Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair

Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

Original Article

Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair Yuan Zhang,1,2,5 Xing Li,1,2,5 Bogoljub Ciric,1 Cun-Gen Ma,3 Bruno Gran,4 Abdolmohamad Rostami,1 and Guang-Xian Zhang1 1Department

of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA; 2Key Laboratory of the Ministry of Education for Medicinal Resources and Natural

Pharmaceutical Chemistry, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, China; 3Institute of Brain Science, Department of Neurology, Shanxi Datong University Medical School, Datong 037009, China; 4Clinical Neurology Research Group, Division of Clinical Neuroscience, University of Nottingham School of Medicine, Nottingham NG7 2RD, UK

Inflammatory demyelination and axonal damage of the CNS are hallmarks of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Fingolimod (FTY720), the first FDA-approved oral medication for MS, suppresses acute disease but is less effective at the chronic stage, and whether it has a direct effect on neuroregeneration in MS and EAE remains unclear. Here we show that FTY720, at nanomolar concentrations, effectively protected survival of neural stem cells (NSCs) and enhanced their development into mature oligodendrocytes (OLGs) in vitro, primarily through the S1P3 and S1P5 receptors. In vivo, treatment with either FTY720 or NSCs alone had no effect on the secondary progressive stage of remitting-relapsing EAE, but a combination therapy with FTY720 and NSCs promoted significant recovery, including ameliorated clinical signs and CNS inflammatory demyelination, enhanced MBP synthesis and remyelination, inhibited axonal degeneration, and reduced astrogliosis. Moreover, FTY720 significantly improved incorporation and survival of transplanted NSCs in the CNS and drove their differentiation into more OLGs but fewer astrocytes, thus promoting remyelination and CNS repair processes in situ. Our data demonstrate a novel effect of FTY720 on NSC differentiation and remyelination, broadening its possible application to NSC-based therapy in the secondary progressive stage of MS.

INTRODUCTION Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS that results in oligodendrocyte (OLG) loss and neuronal dysfunction.1,2 It is believed that at the chronic stage of MS and of its animal model, experimental autoimmune encephalomyelitis (EAE), the main processes underlying disease progression are OLG and neuron death, axonal loss, and myelin destruction.3,4 In the last few decades, immunomodulatory drugs for MS treatment have been developed that blunt the early inflammatory phase, reducing relapses and delaying progression. However, strategies for remyelination by generating new OLGs, in particular at the chronic or second-

ary progressive stage, when chronic lesions in the CNS have been established, remain a major challenge in this field.5 Developments in neural stem cell (NSC) research have revived the idea of cell-based therapy for remyelination and slowing or preventing axonal degeneration. Endogenous NSCs, residing in specific germinal niches in the CNS, are thought to develop into new OLG precursor cells (OPCs), thus contributing to myelin repair after a demyelinating relapse. In chronic EAE, however, remyelination is ineffective due to the poor survival and low differentiation efficacy of endogenous NSCs.6 Transplantation of exogenous NSCs may be an effective therapeutic approach to accomplish remyelination in MS. A number of studies show that grafting NSCs does have a beneficial effect for the repair and reconstitution of brain tissues in EAE and other neurodegenerative disorders.7–9 Yet, in most cases, NSCs alone had only mild to moderate efficacy in EAE,9–14 and their beneficial effects were mainly by weak immunomodulatory and neuroprotective mechanisms.6,9,13,15–17 Concerns about the efficacy of NSC therapy for MS have been raised because of the likelihood that the hostile milieu generated by chronic inflammatory processes affects the migration and survival of the transplanted cells and inhibits their differentiation into remyelinating OLGs.18–21 Furthermore, we have found in pilot experiments that, while NSCs effectively suppressed acute EAE, these cells failed to do so when they were injected at the chronic stage of EAE (e.g., day 60 post-immunization [p.i.]; data not shown). In view of this, it might be assumed that NSC-based therapeutic strategies, especially those intended for the chronic stage of MS, would benefit from supplemental treatment with both immunomodulatory and OLG differentiation-promoting agents.

Received 18 August 2016; accepted 2 December 2016; http://dx.doi.org/10.1016/j.ymthe.2016.12.008. 5

These authors contributed equally to this work.

Correspondence: Guang-Xian Zhang, Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA. E-mail: [email protected]

Molecular Therapy Vol. 25 No 2 February 2017 ª 2016 The American Society of Gene and Cell Therapy.

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Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

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Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

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Fingolimod (FTY720, Gilenya), a sphingosine 1-phosphate (S1P) receptor modulator, is the first oral therapy for MS to be approved by the U.S. Food and Drug Administration (FDA).22–24 FTY720 primarily reduces CNS infiltration and alleviates MS severity, especially in remitting-relapsing (RR) MS, while it lacks efficacy when the disease reaches the secondary progressive stage (SP) of MS.22,25,26 The lipophilic nature of FTY720 allows it to easily cross the blood-brain barrier (BBB) and reach physiologically meaningful concentrations in CNS tissue.27,28 Because S1P receptors are expressed on all cell types within the CNS, FTY720 has the potential to exert effects directly on CNS cells.27–30 The effects and underlying mechanisms of FTY720 on immunomodulation have been well studied; however, its effects in NSC differentiation and OLG generation or remyelination are not clear. Whether FTY720 can induce a growth-promoting environment and enhance the therapeutic effect of transplanted NSCs in MS and EAE remains to be determined.

RESULTS Effect of FTY720 on NSC Differentiation In Vitro

NSCs were isolated and cultured from the subventricular zone (SVZ) of adult Swiss Jim Lambert (SJL) mice as described in Materials and Methods. NSCs at passages 10–15 were used in all in vitro experiments. Single NSCs transduced with GFP were used in all in vivo experiments. Neurospheres and dissociated single NSCs were positive for the NSC markers nestin and SOX2 (Figures S1A and S1B). To determine the optimal dose of FTY720 for NSC treatment in vitro, we first measured cell viability and apoptosis at different concentrations. FTY720 did not affect cell viability at doses up to 10 nM, but it decreased cell viability at a dose of 100 nM and above (Figure S1C). In addition, 1–10 nM treatment of FTY720 did not significantly affect the percentage of apoptotic cells (Figures S1D and S1E). These results show that FTY720 is not cytotoxic for NSC in vitro at concentrations up to 10 nM. A crucial property of NSCs is the capacity to differentiate into specialized neural cells. To study the effect of FTY720 on NSC differentiation, we cultured NSCs in specific differentiation culture media with different doses of FTY720. After 6–14 days, GFP+ NSCs changed morphology and developed into various CNS cell phenotypes, neurons (NF-H+), astrocytes (GFAP+), OPCs (A2B5+), and mature OLGs (MBP+), as determined by immunostaining (Figure 1). While the number of NF-H+ neurons was not significantly increased by 1 nM FTY720, the number of these cells was significantly increased

at 10 nM (Figures 1A and 1C). At the same time, FTY720 reduced numbers of GFAP+ astrocytes in a dose-dependent manner (0–10 nM) (Figures 1B and 1C). For OPC and OLG lineage differentiation of NSCs, FTY720 increased numbers of OPCs (A2B5+) in a dose-dependent manner compared with untreated cultures (Figures 1D and 1E). We then investigated whether FTY720 affects the differentiation of NSCs into mature OLGs. NSCs exhibited a concentration-dependent biphasic response to FTY720 (Figure 1F). FTY720 at a high concentration (10 nM) inhibited NSC differentiation into OLGs and induced OLG death, while at low concentrations (0.01–1 nM), it enhanced NSC differentiation into mature CNPase+ OLGs, with myelin sheath and complex membranes (Figures 1F and 1H). To quantify the effects of FTY720 on the OLG processes, the branch complexity of CNPase+ OLGs was scored using the Sholl analysis plugin for ImageJ software (Figure 1I). FTY720 (0.01–1 nM) increased branching of the processes compared with other groups (Figure 1J), indicating that FTY720 treatment strongly promoted NSC differentiation into mature OLGs. Based on these findings, we concluded that 1 nM FTY720 has the best effects in driving NSCs to differentiate into OPCs or OLGs, but fewer astrocytes; meanwhile, at a high concentration (i.e., 10 nM), FTY720 induces cell death and blocks OLG differentiation from NSCs. We then determined concentrations of neurotrophic factors NT-3, BDNF, and glial cell line-derived neurotrophic factor (GDNF) in the supernatants (SNs) of NSCs treated with FTY720 (1 nM) under OLG differentiation conditions. No significant difference was observed in the production of these neurotrophic factors (data not shown), indicating that FTY720-induced OLG differentiation is not through neurotrophic factor induction. Mechanism of FTY720 Mediating OLG Development via the S1P5/ERK Signaling Axis

To investigate the signaling pathway through which FTY720 promotes differentiation of NSCs into OLGs, we performed loss-offunction experiments on OLG generation by employing specific small interfering RNAs (siRNAs) and then adding FTY720. The focus was on S1P1 and S1P5, given the relatively high abundance of mRNA of these subtypes in OPCs and OLG lineage cells, the lack of binding of FTY720 to S1P2, and the very low expression of S1P3 and S1P4 in cultured OLGs.29,31 FTY720-induced S1P1 and S1P5 expression during NSC differentiation to OLGs was confirmed by

Figure 1. Effect of FTY720 on NSC Differentiation In Vitro Dissociated single NSCs were cultured in specific NSC differentiation medium, followed by immunostaining. (A) NSCs differentiated into NF-H+ neurons. (B) NSCs differentiated into GFAP+ astrocytes. (C) Quantitative analysis of numbers of NSCs differentiated into each type of CNS cell per field. (D–H) Staining of OLG-specific markers indicated that NSCs differentiated into A2B5+ OPCs (D) and CNPase+ mature OLGs (F). Scale bars, 20 mm. After treatment with FTY720, OLG death was detected by TUNEL staining. Nuclei were stained with DAPI (blue). (G) Quantitative data of TUNEL+ cells. Quantitative analysis was performed for numbers of NSCs differentiated into OPCs (E) and mature OLGs, i.e., CNPase+ cells with myelin sheath or with complex membranes (H) per field. (I) Demonstration of the method to quantify branching of oligodendrocyte process extension. Concentric circles separated by 15 mm were drawn around the cell bodies of the microphotographed oligodendrocytes, and the number of intersections that oligodendrocyte processes made with the concentric circles was defined as the branching score using the Sholl analysis plugin for ImageJ software. (J) CNPase+ branch score of differentiated OLGs with or without FTY720 treatment. Data are shown as mean values ± SD (n = 5 each group) and are representative of three experiments. **p < 0.01, ***p < 0.001, compared to the untreated group, one-way ANOVA with Tukey’s multiple comparison test.

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immunofluorescence, as shown in Figure 2A. NSCs transfected with specific siRNAs resulted in a significant reduction in expression of S1P1 or S1P5, as detected by real-time PCR (Figure S2A). While NSCs treated with FTY720 (1 nM) in OLG differentiation medium led to an increase in the numbers of OLGs (CNPase+), which exhibited a ramified morphology with complex processes, these effects were significantly abrogated by S1P5 knockdown (Figures 2B and 2C). Pre-treatment of cultures with S1P5 siRNA, but not S1P1 or control siRNA, reversed the FTY720-mediated increase in OLG differentiation observed with FTY720 alone (from 35% to 22%, p < 0.001) (Figure 2C). This was confirmed by pre-treating cultures with antagonists of specific S1P receptor subtypes. Pre-treatment of cultures with S1P5 pathway antagonist suramin, but not with S1P1 receptor antagonist W123, inhibited the OLG differentiation induced by addition of FTY720 (Figures S2B and S2C). In support of this, S1P1 agonist SEW2871 (100 nM) alone did not change the numbers of CNPase+ cells (Figures S2B and S2C). Because differentiation is often correlated with cell-cycle exit, we observed that the percentage of Ki67+ cells inversely correlated with the number of differentiated OLGs (Figure S2D). Altogether, our data suggest that FTY720 enhanced NSC differentiation to OLGs in the cultures specifically through the S1P5 receptor subtype. We further investigated the downstream signaling events involved in FTY720 action through S1P5 during NSC differentiation into OLGs. Given that the functional consequences of S1P5 modulation are associated with activation of signaling pathway molecules such as extracellular signal-regulated kinases 1 and 2 (ERK1/2),32 we tested whether ERK1/2 plays a role in the effects of FTY720. FTY720 significantly stimulated the phosphorylation of ERK1/2 (P-ERK1/2) during NSC differentiation into OLGs, as confirmed by both immunofluorescence (Figure 2D) and western blot (Figures 2F and 2G). ERK1/ 2-specific siRNAs (siERK1/2) were also examined for their ability to alter the effects of FTY720 on NSC differentiation to OLGs (Figures 2D–2G). As shown in Figures 2D and 2F, ERK1/2 siRNA could specifically and efficiently suppress ERK1/2 expression compared to cells treated with control siRNA and untreated cells. Downregulation of ERK1/2 significantly blocked FTY720-induced differentiation of NSCs to CNPase+ OLGs, indicating the critical role of ERK1/2 in FTY720-induced NSC differentiation into OLGs. We also observed the similar result that FTY720-evoked ERK1/2 was blocked by

MEK inhibitor U0126 (data not shown). Furthermore, FTY720induced ERK1/2 expression was significantly reversed by pretreatment with S1P5-specific siRNA (from 2.16-fold to 1.09-fold compared with the PBS control, p < 0.001) (Figure 2D–2G), indicating that the effects of FTY720 on ERK1/2 activation during NSC differentiation to OLGs were S1P5 dependent. There results were confirmed by pre-treating NSCs with SCH772984, an inhibitor of ERK1/2, for 30 min before treatment with or without FTY720 (Figures S2E–S2G). Collectively, these results demonstrate that the underlying mechanism of FTY720 on NSC differentiation into OLGs is mediated by the S1P5/ERK signaling axis, which mediates many processes, such as NSC proliferation and OLG differentiation and survival (Figure 2H). Effect of FTY720 in NSC Differentiation into OLGs under Pathological Conditions

A previous study demonstrated that during an inflammatory attack in MS or EAE, OLG death and demyelination are significantly exacerbated.33 To test whether FTY720 has the capacity to protect NSC differentiation into OLGs under such pathological conditions in vitro, NSCs were treated with culture supernatants (SNs) of proteolipid peptide (PLP)-specific splenocytes derived from EAE mice under OLG-specific differentiation culture conditions. Pro-inflammatory cytokines (e.g., interleukin-17 [IL-17] and interferon g [IFN-g]) released by splenocytes of EAE mice (Figure S3A) are reported to have an inhibitory effect on NSC proliferation and neural cell differentiation.34 Our results here showed that NSC differentiation to OLGs was significantly suppressed in the presence of culture supernatants from PLP-specific splenocytes, as shown by decreased cell survival (Figure S3B), reduced OLG number (CNPase+ cells) (Figure S3C), and less OLG arborization (Figures S3D and S3E) compared with PBS-treated controls. However, this impairment of NSC differentiation was overcome by FTY720 (1 nM) (Figure S3), demonstrating the protective effect of FTY720 during NSC differentiation into OLGs under pathological conditions and possibly during remyelination of already-damaged CNS tissues in MS. FTY720 in Combination with Transplanted NSCs Significantly Alleviated the Chronic Stage of RR-EAE

The enhancing effect of FTY720 on NSC differentiation into OLGs under pathological conditions in vitro prompted us to investigate

Figure 2. Mechanism of FTY720 Mediating OLG Development via the S1P5/ERK Signaling Axis (A) Dissociated single NSCs were cultured in differentiation medium for 6 days with or without FTY720 (1 nM). OLGs were stained by the specific marker CNPase (green) and corresponding antibodies against S1P1 or S1P5 (red). Nuclei were stained with DAPI (blue). One of five representative experiments is shown. (B) Dissociated single NSCs cultured in differentiation medium were treated for 3 days with FTY720 (1 nM) alone or pre-treated for 2 hr with siRNA targeting S1P1 or S1P5 or with control siRNA before addition of FTY720, followed by immunofluorescence staining. (C) Quantitative analysis was performed for numbers of NSCs differentiated into mature OLGs (CNPase+ cells with myelin sheath or with complex membranes) per field and CNPase+ branch score of differentiated OLGs. (D) Dissociated single NSCs cultured in differentiation medium were pre-treated for 2 hr with siRNA targeting S1P5 or ERK or with control siRNA and then co-treated with or without FTY720 for 72 hr followed by immunofluorescence staining. (E) Quantitative analysis was performed for numbers of NSCs differentiated into mature OLGs per field and CNPase+ branch score of differentiated OLGs. (F) Western blot analysis was performed for detection of activated ERK1/2 (P-ERK1/2) with phospho-specific antibodies. (G) The amount of phosphorylated protein was standardized using b-actin, and quantification is presented in each panel. (H) Putative mechanism of how FTY720 regulates NSC differentiation into OLGs via the S1P5/ERK signaling axis, which mediates many processes such as cell proliferation, differentiation, and migration. Data are shown as mean values ± SD (n = 5 each group) and are representative of three experiments. Scale bars, 20 mm. **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparison test.

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Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

Molecular Therapy

Figure 3. FTY720 in Combination with Transplanted NSCs Alleviated the Chronic Stage of RR-EAE, Decreased CNS Inflammation, and Promoted an M2 Phenotype in Macrophages and Microglia (A) Schematics of treatment strategies for SP-EAE. EAE mice were injected intravenously with dissociated single NSCs (1.0  106 cells/mouse) at day 60 p.i. and treated or not with FTY720 (fed at 0.3 mg/kg/day) from the day of cell injection. EAE mice that received NSCs or were fed with FTY720 alone served as single-treated controls; mice that (legend continued on next page)

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whether FTY720 could enhance the therapeutic potential of NSCs at the chronic stage of EAE. To this end, treatment with NSCs and/or FTY720 was initiated at the late stage (day 60 p.i.), when there is no longer remission but only progression from RR-EAE to SP-EAE and when myelin and axonal damage has been established. Experimental design and treatment strategies are shown in Figure 3A. Dissociated single NSCs (1.0  106 cells/mouse) were injected intravenously (i.v.) at day 60 p.i. with or without FTY720 (fed at 0.3 mg/kg/day) from the day of cell injection. EAE mice that received NSCs or FTY720 alone were single-treated controls, and mice that received the same volume of PBS were sham-treated controls. No obvious clinical differences were observed between the PBS-treated and the FTY720-treated groups (Figure 3B). Animals transplanted with NSCs exhibited a milder disease course after injection but were not statistically different from the control group. A significantly reduced clinical severity in response to co-treatment with FTY720 and NSCs was observed and sustained until the mice were sacrificed. The cumulative score from days 60 to 120 p.i. was significantly decreased in the co-treatment group (57.65 ± 11.22, p = 0.006) when compared to the PBS-treated control group (102.63 ± 20.16), while the single-treated group (FTY720 or NSCs only) had no significant differences with the PBS-treated control group. These findings indicate that NSCs or FTY720 alone is inefficient in therapy of the secondary stage of EAE, while FTY720 combined with NSCs have neural repair potential.

specific sites within the dorsal column of the lumbar spinal cord (at L3) and corpus callosum of the brain, because infiltration and demyelination lesions were consistently observed in these areas. H&E staining was performed to detect the extent of CNS inflammation (Figure 3C). Consistent with clinical observation, mice co-treated with FTY720 and NSCs exhibited slightly, though significantly (p = 0.035), reduced inflammatory infiltration compared with that of single- or sham-treated control mice, while no significant inhibition was observed between other groups (p = 0.069) (Figure 3D).

It has been previously shown that while FTY720 was effective when administered at the beginning of RR-EAE induction or at the first relapse, mice suffered a severe relapse when FTY720 was discontinued.35 We investigated whether this would also be the case when treatment was started at the chronic stage, when FTY720 alone has no effect. In a separate experiment, FTY720 was administered starting at day 60 p.i. and was discontinued at day 120 p.i. FTY720 alone remained ineffective, and there was no relapse in the 30 days following discontinuation of FTY720 (up to day 150 p.i.); relapse also was not observed in FTY720 and NSC co-treated mice (Figure S4). These results indicate that FTY720 is ineffective when treatment is started at the chronic stage, and the effect of FTY720 and NSC co-treatment would be primarily attributable to the neuroregeneration component of FTY720 and NSC co-treatment, while its role in anti-inflammation may be secondary.

FTY720 in Combination with NSCs Reduced Demyelination and Promoted Remyelination at the SP Stage of RR-EAE

To determine the effects of combined treatment with FTY720 and NSCs on CNS inflammation at the chronic stage of EAE, spinal cord and brain were harvested for pathological studies and immunostained at day 60 after treatment. All pathological and immunohistochemical studies were focused within standard 500 mm2 fields at

Previous studies showed that microglia and infiltrating macrophages play an important role in CNS inflammation during EAE chronicity, which exhibit either pro-inflammatory (type 1 phenotype, M1) or anti-inflammatory (type 2 phenotype, M2) properties, depending on the disease stage and the signals they receive.36 Here, we determined expression of inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg1), primary markers of M1 and M2, respectively, by macrophages and microglia (Iba1+) in the dorsal column of the lumbar spinal cord (Figure 3E). A reduction in M1 microglia and macrophages (iNOS+Iba1+) and increase in M2 (Arg1+Iba1+) cells were found in the NSC single-treated mice compared to the PBS-treated group (p = 0.022) (Figure 3F). In the co-treatment group, FTY720 enhanced this effect of NSCs (p = 0.004), while FTY720 single treatment did not show significant difference with PBS controls (Figure 3F).

We then evaluated the extent of myelin loss over the course of RR-EAE and SP-EAE and investigated whether FTY720 and NSC co-treatment reversed clinical disease by protecting axons from demyelination and promoting myelin regeneration. Luxol fast blue (LFB) staining showed pronounced myelin loss and a significantly larger demyelination area (18.2% ± 2.6%) in the spinal cord white matter of mice treated with PBS at day 120 p.i. In contrast, spinal cords of mice treated with combination of FTY720 and NSCs, but not either one alone, exhibited rare demyelination foci (3.6% ± 1.5%) (Figures 4A and 4B). These results indicated that untreated EAE mice underwent progressive demyelination, which was blocked by FTY720 and NSC co-treatment (p = 0.007), while treatment with NSCs or FTY720 alone failed to promote remyelination at the SP stage of RR-EAE. We next analyzed the extent of demyelination and occurrence of remyelination in EAE lesions in all experimental groups using toluidine blue staining and MBP immunohistochemistry. EAE mice sacrificed at day 60 p.i. were a baseline for demyelination and compared to the

received the same volume of PBS served as sham-treated controls. All mice were sacrificed and their spinal cords were harvested at day 120 p.i. (B) Clinical scores were checked daily by two researchers blindly, according to a 0–5 scale. (C) EAE mice were sacrificed at day 120 p.i., and the dorsal column of the lumbar spinal cord (at L3) was harvested for H&E staining to detect inflammation. (D) Mean score of inflammation in H&E staining. (E) Spinal cords were immunostained against iNOS and Arg1 on infiltrating macrophages and microglia (Iba1+). (F) iNOS+Iba1+ or Arg1+Iba1+ intensity was measured in the dorsal column of the lumbar spinal cord using Image-Pro. Representative images are shown, and quantitative data refer to mean ± SD (n = 5 mice/group). Scale bars, 40 mm in (C) and 10 mm in (E) and in the insets of (C). *p < 0.05, **p < 0.01, ***p < 0.001, compared to the PBS-treated group, one-way ANOVA with Tukey’s multiple comparison test. One representative of three independent experiments is shown.

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Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

Molecular Therapy

Figure 4. FTY720 in Combination with NSCs Reduced Demyelination and Promoted Remyelination in SP-EAE The lumbar regions of spinal cords of EAE mice were harvested before treatment (day 60 p.i.) and 120 days p.i.. (A) Transverse sections of the lumbar spinal cord were stained with LFB for detection of demyelination; lesions are outlined. (B) Demonstration of the method used to quantify demyelination using Image-Pro Plus software. Areas with demyelination (blue) were divided by the total white matter area of the column (outlined in red). Demyelination is expressed as a percentage of white matter area that has been demyelinated. (C) Toluidine blue staining of 1 mm sections to visualize myelination in the EAE groups mentioned earlier. (D) All intact axons, regardless of their myelination state, whose axoplasm was intercepted by a sampling line, were tagged and counted. (E) Mean g ratio (axon diameter divided by the entire myelinated fiber diameter) was determined using Image-Pro Plus software. (F) Confocal image of MBP (red; for myelin) immunostaining. Dash lines mark the edges of spinal cord. (G) MBP intensity was measured in spinal cord lesions using Image-Pro. Representative images are shown, and quantitative data refer to mean ± SD (n = 5 mice/group). Groups designated by the same letter are not significantly different, while those with different letters (A, B, C, or D) are significantly different (p < 0.05–0.01). *p < 0.05, **p < 0.01, compared to the PBStreated group. One-way ANOVA with Tukey’s multiple comparison test. Scale bars, 40 mm in (A) and 10 mm in the insets of (A) and (C). One representative of three independent experiments is shown.

FTY720-treated and/or NSC-treated groups. Naive mice were a control for normal myelin. Loss of myelin was apparent in the sham controls of chronic EAE, whereas a greater number of myelinated and/or remyelinated axons was found in co-treated mice (Figures 4C–4E). These newly formed myelin sheaths were thinner than in the naive group. Consistent with the neuropathy phenotype, the g ratio (the measurement of the size ratio between axon and total fiber diameters) of the FTY720 and NSC co-treated group was significantly higher than that of the naive mice, a feature of enhanced remyelination, but lower than that of the PBS- or single-treated group (Figure 4E). Similarly, significant demyelination had occurred before treatment of EAE (day 60 p.i.), and this pathologic process deteriorated in the late phase (day 120 p.i.) in untreated mice, indicating disease progres-

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sion. At this point, the PBS- or single-treated control group had large demyelinated lesions filled with MBP+ myelin debris, whereas cotreated mice exhibited much smaller demyelinated areas and higher MBP intensity compared to baseline (before treatment) (Figure 4F). Quantification of MBP staining density in PBS- or single-treated EAE mice revealed a significant reduction (35% to 41%, p = 0.0006) in myelin density compared with naive controls (Figure 4G). In contrast, the co-treated group showed a trend toward increased MBP intensity (70% of naive mice, p = 0.008), which differed significantly from the EAE- or single-treated group (Figure 4F), as well as from baseline (day 60 p.i.; before treatment). These data indicate that the anti-inflammatory effects of FTY720 or the neuroprotection by NSCs alone is unable to induce recovery at the late stage of EAE;

Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

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Figure 5. FTY720 in Combination with NSCs Inhibited Axon Degeneration and Reduced Astrogliosis in SP-EAE EAE mice were sacrificed and their spinal cords were harvested at day 120 p.i. (A) Double immunostaining of MBP (white) and TUJ1 (red; for axons) showed a significant increase in myelinated axons in the dorsal column of the spinal cord (MBP+TUJ1+). (B) GFAP staining of spinal cord sections for detection of astrogliosis. (C) Quantification of myelinated axons (MBP+TUJ1+) among total axons (TUJ1+). (D) Quantification of total axons (TUJ1+). Groups designated by the same letter are not significantly different, while those with different letters (A, B, C, or D) are significantly different (p < 0.05–0.01). One-way ANOVA with Tukey’s multiple comparison test. (E) Quantitative analysis of GFAP expression. GFAP intensity was measured in spinal cord lesions using Image-Pro. (F) FTY720 inhibits astrocyte activation in vitro. Dissociated single NSCs were cultured in astrocyte differentiation medium with or without FTY720 at indicated concentrations. Three days later, real-time PCR analysis of GFAP, Vimentin, and N-cadherin in astrocyte activation was performed. Gapdh was used as an internal control. Representative images are shown, and quantitative data refer to mean ± SD (n = 5 mice/group). *p < 0.05 compared to PBS-treated group, one-way ANOVA. Groups designated by the same letter are not significantly different, while those with different letters (A or B) are significantly different (p < 0.05–0.01), as determined by one-way ANOVA comparison with Tukey’s multiple comparison test. Scale bars, 40 mm in (A) and (C) and 10 mm in the insets of (A). One representative of three independent experiments is shown.

instead, a combination of FTY720 and NSCs could reduce demyelination and/or promote remyelination at the later disease stage. FTY720 in Combination with NSCs Inhibited Axonal Degeneration and Reduced Astrogliosis

To determine the effect of FTY720 and NSCs on axonal loss within spinal cord lesions, sections were labeled with anti-neurofilament antibody (TUJ1; axonal marker) in conjunction with antibody against MBP (myelin marker). This labeling showed that in the PBS- and single-treated groups (either FTY720 or NSCs), there were areas of demyelination (TUJ1+/MBP ), as well as areas of white matter lack-

ing axons and myelin (TUJ1 /MBP ) (Figure 5A). In regions of myelin damage (MBP low or negative), TUJ1 staining showed sparse processes, axonal transection, and fiber deterioration, indicative of the axonal pathology that accompanies the demyelination process. In contrast, mice co-treated with FTY720 and NSCs exhibited a significantly greater number of myelinated axons (red) surrounded by MBP+ rings (white) in the white matter of the spinal cord (Figures 5A–5C). Quantification of myelinated axons in PBS- and singletreated mice reduced to 32%–46% of those in naive mice, whereas co-treated mice showed myelinated axons that reached 79% of naive controls (Figure 5C).

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Molecular Therapy

There is increasing evidence that astrogliosis is a principal consequence of the chronic immune response in the CNS in EAE and MS.37 A significant increase in GFAP+ (an astrocyte marker) immunoreactivity was observed throughout the gray and white matter of spinal cords from PBS-, FTY720-, or NSC-treated EAE mice (Figure 5B). Quantification of GFAP immunoreactivity revealed a 2.8-fold reduction in reactive astrogliosis after treatment with FTY720 in combination with NSCs (Figure 5E). These findings are consistent with our in vitro data, which showed that addition of FTY720 significantly suppressed the expression of astrocyte genes, including GFAP, Vimentin, and N-cadherin, under NSC differentiation conditions (Figure 5F). Collectively, these results indicate that FTY720 combined with NSCs block chronic RR-EAE progression by protecting axons from damage and reducing astrogliosis and, more importantly, that their combined effects reverse EAE progression by promoting neuroregeneration. FTY720 Improved Survival of Transplanted NSCs and Promoted Development of OPCs and OLGs In Vivo

To investigate the effects of FTY720 on the in vivo fate of transplanted NSCs, brain sections of EAE mice in each group were harvested at day 120 p.i. and immunostained with neural cell-specific antibodies to evaluate distribution and differentiation of NSCs. Transplanted cells (GFP+) were located at demyelinated areas of brain parenchyma, as previously described.14,38 FTY720 treatment significantly increased the number of transplanted NSCs, i.e., as quantitative analysis showed that approximately 48 GFP+ cells/mm2 were found in the brain of NSCs with FTY720 co-treated mice while approximately 13 GFP+ cells/mm2 were found in mice treated with NSCs alone (Figure 6A). Co-localization of GFP+ and neural-specific markers revealed the differentiation of transplanted NSCs into APC+ and MBP+ mature OLGs, NeuN+ neurons (Figures 6B and 6C), GFAP+ astrocytes, and A2B5+ OPCs or that NSCs remained undifferentiated (SOX2+) (Figure S5). High-magnification confocal images further showed that GFP (green) was frequently co-localized with the OLG marker APC (red) or the neuronal marker NeuN (red) in the NSC and FTY720 co-treated group (Figure 6D). Similar to the spinal cord, as shown in Figure 4F, multifocal myelin loss with markedly decreased MBP immunoreactivity was detected in the corpus callosum in PBS, FTY720, and NSC single-treated EAE mice (Figure 6C). MBP expression was enhanced in the NSC and FTY720 co-treated group (Figure 6E), and some of the GFP+ NSCs co-localized with MBP (Figure 6C), indicating remyelination by transplanted NSCs. However, most of the MBP+ area had no GFP+ NSCs, indicating remyelination by endogenous cells (Figure 6C). Quantitative analysis of transplanted cells showed that in the NSC plus FTY720 co-treated group, the percentages of APC+ OLGs and A2B5+ OPCs derived from transplanted GFP+ NSCs (40.21% and 18.22%, respectively) were significantly higher than those in control NSC-treated mice (18.37% and 10.01%, respectively) (Figures 6B

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and 6F), while the percentage of NSC-derived GFAP+ astrocytes in NSC plus FTY720-treated mice (3.26%) was much lower than that in control NSC-treated mice (19.68%). Although our in vitro data showed that FTY720 at high concentrations (10 nM) promoted NSC differentiation into neurons (Figures 1A and 1B), there were no obvious differences between the percentages of NeuN+GFP+ neurons in the two groups (Figures 6B and 6C). Consistent with previous reports,14,38 approximately 25% of GFP+ NSCs retained undifferentiated SOX2+ in the NSC-treated group, and a smaller percentage of these cells was observed in co-treated EAE mice (Figure 6B). These results indicate that FTY720 not only promotes survival of transplanted NSCs but also drives them to differentiate into more OPCs and OLGs but fewer astrocytes in vivo, thus promoting remyelination and axonal growth and inhibiting astrogliosis.

DISCUSSION In the present study, we provide an effective therapy combining FTY720 and transplanted NSCs that facilitates clinical recovery from the chronic stage of RR-EAE, i.e., SP-EAE. The therapeutic effects correlated not only with the reduction in several deleterious parameters of the EAE pathological process, including inflammation, axon degeneration, astrogliosis, and demyelination, but also with the efficient promotion of transplanted NSC survival, OLG and neuron differentiation, and neural repair of established chronic CNS damage. In view of the weak self-regeneration in the CNS, the notion of introducing new cells with myelin-producing potential is a promising strategy for reconstruction of injured CNS tissue.7 NSCs are considered an attractive candidate for treating neurodegenerative diseases because of their capacity for immunomodulation, neurotrophic production, and neural repopulation.14,38 However, at the late stage of chronic EAE, using NSC alone as a therapy resulted in limited clinical and pathological improvement. The major obstacles for stem cell therapy in the chronic stage of EAE are the unfavorable microenvironment (long-term inflammation stress and loss of trophic support), weak neuron and OLG differentiation capacity, and intrinsic deficits (e.g., relatively weak immunoregulatory capacity) of transplanted NSCs in CNS lesions.39,40 It has been demonstrated that even under the best circumstances, transplanted cell survival in the CNS is only 10%,41 and fewer cells differentiate into mature neural phenotypes.38,40,42 The sustained inflammation stress in the CNS of chronic EAE may provide a hostile environment for transplanted NSCs or other multipotent stem cells.43 Strategies targeted at both relieving inflammation and augmenting differentiation could therefore make the environment supportive for engrafted NSCs, promote cell differentiation toward the OLG lineage, and effect substantial improvement in the late stage of chronic EAE. In this context, the treatment combining NSCs with FTY720, an approved immunomodulation therapy for MS, is of special significance. Our findings showed that in combination with FTY720, the number of NSCs in the brain parenchyma that survived for 60 days after transplantation was 3.7-fold higher than in mice treated with NSCs alone. FTY720 also augmented NSC differentiation toward the neuron and OPC or OLG lineages.

Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

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Figure 6. FTY720 Improved Survival of Transplanted NSCs and Promoted Development of Neurons and OLGs In Vivo Brains of EAE mice were harvested on day 120 p.i. and immunostained with anti-neural cell-specific antibodies. (A) Quantitative analysis of transplanted NSCs (GFP+) in the parenchyma of corpus callosum. (B) Quantitative analysis of differentiation of transplanted NSCs in the CNS, as shown in (C). (C) Immunofluorescence images of the brain of EAE mice. Cells co-labeled with GFP and neural-specific markers (red) were identified as differentiated cells derived from NSCs (arrows), and cells positive only for neuralspecific markers (red) were endogenous cells (dashed arrows). GFP single-positive cells (arrowheads). MBP+, myelin; APC+, OLGs; NeuN+, neurons. Scale bars, 20 mm in (C) and 5 mm in the insets of (C). (D) High-magnification confocal images show that GFP was co-localized with the OLG marker APC (red) or the neuronal marker NeuN (red). (E) MBP intensity was measured in spinal cord lesions using Image-Pro. (F) Quantification of total APC+ cells. Representative images are shown, and quantitative data refer to mean ± SD (n = 5 mice/group). **p < 0.01, ***p < 0.001, Student’s t test (A) and one-way ANOVA with Tukey’s multiple comparison test (D and E). One representative of three independent experiments is shown.

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Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

Molecular Therapy

These findings suggest that the therapeutic effect of the NSC and FTY720 combination comes from converting a hostile environment for neural cells into a supportive one, thus promoting both exogenous and endogenous remyelination and neural repair. We also found that at the chronic stage of EAE, FTY720 alone is not sufficient to exert its immunomodulatory effects, while a single NSC treatment resulted in slight, but significantly less, improvement than in the FTY720 and NSC co-treated group. These results indicate that the decreased CNS inflammation, reduced M1 phenotype, and promotion of the M2 phenotype of macrophages and microglia by FTY720 and NSC co-treatment are most likely the effect, rather than the cause, of neural repair. Thus, reduced release of myelin tissues or antigens would result in low levels of microglia activation and inflammatory infiltration, which, in turn, promotes neuroregeneration.36 Unlike other systemic immunomodulators used in MS, the effects of FTY720 extend beyond the reduction in inflammation, because FTY720 has direct neuroprotective effects on neural cells.44 For example, in vitro studies have shown the dose-dependent effect of FTY720 on axon preservation, neural cell survival, OPC process extension and differentiation, and cytoskeletal dynamics, all of which are frequently linked to improved remyelination.28,29,45 Similarly, FTY720 treatment induced production of neurotrophic factors BDNF and GDNF, promoted NSC proliferation, and drove embryonic stem cells to differentiate into the OLG lineage.28,46–48 Furthermore, it has also been shown that FTY720 can enhance remyelination in organotypic slice cultures.27,49,50 In vivo studies demonstrated that FTY720 is effective at treating acute EAE by promoting OPC proliferation and differentiation and by stimulating the sonic hedgehog (Shh) signaling pathway.51 These results, together with our findings, indicate that FTY720 has both anti-inflammatory and neuroregenerative effects. Contradicting these promising results, it has been reported that remyelination was not affected, or was even blocked, by FTY720 treatment in cuprizone- and lysophosphatidylcholine-induced demyelination models.52 We speculate that the contradictory results are most likely due to the pleiotropic actions and dose-dependent responses of OLGs and other neural cells to FTY720. High doses or concentrations of FTY720 blocked myelination or remyelination in vivo and in vitro.45 In the previously mentioned chemical-induced demyelination models, FTY720 had been either directly injected into the demyelination foci, in which an extremely high concentration in situ (2 mg/mL; 6.7 mM) of FTY720 would be toxic and kill OPCs and OLGs, or it was administered as an oral treatment at a dosage of 1 mg/kg/day.52 In in vitro studies, Oyama et al. showed that FTY720 treatment was cytotoxic to neurons at high concentrations (>30 mM),53 while Miron et al. reported that low doses of FTY720 (0.1–1 nM) induced OLG membrane elaboration, with higher doses (10 nM to 1 mM) causing the opposite modulation of membrane dynamics.45 Consistent with these findings, our in vitro results showed that low doses of FTY720 (%5 nM) are safe and have positive effects on NSC proliferation, driving NSCs to differentiate into greater numbers of neurons, OPCs, and OLGs but fewer as-

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trocytes, while at high concentrations (e.g., 10 nM) FTY720 reduced numbers of OLGs and even induced OLG death. In in vivo studies, at the lower dosage (oral, 0.3 mg/kg/day) used in a previous report51 and in our study, FTY720 treatment enhanced MBP expression and remyelination. These consistent results suggest that FTY720 induces OLG death only at high concentrations, while it promotes OLG differentiation at low concentrations (e.g., %5 nM in vitro and 0.3 mg/kg/day in vivo). It is known that the anti-inflammatory effects of FTY720 were not effective in slowing disease progression in primary progressive MS.54 It has also been reported that FTY720 did not promote remyelination when it was given at the recovery phase after a 3-week cuprizone diet.55 These data suggest that the neuroprotective capacity of FTY720 at the later disease stage is not sufficient to induce endogenous neurogenesis but that the ability of FTY720 to augment exogenous OLG generation by transplanted NSCs, as demonstrated in the present study, could be highly significant. Because FTY720 is widely used in MS, these results may point to its potential advantage as a complementary treatment to cell therapy in MS. Although the neurotrophic factors NT-3, BDNF, and GDNF are reported to be induced or to interact with FTY720 during OPC differentiation,47,56 no significant difference was observed in our study, indicating that FTY720-induced NSC differentiation is not through induction by these neurotrophic factors. We then focus on the S1P receptors, the designated interaction partners of phosphorylated FTY720, and their downstream signaling during NSC differentiation. Given that S1P receptors are expressed in various types of CNS resident cells,28 the functional complexity and interactions between FTY720 and S1P receptors are dependent on species, stage of development, treatment dose, and duration in terms of cell survival, process dynamics (extension or retraction), cell migration and differentiation, cross-talk with neurotrophins, and remyelination.27,30,31,45 The role of FTY720 on S1P receptor modulation in organotypic cerebellar slices has been shown,27 and among S1P receptor subtypes, S1P5 is highly expressed on OLG lineage cells, where it mediates MEK-ERK signaling pathways.57,58 While the importance of S1P5 for OLG development has been recognized, whether S1P signaling is involved in the effect of FTY720 on NSCs and the identification of the specific intracellular signaling pathways that transduce these signals remains to be elucidated. Our present study provides direct evidence that FTY720 enhances OLG differentiation from NSCs primarily via the S1P5, but not the S1P1, signaling pathway. Our loss-of-function studies also show that the sustained activation of ERK1/2 by FTY720 is downstream of S1P5, which plays an essential role in FTY720-induced NSC differentiation into OLGs. Altogether, these results indicate that the underlying mechanism, whereby FTY720 promotes NSC differentiation to OLGs, is mediated by the S1P5/ERK signaling axis. In conclusion, our data demonstrate a novel mechanism of FTY720 action in NSC differentiation and remyelination and broaden its application as a potential drug to increase the efficiency of conventional NSC-based therapy at the SP stage of MS.

Please cite this article in press as: Zhang et al., Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair, Molecular Therapy (2016), http://dx.doi.org/10.1016/j.ymthe.2016.12.008

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MATERIALS AND METHODS NSC Generation and GFP Transduction

NSCs were generated from the SVZ area of SJL/J mice (Jackson Laboratory), 6–8 weeks of age, as described previously.14,38 Briefly, the SVZ region of fresh brain was harvested under sterile conditions and placed in DMEM media. The dissected tissue was cut into 1 mm3 pieces and dissociated using the Neural Tissue Dissociation Kit (Miltenyi Biotec). Cells were resuspended in DMEM/F-12 (Invitrogen) and supplemented with 2% B27, 20 ng/mL epidermal growth factor (EGF), and 20 ng/mL basic fibroblast growth factor (bFGF), along with antibiotics. Cells were plated at a density of 1  106 cells/well, and media was changed every 4 days. After 3–5 days in culture, a proportion of individual cells proliferated to form distinct neurospheres. After 3–4 weeks, the neurospheres were collected, dissociated to single cells by Accutase (Invitrogen), and replated at 1.0  105 cells/mL for the next passage. Expression of neural-specific markers Nestin and Sox2 was determined by immunocytochemistry. Single NSCs at the fourth passage were infected with the lentiviral vector expressing hygromycin and GFP according to our previous study.42 Hygromycin is the selected marker of the lentiviral vectors, and GFP is used for tracing transplanted NSCs in future in vivo studies. Under hygromycin selection, GFP+ cells having reached more than 99% purity were transferred into growth medium for the next passage. GFP+ NSCs at passages 5–15 were used in all in vivo experiments.

In Vitro Differentiation of NSCs

The stock solution of the active phosphorylated form of FTY720 (Sigma-Aldrich) was prepared in DMSO/50 mM HCl. FTY720 was then diluted in culture media to meet the final concentration (0–10 nM). To evaluate the differentiation of NSCs under FTY720 treatment, dissociated single cells were plated on poly-D-lysine/ laminin-coated coverslips at a density of 2.0  104 cells/mL and cultured in specific NSC differentiation medium. In brief, for neuron differentiation, Neurobasal medium was supplemented with 2% B-27, 2 mM GlutaMax-I, and 0.5 mM cyclic AMP (cAMP). For astrocyte differentiation, DMEM was supplemented with 1% N-2, 2 mM GlutaMax-I, and 1% fetal bovine serum (FBS). The OLG differentiation medium requires Neurobasal medium supplemented with 2% B-27, 2 mM GlutaMax-I, and 20 ng/mL T3. Cell proliferation was tested by Ki67 (Abcam) staining in vitro. The proliferation was quantified as the percentage of Ki67-positive cells. Cell death was tested by In Situ Cell Death Detection Kit (Roche) staining in vitro. Cell death was quantified as the percentage of TUNEL-positive cells. FTY720 is an activator of S1P1, S1P4, and S1P5 receptor subtypes, with partial agonism on S1P3 but no activity on S1P2. To determine the mechanism of FTY720-induced effects, cultures were supplemented, 30 min before treatment with or without FTY720 (1 nM), with optimal concentrations of S1P1 antagonist W123 (1 mM), S1P1 receptor agonist SEW2871 (100 nM), and S1P5 pathway antagonist suramin (100 nM; all from Cayman Chemicals) following a previous study,25 as well as MEK inhibitor U0126 (Promega), ERK1/2 inhibitor

SCH772984 (Selleckchem), or culture supernatants (10%) described in Figure S3. After 6–14 days, NSCs in different culture conditions changed morphology and developed markers of neurons, astrocytes, and OLGs, which were detected by immunocytochemistry staining, western blot, and/or real-time PCR. The percentage of positive cells labeled with a specific neural marker in the total number of DAPI+ cells was expressed as the mean value of specific neural cell differentiation. RR-EAE and SP-EAE Induction and Treatment

Female SJL/J mice, 8–10 weeks of age, were purchased from the Jackson Laboratory. All experimental procedures and protocols were approved by the Institutional Animal Care and Committee of Thomas Jefferson University and were carried out in accordance with the approved institutional guidelines and regulations. Mice were immunized subcutaneously on the back with 200 mg of murine proteolipid peptide (PLP139-151; HSLGKWLGHPDKF) emulsified in complete Freund’s adjuvant (CFA; Difco) containing 4 mg/mL Mycobacterium tuberculosis H37Ra (Difco). Then, 200 ng of pertussis toxin (List Biological Lab) were given intraperitoneally on days 0 and 2 post-immunization (p.i.). Clinical scores were assigned blindly by two researchers daily according to a 0–5 scale as follows:51 1, limp tail or waddling gait with tail tonicity; 2, waddling gait with limp tail (ataxia); 2.5, ataxia with partial limb paralysis; 3, full paralysis of one limb; 3.5, full paralysis of one limb with partial paralysis of the second limb; 4, full paralysis of two limbs; 4.5, moribund; and 5, death. A relapse is defined as an increase in at least one clinical grade sustained for at least 2 consecutive days after animals have previously improved at least a full clinical grade and stabilized, while SP-EAE is defined as disease without remissions.59,60 Experiment design and treatment strategies are shown in Figure 3A. EAE mice were randomly enrolled into the following treatment groups: (1) sham-treated control group, in which EAE mice were gavaged with PBS; (2) NSC single-treated group, in which dissociated single NSCs (1.0  106 cells/mouse) were injected i.v. at the chronic stage, day 60 p.i. of RR-EAE; (3) FTY720 single-treated group, in which FTY720 (Sigma-Aldrich) at dosages of 0.3 mg/kg/day was gavaged once a day for 60 consecutive days from the day of cell injection and the dosage used was adapted from human safety dosage studies;29,44 and (4) combination of FTY720 and NSC treatment group, in which NSCs (1.0  106 cells/ mouse) were injected i.v. at day 60 p.i. with FTY720 (fed at 0.3 mg/kg/day) from the day of cell injection. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 6 software. Data are presented as mean ± SD. When comparing multiple groups, data were analyzed by ANOVA with Tukey’s multiple comparison test. A significance criterion of p < 0.05 was used for all statistical analysis.

SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Materials and Methods and five figures and can be found with this article online at http://dx.doi.org/10.1016/j.ymthe.2016.12.008.

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Molecular Therapy

AUTHOR CONTRIBUTIONS Y.Z., X.L., and G.-X.Z. conceived and designed the experiments, analyzed data, and wrote the manuscript. Y.Z. and X.L. carried out the experiments. C.-G.M. and B.G. designed the experiments and interpreted the data. B.C. and A.R. co-supervised the study and wrote the paper. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

ACKNOWLEDGMENTS This study was supported by Novartis Pharma (Basel, Switzerland) and the NIH. Y.Z. and X.L. are partly supported by the Chinese National Natural Science Foundation (81501062), the Overseas Scholarship Program of Shaanxi Normal University, and the Fundamental Research Funds for the Central Universities (GK201603114). We thank Katherine Regan for editorial assistance.

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