Differentiation and characterization of NSCs-derived from primitive MSCs. We first differentiated the primitive MSCs into NSCs (18), which displayed typical neural extension morphology, significant loss of MSC markers and increased expression of the neural genes, NESTIN, TUJ1, VIMENTIN, and PAX6, as well as neurotrophic factors, CNTF, BMP2, PDGF, BDNF, GDNF, IGF, EGF, and FGF (Fig. 1A-1E and Additional file 2). They also expressed neural proteins, NESTIN, TUJ1, VIMENTIN, and PAX6 (Fig. 1F-1I and Additional file 2). This protocol reproducibly yielded 73% differentiation of MSCs into NSCs (Fig. 1J).
Effect of cell transplantation in EAE mice. We then investigated the effects of MSCs and NSCs on the disease process in EAE mice. EAE was induced by myelin oligodendrocyte glycoprotein (MOG) peptide immunization in animals using an established protocol (16) and disease progression was followed by neurobehavioral analysis (Fig. 2A) (19). The first signs of EAE induction in animals were noticed 11–12 days after MOG injections. Then EAE stage 1 and 2 animals were intravenously injected with PKH26 labeled 106 cells and animals were evaluated for neurobehavioral changes twice a day until they were sacrificed for post-transplantation analysis. Neurobehavioral analyses results showed that average clinical score of the EAE control (4.5 ± 0.5) was significantly higher than the average score of mice transplanted with MSCs (1.7 ± 0.3) and NSCs (0.8 ± 0.1) at stage 1 and MSCs (2.4 ± 0.2) and NSCs (2.2 ± 0.1) at stage 2 (Fig. 2B and Additional file 3). In addition, the average weight of the untreated EAE control (13g ± 0.6g) was significantly lower than the average weights of the mice transplanted with MSCs (15.7g ± 0. 7g) and NSCs (16g ± 0.4g) at stage 1 and MSCs (14g ± 0.7g) and NSCs (14.6g ± 0.7g) at stage 2 (Fig. 2C and Additional file 3). When MSCs were transplanted at EAE stage 1, they slowed and halted the disease progression. However, NSCs not only slowed but also reversed the disease process, and animal conditions were improved to near normal levels. Overall, NSCs showed greater efficacy than MSCs when transplanted at EAE stage 1 than stage 2. Animals were sacrificed two weeks post-transplantation, and blood, lungs, lymphatic and CNS tissues were collected for histological, biochemical, immunological and molecular analyses.
Homing of transplanted cells to the blood, lymphatic, and CNS systems. The labeled transplanted cells homed to the blood, spleen, brain, and spinal cord (Fig. 3A). Only an insignificant number of transplanted cells were found in the lungs of all animals, suggesting minimal lung trapping. Interestingly, significantly more transplanted MSCs (10.0%) than NSCs (6.7%) were observed in the blood of healthy animals, suggesting greater survivability of MSCs in the vascular system. In the blood of EAE stage 1 animals, the number of transplanted MSCs and NSCs was 9.3% and 10.3%, respectively. Strikingly, a very low number of MSCs and NSCs (1.2% and 2.0%, respectively) were found in the blood of EAE stage 2 animals. Nearly half of transplanted cells were found in the spleen in all animals. Importantly, a significant number of transplanted cells were present in the CNS of EAE animals but not in healthy controls. In the case of EAE stage 1 animals, a similar number of transplanted MSCs and NSCs were found in the brain (7.0% and 7.5%, respectively) but significantly more NSCs than MSCs (10.5% and 12.3%, respectively) were found in spinal cord. Overall, the number of transplanted labeled MSCs and NSCs found in the CNS of EAE stage 1 animals were more than Stage 2. Notably, NSCs were significantly more in the spinal cord than MSCs. Overall, NSCs exhibited greater survivability and/or proliferation in EAE stage 1 animals than MSCs.
Improvement in the histopathology of the brain and spinal cord of EAE mice by transplanted cells. EAE induction mediates T cell activation resulting in increased infiltrates and demyelination as well as permeation of the BBB (20). The brain and spinal cord (Fig. 3B and 3D) showed presence of cell infiltrates in EAE animals but not in heathy controls. The number of infiltrates in both brain and spinal cord were significantly decreased in EAE stage 1 compared to stage 2 animals treated with MSCs and NSCs. Furthermore, the LFB staining of the brain and spinal cord (Fig. 3C and 3E) was greatly improved in NSC treated animals compared to those treated with MSCs. The stain intensity was significantly increased in the cortex and spinal cord of EAE stage 1 (less in stage 2) animals treated with the cells suggesting endogenous remyelination. Taken together, these results suggest that transplanted NSCs not only reduced cell infiltrates, but also showed increased remyelination in the CNS at EAE stage 1 compared to the stage 2.
Modulation of the immune response by transplanted cells. The main effector cells of cell mediated immune response in EAE and MS are Treg (CD4 + and CD25+) and Th17 (CD4 + and IL17A+) (20). We investigated the level of these cells in the blood, spleen, and CNS by flow cytometry. Treg cells were significantly reduced to 8.6% and 7.3% in EAE animals from 35.6% and 43.0% in healthy controls in both the blood and spleen (Fig. 4A). In contrast, Treg cells were substantially increased in EAE animals transplanted with cells in the blood and spleen. In EAE stage 1 animals treated with MSCs, Treg cells were 27.4% and 41.1% in the blood and spleen, respectively. Whereas in EAE stage 2 animals treated with MSCs, Treg cells were 15.8% and 37.5% in the blood and spleen, respectively. On the other hand, in EAE stage 1 animals treated with NSCs, Treg cells were 39.3% and 42.0% in the blood and spleen, respectively. In EAE stage 2 animals treated with NSCs, Treg cells were 22.3% and 42.6% in the blood and spleen, respectively. In conclusion, Treg cell numbers increased in animals treated with both MSCs and NSCs; however, the improvements were more significant in the case of NSCs and their efficiency of restoring Treg levels was greater in EAE stage 1 than stage 2 animals. In the case of the CNS, Treg levels were 14.6% and 22.6%, respectively, in EAE animals. While cell transplantation did not affect the infiltrated Treg cells in the brain, Treg cells were significantly reduced to 14.8% and 10.0% in the spinal cord of EAE stage 1 animals treated with MSCs and NSCs, respectively. Cell transplantation in EAE stage 2 animals was less effective, as Treg cells reduced only to 17.0% in both MSCs and NSCs. Taken together, NSCs showed greater efficacy than MSCs in reducing Treg cells in the spinal cord in EAE stage 1 animals. It is known that Treg cells increase during the chronic late stages of EAE (21). So, reduced Treg levels in animals treated with cells could contribute to improved disease symptoms.
As expected, Th17 cells were significantly increased from 10.3% and 6.9% in the blood and spleen of healthy controls to 15.3% and 13.7% in EAE animals, respectively. When treated with MSCs, Th17 cell levels were not significantly affected in the blood (14.9%) but were reduced to normal levels (10.1%) in the spleen in EAE stage 1 animals; however, no effect was noticed in the case of EAE stage 2 (Fig. 4A). Importantly, NSC transplantation reduced Th17 in EAE stage 1 and stage 2 animals to levels similar to the healthy control animals. Both the brain and spinal cord of EAE animals had significantly high levels of Th17 cells (22.5% and 30.5%, respectively) as compared healthy control animals (1.6% and 0.9%, respectively). Upon MSC transplantation, Th17 cells were significantly reduced in the brain and spinal cord of EAE stage 1 animals (12.6% and 20.3%, respectively) but not in EAE stage 2 animals. On the other hand, NSC transplantation had a more prominent effect on reducing Th17 cells in both the brain and spinal cord in EAE stage 1 (11.5% and 13.7%, respectively) and in stage 2 (19.4% and 22.2%, respectively) animals. However, the NSC effect was less significant in EAE stage 2 animals.
Effect of transplanted cells on immune infiltrates in the CNS. To investigate the effect of transplanted cells on the inflammatory response in EAE mice, we analyzed the expression of CD45, CD68, and CD3E markers, representing leukocytes, macrophages, and T cells, respectively. The results depicted in Fig. 4B to 4D show high expression of the markers in the brain and spinal cord of EAE animals but not in healthy controls. Importantly, MSCs and NSCs were more effective in reducing the expression of all three markers in the spinal cord of EAE stage 1 but not stage 2 animals. Clearly, NSCs exert a greater anti-inflammatory response, particularly in EAE stage 1 animals potentially by reducing the pro-inflammatory cells, macrophages, leukocytes, and T cells.
Restoration of myelination and inhibition of astrogliosis by MSCs and NSCs. We next examined the effect of transplanted cells on the proteins associated with myelination and neural regeneration. We observed a significant decrease in the expression of MOG, MBP, TUJ1, and NESTIN in the brain of EAE animals (Fig. 5A and 5C). The expression of MOG and MBP was significantly increased in the brain upon transplantation of NSCs in both stages of EAE. However, MSCs had a significant increase in EAE stage 1 animals only. Similarly, the expression of TUJ1 was significantly increased in the brain of both EAE stage 1 and stage 2 animals transplanted with NSCs, but this was only seen in MSC treated stage 1 animals. Upon induction of EAE, the expression of GFAP increased (Fig. 5A and 5C), due to increased astrogliosis in the brain of EAE animals (22). However, GFAP expression was reduced in both EAE stage 1 and stage 2 animals transplanted with cells. The trend in the expression of MOG, MBP, TUJ1, and NESTIN in the spinal cord of EAE animals treated with cells was similar to the brain. However, NESTIN was significantly increased in EAE stage 1 animals treated with NSCs and the decrease of GFAP in the spinal cord of transplanted animals was not significant (Fig. 5B and 5D). Overall, NSCs promoted greater remyelination and neural recovery as well as reduction of astrogliosis in the EAE stage 1 animals.
Effect of cells on the transcription for inflammatory, astrogliosis, neuroprotection, and myelination genes. To determine the effects of transplanted cells on the expression of inflammatory, neuroprotection, and myelination genes, transcriptional analysis of the spleen, brain, and spinal cord tissues was performed. As expected, higher expression of the inflammatory markers, Cd3e, Il-17a, Il-2, Il1β, Il-6, Tnfα, and Ifnγ was observed in the brain, spinal cord, and spleen, respectively, in EAE animals compared to the healthy controls (Fig. 6A and 6B, Additional file 4, and Additional file 5). Expression of these markers was significantly reduced in the brain of MSC and NSC treated EAE stage 1 and stage 2 animals, except Il-17a in MSC treated Stage 2 animals. In contrast, expression of the inflammatory markers were significantly reduced in the spinal cord when NSCs and MSCs were transplanted at EAE stage 1 but not stage 2. Similar to the brain, expression of these markers was significantly reduced in the spleen of animals transplanted with both types of cells at EAE stage 1 and stage 2, except for the Cd3e, Il-17a, Il-2, and Il-1β markers, in MSCs transplanted in EAE stage 2 animals.
Cytokine storm is elicited in MS. Although many cytokines take part in the “cytokine storm”, IL-6 plays a crucial role. Dysregulated continual release of IL-6 has pleiotropic effects on chronic inflammation and autoimmunity, and also lead to the onset or development of various diseases including MS, acute respiratory distress syndrome, and multiple organ dysfunction syndrome (23). In EAE, IL-6 aggravates clinical symptoms and spinal cord pathology mainly by promoting pathogenic Th17 cells, which initiate and perpetuate inflammation and demyelination (24–26). IL-6 also affects autoreactive effector T cells in MS. IL-6 signaling supports T effector cell resistance to regulation by Treg cells, which may contribute to disease aggravation (27). Our results show that cell transplantation was effective at countering the induction of Il-6 gene expression in spleen and CNS tissues. In addition, NSCs showed more efficacy in regulating Treg cells when compared to MSCs, suggesting that NSCs are more potent in eliciting anti-inflammatory effects than MSCs in the CNS.
As anticipated, the expression of neural markers, Tuj1 and Nestin, was downregulated in the CNS in EAE animals but was significantly increased in transplanted animals (Fig. 6A and 6B and Additional file 5). Studies have shown that upregulation of Gfap and Ccnd1 is associated with reactivation of astrocytes (astrogliosis) (22, 28). Overall, EAE stage 1 animals transplanted with NSCs displayed reduction of astrogliosis genes in the brain and spinal cord. Our results also suggest that expression of Gfap and Ccdn1 is likely to be regulated by the JAK/STAT pathway and promote the transcription of astrogliosis genes such as Gfap, Cntfrα, and Nfl-b in EAE animals (Fig. 6A and 6B and Additional file 5). Our results demonstrated that there was an upregulation of these astrogliosis genes via activation of the JAK/STAT pathway leading to an increase of astrogliosis in EAE mice. However, in EAE stage 1 animals treated with NSCs, these genes were significantly downregulated causing a decrease in astrogliosis in the CNS.
We determined that several paracrine neurotrophic factors were significantly upregulated in NSCs versus MSCs in vitro (Fig. 1E). Predictably, these factors contributed to the greater efficacy of NSCs for neuroprotection and the reversal of EAE symptoms. The CNS also showed higher expression of several neurotrophic factors such as Igf, Bdnf, and Trkb, which are pertinent to neuroprotection (Fig. 6A and 6B and Additional file 5). The fact that the expression of these factors is improved more significantly in EAE animals transplanted with NSCs, and that the animals recovered to near normal provide a ‘proof of concept’ that the use of NSC therapy in MS patients would more effective than using MSCs.
Factors controlling myelination have been reported to synergistically induce the expression of Krox-20, which is involved in activation of myelin genes such as Mpz and Mbp (29, 30). Our results showed that several genes, Erk2, Krox-20, Oct-6, Mpz, Mbp, and Mog, associated with myelination were downregulated in EAE animals. Expression of these genes was significantly increased in the brain and spinal cord of EAE stage 1 animals injected with both types of cells, but NSCs provided a greater improvement compared to EAE stage 2 (Fig. 6A and 6B and Additional file 5).
Mechanism of neural protection and regeneration. Our results showed downregulation in EAE animals but increased expression of Oct-6 and Krox-20 in transplanted animals, which suggests that the MAPK pathway may be involved in the regulation of these genes, because inhibition of MAPK signaling has shown to upregulate myelination associated genes (31). Chronic demyelination favors neurodegeneration of denuded axons, which causes irreversible neuronal deficits and disability in MS patients (32). Furthermore, transplanted cells promoted neural survival and regeneration responsible for increased LFB stain intensity, indicative of an increase in remyelination of denuded axons. Taken together, results of this investigation suggest that NSCs influence not only MAPK but also several other signaling pathways, particularly MAPK, JAK/STAT, and PI3K/AKT as illustrated in Fig. 6C.