Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). MS affects millions of people and causes a great economic and societal burden. Currently used treatment drugs have side effects and only address the symptoms but not the causes of MS. In this study, a novel approach of transplanting neural stem cells (NSCs) derived from human primitive mesenchymal stem cells (MSCs) was investigated in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS.
Primitive MSCs were differentiated into NSCs using selective media. The cells were labeled with PKH26 and injected into the tail vein of EAE mice. The animals were evaluated for changes in neurobehavior and weight twice daily. Two weeks following cell transplantation, the animals were sacrificed to collect the blood, lymphatic and CNS tissues for analysis. FACS analysis was used to track labeled cells and infiltrates. Histochemical analysis was performed to determine the levels of myelination. Expression of inflammation, neural, astrogliosis, neuroprotection, and myelination markers was investigated by using immunohistochemical and qRT-PCR analyses.
Neurobehavioral assays showed that EAE disease process was halted by transplantation of both MSCs and NSCs. However, NSCs showed greater efficacy in reversing the disease symptoms, which resulted in near complete recovery of EAE animals. Post-transplantation analyses also showed homing of transplanted cells into the CNS with concomitant induction of anti-inflammatory response resulting in reduction of immune infiltrates. Luxol fast blue staining intensity of CNS tissues was significantly improved in treated mice as compared to EAE animals, suggesting endogenous remyelination. NSC transplantation also modulated Treg and Th17 cells in EAE mice to levels comparable to healthy controls. In addition, several of the markers associated with neuroprotection (i.e. Igf, Bdnf, and Trkb), myelination (i.e. Erk2, Krox-20, Oct-6, Mpz, Mbp, and Mog) and neurogenesis (i.e. Tuj1 and Nestin) were upregulated, suggesting endogenous regeneration in treated animals.
Cell transplantation was more effective at an earlier point of EAE disease (EAE stage 1) than later (EAE stage 2). These promising results provide basis for large-scale clinical studies to treat MS using NSCs derived from primitive MSCs.
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This is a list of supplementary files associated with this preprint. Click to download.
List of Primer Sequences Used in qRT-PCR
Expression of neural and neurotrophic genes and neural proteins determined by qRT-PCR and immunostaining, respectively.
Clinical parameters of the EAE mice prior to and after treatment with primitive MSCs or NSCs
Transcriptional analysis of spleen samples of EAE mice transplanted with cells. Spleens were homogenized and analyzed for mRNA expression of the mouse inflammation genes. Gene expression was normalized to Gapdh and Actin and error bars represent the SEM of triplicate measures (**p ≤ 0.01).
Expression of inflammatory, neural, astrogliosis, neurotrophic and myelination genes in the brain and spinal cord, respectively, determined by qRT-PCR
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Posted 03 Mar, 2021
Posted 03 Mar, 2021
Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). MS affects millions of people and causes a great economic and societal burden. Currently used treatment drugs have side effects and only address the symptoms but not the causes of MS. In this study, a novel approach of transplanting neural stem cells (NSCs) derived from human primitive mesenchymal stem cells (MSCs) was investigated in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS.
Primitive MSCs were differentiated into NSCs using selective media. The cells were labeled with PKH26 and injected into the tail vein of EAE mice. The animals were evaluated for changes in neurobehavior and weight twice daily. Two weeks following cell transplantation, the animals were sacrificed to collect the blood, lymphatic and CNS tissues for analysis. FACS analysis was used to track labeled cells and infiltrates. Histochemical analysis was performed to determine the levels of myelination. Expression of inflammation, neural, astrogliosis, neuroprotection, and myelination markers was investigated by using immunohistochemical and qRT-PCR analyses.
Neurobehavioral assays showed that EAE disease process was halted by transplantation of both MSCs and NSCs. However, NSCs showed greater efficacy in reversing the disease symptoms, which resulted in near complete recovery of EAE animals. Post-transplantation analyses also showed homing of transplanted cells into the CNS with concomitant induction of anti-inflammatory response resulting in reduction of immune infiltrates. Luxol fast blue staining intensity of CNS tissues was significantly improved in treated mice as compared to EAE animals, suggesting endogenous remyelination. NSC transplantation also modulated Treg and Th17 cells in EAE mice to levels comparable to healthy controls. In addition, several of the markers associated with neuroprotection (i.e. Igf, Bdnf, and Trkb), myelination (i.e. Erk2, Krox-20, Oct-6, Mpz, Mbp, and Mog) and neurogenesis (i.e. Tuj1 and Nestin) were upregulated, suggesting endogenous regeneration in treated animals.
Cell transplantation was more effective at an earlier point of EAE disease (EAE stage 1) than later (EAE stage 2). These promising results provide basis for large-scale clinical studies to treat MS using NSCs derived from primitive MSCs.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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