---|---|---|
GAPDH-f | GCTGGGGCTCATTTGCAGG | 258 bp |
GAPDH-r | CGGAGGGGCCATCCACAGT | 258 bp |
TH-f | TGT CAC GTC CCC AAG GTT CAT | 276 bp |
TH-r | CGT GGG ACC AAT GTC TTC AGT G | 276 bp |
NURR1-f | TCC CGG AGG AAC TGC ACT TCG | 683 bp |
NURR1-r | TGG TGA GGG TTG AGG TTT | 683 bp |
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.
Colony formation and expansion during the second subculture were maximal when the cells were cultured in α-MEM containing 10% FBS (Fig.
Phase-contrast images of rBMSCs at different passages. (A) The cultured cells formed colonies after the first subculture (200×); (B) The cells at passage 3 (200×); (C) Different morphologies of rBMSCs at passage 4: Round and proliferating cells (Red arrow), Fibroblast cells (Blue arrow), and Flat cells (Green arrow) (200×).
Rat BMSCs demonstrated a strong capacity for differentiation into adipogenic and osteogenic lineages. Control cells at passage 4 were shown in Fig.
Rat BMSCs differentiation into osteoblasts or adipocytes. (A) Control (200×); (B) Alizarin Red stained the cells of the control group after 21 days (200×); (C) Differentiation of MSCs into osteoblasts and detection of calcium deposits (mineralization) after 21 days (400×); (D) Calcium deposits stained with Alizarin red (400×); (E) Differentiation of MSCs into adipocytes and production of lipid droplets after 21 days (400×); (F) Lipid droplets stained with oil red (400×).
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.
About 70% of cells in the control group and more than 90% of 10 -8 selegiline-treated cells were nestin-positive (Fig.
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
The expression of the TH and NURR1 genes in the cells treated with selegiline was significantly increased compared to the control (Fig.
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. 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 (
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 (
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.