Neural Stem Cells Engineered to Express Three Therapeutic Factors Mediate Recovery from Chronic Stage CNS Autoimmunity.

Treatment of chronic neurodegenerative diseases such as multiple sclerosis (MS) remains a major challenge. Here we genetically engineer neural stem cells (NSCs) to produce a triply therapeutic cocktail comprising IL-10, NT-3, and LINGO-1-Fc, thus simultaneously targeting all mechanisms underlie chronicity of MS in the central nervous system (CNS): persistent inflammation, loss of trophic support for oligodendrocytes and neurons, and accumulation of neuroregeneration inhibitors. After transplantation, NSCs migrated into the CNS inflamed foci and delivered these therapeutic molecules in situ. NSCs transduced with one, two, or none of these molecules had no or limited effect when injected at the chronic stage of experimental autoimmune encephalomyelitis; cocktail-producing NSCs, in contrast, mediated the most effective recovery through inducing M2 macrophages/microglia, reducing astrogliosis, and promoting axonal integrity and endogenous oligodendrocyte/neuron differentiation. These engineered NSCs simultaneously target major mechanisms underlying chronicity of multiple sclerosis (MS) and encephalomyelitis (EAE), thus representing a novel and potentially effective therapy for the chronic stage of MS, for which there is currently no treatment available.

Introduction

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating disorders of the central nervous system (CNS).[1,2] MS is thought to begin when peripherally activated myelin-reactive T cells infiltrate the CNS, followed by other immune cells, including monocytes, B cells, and neutrophils. In the CNS, these cells activate microglia and astrocytes and produce IFN-γ, IL-17, Granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-23, which promote inflammatory responses,[3] while immunoregulatory cytokines like IL-4, IL-10, and IL-27 may be protective.[4,5,6] Three major mechanisms contribute to the chronic progression in MS: (i) persistent CNS inflammation[7]; (ii) loss of trophic support for both oligodendrocytes and neurons[8]; and (iii) accumulation of CNS regeneration inhibitors such as Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp).[9] Persistent CNS inflammation results in continuous CNS tissue damage, i.e., demyelination, axonal degeneration, and neuronal dysfunction,[10] while myelin proteins from the damaged tissue, in turn, perpetuate chronic inflammation and the production of neuroregeneration inhibitors in the disease foci.[8,9] This vicious cycle results in failure of spontaneous remyelination, axonal loss, and disease progression.[11]

Current MS therapies mainly target dysfunctional immune response, one of the above-mentioned three pathogenic mechanisms; they are thus only partially effective in mitigating disease progression and are less effective at the chronic stage.[12] These immunomodulatory agents have little or no effect on the lack of neurotrophic factors and/or the accumulation of neuroregeneration inhibitors in disease foci. On the other hand, delivery of a neurotrophic factor may primarily modulate myelination in the CNS and promote oligodendrocyte precursor (OPC) proliferation, survival, and differentiation,[13] while its effects will likely be diminished by inflammation. Blocking neuroregeneration inhibitors in the CNS partially suppresses EAE, while having no impact on CNS inflammation.[14] An MS therapy that targets these three pathogenic mechanisms at the same time would therefore be most beneficial.

In view of the beneficial effects of neural stem cells (NSCs) in EAE treatment, including their capacity for neuroprotection, immunomodulation, trophic function, and possibly neuro-repopulation, they appear to be an excellent candidate for cell-based therapy. Indeed, NSCs, even without any modification, suppress EAE development.[15,16,17,18,19,20] However, NSC therapy in EAE has resulted in only marginal improvement in clinical score for acute EAE,[15,16,21,22] while their effect on the chronic stage of disease has not been studied. Our pilot experiments showed that unmodified NSCs failed to induce recovery from EAE when injected at day 60 postimmunization (p.i.) (data not shown). The main reasons for this lack of therapeutic effect may be the poor survival and low differentiation potential of transplanted NSCs in CNS lesions, and their suboptimal immunoregulatory capacity.[6,16,22] An approach that significantly enhances therapeutic capacity of NSCs in chronic stage of EAE is, therefore, crucial. Further, given that transplantated NSCs migrate only into the CNS inflamed foci,[23] these cells have been a useful vehicle to deliver therapeutic molecules to the disease foci for EAE treatment.[16,21]

In the present study, we have tested the effect of a novel system on the chronic stage of EAE using adult NSCs that were engineered to simultaneously produce a therapeutic cocktail (cocktail-NSCs) containing IL-10, an effective immunoregulatory cytokine; neurotrophin 3 (NT-3), a potent neurotrophic factor; and soluble LINGO-1 protein (LINGO-1-Fc), an antagonist of LINGO-1, a key part of the common receptor complex for neuroregeneration inhibitors,[24] under the control of the Tet-On system. These NSCs target the three major mechanisms underlying the pathogenesis of chronic/progressive EAE and would thus be a highly effective approach for EAE therapy. This cocktail, continuously produced by autologous NSCs on the CNS disease foci, would also avoid potential side effects caused by repeated systemic administration of a large amount of drugs, or harmful local administration.[25]

Results

Generation and characterization of bone marrow-derived NSCs

BM cells were isolated from femurs of adult B6 mice as described in Materials and Methods and our previous report.[22] After 1 week in culture, individual cells exhibited obvious proliferative ability, as shown in Supplementary Figure S1a. Two weeks later, individual cells proliferated to form various sizes of neurospheres (Supplementary Figure S1b) and, at 3–4 weeks, the neurospheres increased in size and gradually detached from the bottom of the culture plate (Supplementary Figure S1c). These neurospheres were collected, dissociated to single cells, and reseeded at 1.0 × 105 cells/ml for the next round of expansion. Neurospheres at the 5th passage were dissociated into single cells, transferred onto poly-D-lysine and laminin precoated coverslips and used in all in vitro experiments. These cells were positive for NSC markers Nestin and Sox2 (Supplementary Figure S1d,e), and their NSC nature was further confirmed by the capacity to proliferate and differentiate into neural cell lineages ().

Transduction of NSCs with inducible NT-3, IL-10, and/or LINGO-1-Fc

The most recent Lenti-X Tet-On Advanced Inducible Expression System was used to transduce tetracycline-controlled transactivator, NT-3, IL-10, LINGO-1-Fc and copGFP into NSCs cells. Innovatively, we inserted NT-3, IL-10, and LINGO-1-Fc genes individually into separate vectors and transduced them into cells sequentially. This method avoids the drawback of limited space for insertion of several genes in a lentiviral vector. We used neomycin, puromycin and hygromycin as selection markers in different vectors to ensure that genes are transduced into the same cell. The structure of the vectors is shown in Supplementary Figure S2a.

To test the expression of these transduced genes in NSCs, we cultured the cells with or without Dox.[26] After 24 hours, supernatants and cells were harvested and analyzed by ELISA and real-time PCR. NT-3, IL-10, and LINGO-1-Fc were strongly expressed only in the presence of Dox (Supplementary Figure S2b). Meanwhile, cells transfected with a single gene (NT-3-NSCs, IL-10-NSCs, and LINGO-1-Fc-NSCs) or two genes (NT-3/IL-10-NSCs, NT-3/LINGO-1-Fc-NSCs, and IL-10/LINGO-1-Fc-NSCs) also produced significant levels of corresponding proteins. Withdrawal of Dox led to a decrease in the production of these proteins after 4 days (Supplementary Figure S2b), confirming the efficacy of the Tet-On/Off system. Furthermore, Tet-On 3G mRNA was present, as detected by real-time PCR, in NSCs transduced with these lentiviruses (Supplementary Figure S2c). These results confirm the success of gene transduction and the Tet-On system.

Proliferation and differentiation potentials of cocktail-NSCs in vitro

The proliferation capacity of cocktail-NSCs was tested by Ki67 immunostaining. Interestingly, the number of Ki67+ cells was significantly increased in all NSCs that carried the NT-3 gene (NT-3-NSCs, NT-3/IL-10-NSCs, NT-3/LINGO-1-Fc-NSCs, and cocktail-NSCs) (,). Adding Dox to mock-transduced control NSCs did not change their proliferation.

To investigate their effect on NSC differentiation, NT-3, IL-10, and/or LINGO-1-Fc-transduced NSCs were cultured in specific differentiation medium with or without Dox treatment. After 2–3 weeks, a number of NSCs had differentiated into astrocytes, neurons, or oligodendrocytes, while adding Dox to the control-NSCs did not change their differentiation profile. NSCs transduced with NT-3 and LINGO-1-Fc differentiated into higher numbers of neurons and oligodendrocytes, but fewer astrocytes (,). These results indicate that NT-3 and LINGO-1-Fc promote differentiation of oligodendrocytes and neurons, while reducing differentiation of astrocytes.

Cocktail-NSCs suppress acute EAE

In order to evaluate the effect of cocktail-NSCs on the development of EAE, we first i.v. injected cocktail-NSCs (1.0 × 106 cell/mouse) into mice at disease onset (day 10 p.i.). The group treated with PBS (Sham-EAE) showed a progressive increase in clinical score from days 10 to 17 p.i., while mice treated with control NSCs, as well as those treated with NSCs transduced with one or two genes, showed a significant reduction in clinical scores at day 28 p.i. compared with the Sham-EAE group (P < 0.05–0.01). Cocktail-NSC-treated mice exhibited the best recovery (P < 0.001), indicating that therapeutic effect of cocktail-NSCs was significantly more potent than that of NSCs transduced with a single gene (P < 0.01) or with two genes (P < 0.05) (,). Consistent with the clinical score, sham-EAE mice showed severe EAE histological alterations, while only few inflammatory infiltrates were present in the spinal cord white matter of the cocktail-NSC-treated mice (P < 0.01; ,), as measured in different white matter areas (Supplementary Figure S3). Meanwhile, mice treated with NSCs transduced with a single gene or two genes also exhibited different degrees of recovery, and the treatment groups with NSCs producing IL-10 recovered better than those without IL-10 (P < 0.05–0.01; ,). These results indicate that overexpression of IL-10 is necessary for the anti-inflammatory effects of cocktail-NSCs.

To assess the therapeutic effect of cocktail-NSCs on the infiltration of pathogenic T cells into the CNS, MNCs were isolated from the CNS and analyzed by flow cytometry. The total number of MNCs was 6.3 × 105 per mouse in the sham-EAE group versus 1.9 × 105 in the cocktail-NSC-treated group. The NT-3/IL-10-NSC-treated group also showed fewer infiltrating cells (2.9 × 105 cell/mouse) compared with the sham-EAE group (). Significantly fewer CD4+ T cells and CD11c+ dendritic cells were observed in the CNS of cocktail-NSC-treated EAE mice than in other groups (, Supplementary Figure S4a). Further, cocktail-NSC treatment significantly reduced the percentages of Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17+) cells in the CNS compared with control (P < 0.01–0.001; , Supplementary Figure S4b). GM-CSF, a critical cytokine in Th17 cell pathogenicity,[27] was also significantly decreased by cocktail-NSC treatment (P < 0.001). Furthermore, the percentages and absolute numbers of IFN-γ+CD4+, IL17+CD4+, and GM-CSF+CD4+ cells were also significantly decreased in all groups treated with NSCs producing IL-10. These results indicate that IL-10 in the cocktail plays an important role in the inhibition of CNS inflammatory infiltration.

We and others have shown that NSCs, upon i.v. injection in EAE mice, were found in almost all peripheral organs within 10 days, and subsequently completely disappeared from them by day 30 after transplantation.[6,16,21,28] Injected NSC migrated exclusively into the CNS inflammed foci.[15] While in the periphery, these cells played an immunomodulatory role, primarily by inducing Th1, but not Th2 responses, and by induction of T-cell apoptosis[21]; transduction with IL-10 significantly enhanced these effects.[6] To study the autoantigen-induced cytokine production of cocktail-NSC-treated mice, splenocytes were harvested 28 days p.i. and stimulated with MOG35–55. As shown in Supplementary Figure S5, concentrations of IFN-γ in cell culture supernatants were dramatically decreased in all groups treated with NSCs overexpressing IL-10. Injection with NT-3/IL-10-NSCs and cocktail-NSCs significantly decreased IL-17 production, and the GM-CSF level was also obviously decreased in mice treated with cocktail-NSCs. Treatment with NT-3-NSCs and NT-3/IL-10-NSCs showed a much higher IL-10 level than in other groups. There was no significant difference in proportions of various immune cell proportions among splenocytes of these groups (data not shown). Overall, cocktail-NSCs inhibited IFN-γ, IL-17, and GM-CSF production.

Cocktail-NSCs significantly alleviate chronic stage of EAE

To define the effect of therapeutic cocktail-NSCs on the chronic stage of EAE, mice were transplanted with cocktail-NSCs by i.v. injection at day 60 p.i. PBS-injected mice exhibited a slight recovery at acute phase, and then remained stable up to day 120 p.i. (), similar to described previously.[29] While control NSCs showed no effect when treatment started at the chronic stage (), NSCs transduced with a single gene (NT-3- or LINGO-1-Fc-NSCs alone, but not IL-10 alone) slightly halted disease progression compared with the sham-EAE group at day 120 p.i. (P < 0.05). The mean clinical score demonstrated that cocktail-NSCs and NT-3/LINGO-1-Fc-NSCs significantly suppressed EAE development at chronic stage EAE. In addition, the cocktail-NSC-treated group also showed significantly inhibited EAE compared with mice treated with NT-3/IL-10-NSCs or IL-10/LINGO-1-Fc-NSCs. These results indicate that, compared to unmodified NSCs or cells transduced with one or two therapeutic molecules, the cocktail-NSCs were the most effective at alleviating EAE when treatment was started at chronic stage, e.g., day 60 p.i.

Given that the autologous property of BM-NSCs makes them of great interest for future clinical use, we focused on these cells in the current study. Similar results have also been observed in NSCs derived from the subventricular zone (SVZ) of brain (data not shown).

Cocktail-NSCs reduce CNS inflammation and promote an M2 phenotype in macrophages/microglia

In order to determine the anti-inflammatory capacity of cocktail-NSCs at the chronic stage of EAE, H&E staining was performed to detect the extent of CNS inflammation. While IL-10-producing NSCs had stronger anti-inflammatory effects than those without IL-10, cocktail-NSC-treated mice showed the lowest inflammation score compared with all other groups (; Supplementary Figure S6a; P < 0.05–0.01).

Microglia/macrophages can exhibit either pro- or anti-inflammatory properties, depending on the disease stage and the signals they receive.[30] Here, we determined expression of inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg1), primary markers of Type 1 (M1; proinflammatory) and Type 2 (M2; immunomodulatory), respectively, by macrophages/microglia (Iba1+) in the spinal cord. Higher numbers of iNOS+Iba1+ (; Supplementary Figure S6b) and lower of Arg1+Iba1+ cells (; Supplementary Figure S6c) were observed in the sham-EAE group compared to other groups, while cocktail-NSC-treated mice had more Arg1+ and fewer iNOS+Iba1+ cells. We also tested whether these engineered NSCs modulate microglial activation induced by lipopolysaccharide (LPS) treatment in vitro. In primary microglial cultures, cocktail-NSCs, as well as other IL-10-producing NSC groups, effectively inhibited expression of major mediators of microglia activation, e.g., TNF-α, IL-1β, and iNOS (Supplementary Figure S7a). These results indicate that cocktail-NSCs can inhibit the proinflammatory M1 phenotype of microglia and switch it to anti-inflammatory or immunoregulatory M2 phenotype, which can stimulate the remyelination process.[31]

Cocktail-NSCs reduce axon degeneration and astrogliosis

It is believed that damage to myelin sheaths in EAE greatly reduces axonal survival,[32] and vice versa, axonal injury precludes remyelination due to the alteration of remyelination signals from axons.[33] Treatment that could preserve axonal integrity in EAE mice may therefore have potential as a neuropreotective agent. Here, axonal pathology of mice treated at the chronic stage of EAE was evaluated by the development of amyloid precursor protein (APP) deposits, a prototypical marker of axonal damage.[34] In comparison with naive mice, sham-EAE mice exhibited a high intensity of APP staining in the white matter of the spinal cord (). Mice that received NSCs transduced with IL-10 or NT-3 alone showed a small but significant decrease in APP staining when compared with sham-EAE mice. All groups treated with NSCs transduced with LINGO-1-Fc had lower APP staining than those treated with NSCs without LINGO-1-Fc (P < 0.05; ). The lowest intensity of APP staining was observed in cocktail-NSC-treated EAE mice, indicating beneficial effects of cocktail-NSCs in axonal protection, in which LINGO-1-Fc played a major role.

Dense astrogliosis is an important factor in plaque formation and MS chronicity.[35] We thus examined the effects of cocktail-NSCs on astrogliosis in EAE spinal cord sections using immunolabeling for GFAP. There was a more than fivefold reduction in reactive astrogliosis in mice that received cocktail-NSCs compared to sham-EAE. Astrogliosis was also significantly attenuated in mice treated with LINGO-1-Fc-NSCs (,). These findings are in agreement with our in vitro data, which showed that LINGO-1-Fc dramatically suppressed expression of astrocyte genes, GFAP, Vimentin, and N-cadherin, in response to LPS activation (Supplementary Figure S7b). Together, these results indicate that cocktail-NSCs effectively reduce axonal injury and astrogliosis.

Cocktail-NSCs promote remyelination

To characterize possible remyelination, EAE mice were treated with different groups of NSCs starting on day 60 p.i., when chronic damage in the CNS was established. Lumbar spinal cords were harvested on day 60 p.i. (before treatment) for Luxol fast blue (LFB) and MBP stainings, which served as a baseline for demyelination. As shown in –, a significant degree of demyelination had occurred by day 60 p.i. (baseline), and demyelination continued to progress in sham-EAE mice up to day 120 p.i. EAE mice that received NSCs expressing either NT-3 or IL-10 alone showed a similar demyelination level compared to the baseline, indicating blockade of further demyelination without remyelination. Importantly, NSCs transduced with LINGO-1-Fc, or any two therapeutic genes, significantly reduced demyelination and enhanced MBP expression compared to the baseline (before treatment), and cocktail-NSCs had the strongest effects, providing evidence for not only blockade of further demyelination but also successful remyelination. Taken together, our data demonstrate that the enhanced myelination observed in mice receiving cocktail-producing NSCs is due to both the blockade of further demyelination and the promotion of remyelination.

Toluidine blue-stained sections were also used to visualize myelinated axons (). Loss of myelin was apparent in spinal cords of sham-treated mice, whereas a greater number of myelinated and/or remyelinated axons was found in cocktail-NSC-treated mice. Newly formed myelin sheaths were thinner than in naive mice (). Consistent with the neuropathy phenotype, the g-ratio (measuring the size ratio between diameter of axon and total fibre) of the cocktial-NSC-treated group was significantly lower than that of the sham-EAE and other NSC-treated groups (), a feature of enhanced remyelination.[6,8] These results clearly indicate that treatment with NSCs that produce a therapeutic cocktail not only prevents further CNS tissue damage but, importantly, also induces remyelination and neurorepair in chronic EAE.

Cocktail-NSC treatment increases the numbers of neurons and oligodendrocytes

We then studied the effect of the cocktail on neural cell differentiation of transplanted NSCs in demyelinated lesions by triple immunostaining. Colocalization of GFP (for transplanted NSCs) and neural specific markers in the striatum and corpus callosum (as shown as Supplementary Figure S8a) revealed that some of the transplanted cells differentiated into NeuN+ and Tuj1+ neurons (Supplementary Figure S8b,c), A2B5+ OPCs (Supplementary Figure S8d), APC+ and MBP+ mature oligodendrocytes (Supplementary Figure S8e,f), GFAP+ astrocytes (Supplementary Figure S7g), while some NSCs remained undifferentiated (Sox2+; Supplementary Figure S8h). Quantitative analysis showed that significantly higher percentages of neurons, OPCs, and oligodendrocytes and a smaller percentage of GFAP+ astrocytes were derived from transplanted cocktail-NSCs than from control-NSCs (P < 0.05–0.01) (–). While up to 30.3 ± 1.1% of transplanted control NSCs (GFP+) retained undifferentiated Sox2+ morphological features (Supplementary Figure S8h), consistent with a previous report,[16] a significantly smaller percentage (20.5 ± 2.0%) of these cells was observed in cocktail-NSCs (), indicating that a large proportion of cocktail-NSCs had differentiated into neural cells. Importantly, the majority of the MBP-stained area and neural cells were GFP-, indicating that the main mechanism of cocktail-induced remyelination and neural repair is via promotion of endogenous repair.

We also hypothesized that a combination of NT-3 and LINGO-1-Fc might enhance MBP synthesis.[36,37] To test this, we used the transwell system to determine their effects on OPC differentiation in vitro. OPCs treated with a combination of LINGO-1-Fc- and NT-3-NSCs had dramatically greater branch range of preoligodendrocytes (O4+ cells) (Supplementary Figure S9a–c) and mature oligodendrocytes (CNPase+ cells) with myelin sheaths (Supplementary Figure S9d,e). In the presence of NT-3/LINGO-1-Fc-NSCs and cocktail-NSCs, the CNPase+ oligodendrocytes exhibited complex membrane morphology compared with other groups (Supplementary Figure S9d), indicating that LINGO-1-Fc directly and strongly promoted OPC differentiation into mature oligodendrocytes and had the best effects in tandem with NT-3. A significantly decreased percentage of Ki67+ OPCs in cultures where NT-3 and LINGO-1-Fc were present (Supplementary Figure S9f,g) indicated an exit from cell cycle and the beginning of oligodendrocyte differentiation.

Discussion

Treatment of chronic, progressive MS remains a major challenge due to the three major mechanisms that contribute to disease progression, including: (i) persistent CNS inflammation[7]; (ii) loss of trophic support for both oligodendrocytes and neurons[8]; and (iii) accumulation of CNS regeneration inhibitors.[9] While unmodified NSCs can suppress EAE when administered at the acute stage (e.g., day 22 p.i.),[6,16] they failed to do so when administered at the chronic stage of disease (e.g., day 60 p.i.) as shown in the present study, most likely due to their weak anti-inflammatory capacity and low neurotrophin production.[6,16,26] To simultaneously target the three major pathogenic mechanisms underlying the failure of neuroregeneration at the chronic stage of EAE, here we have for the first time engineered NSCs expressing a therapeutic cocktail that is triply-effective (): (i) NT-3 supports NSC proliferation and their differentiation into neurons and oligodendrocytes; (ii) LINGO-1-Fc blocks the harmful effect of neuroregeneration inhibitors on OPCs/oligodendrocytes; and (iii) the inhibitory effect of IL-10 on CNS inflammation and the inductive effect on M2 phenotype of microglia/macrophages create a supportive microenvironment for neuroregeneration. Although transplanted NSCs can differentiate into neural cells, the majority of MBP-stained areas and neural cells were GFP-, indicating that promoting development of endogenous cells is the main mechanism of cocktail-induced remyelination and neuroregeneration.[16,21,28]

An important feature of chronic EAE and MS is the loss of both oligodendrocytes and neurons in damaged CNS tissues.[38] An effective treatment for these diseases at the chronic stage would therefore be capable of inducing proliferation and differentiation of endogenous neural precursor cells, e.g., NSCs and OPCs, thus supplying sufficient neural cells for repair. NT-3 is an excellent candidate for this purpose, given its capacity to promote remyelination, axonal regeneration, and functional CNS recovery.[39] Compared to other neurotrophic factors, NT-3 has a significantly greater capacity to provide neuroprotection and reduce astrogliosis,[40] a main cause of MS plaque formation.[35] NT-3 overexpression has been shown to enhance spinal cord injury recovery.[41] Our previous studies showed that NSCs transduced with NT-3 significantly enhanced neuroregeneration in acute EAE.[26] NT-3 acted on donor cells not only in an autocrine, but also in a paracrine fashion to enhance neuronal differentiation of both transplanted and endogenous cells, thus promoting neural repair.[26] Here, NT-3-NSCs significantly promoted NSC proliferation and differentiation towards neurons and oligodendrocytes, both of which are essential for neural repair in the chronic stage of EAE.[42] Although using NT-3 alone at the chronic stage was somewhat effective, in combination with other therapeutic molecules such as IL-10 and/or LINGO-1-Fc, its effect was significantly enhanced. These results indicate that, while immunomodulation and blocking neuroregeneration inhibitors are important, NT-3, together with other therapeutic factors, helps to achieve the maximal therapeutic effect in chronic EAE.

Another pathogenic mechanism underlying the failure of spontaneous remyelination in EAE and MS is the accumulation of neuroregeneration inhibitors, including Nogo, MAG, and OMgp.[9,43] These inhibitors are generated from debris of damaged CNS tissues, and they exert their effect on neural cells through the functional component of the NgR1/p75/LINGO-1 and NgR1/TAJ(TROY)/LINGO-1 receptor complexes that mediate inhibitory signals on axonal growth and oligodendrocyte differentiation.[14] LINGO-1 is a common, key factor in the above-mentioned complexes, and blocking LINGO-1 function using different approaches, including neutralizing antibodies, gene knockout, and LINGO-1-Fc administration, effectively promotes OPC maturation (Supplementary Figure S9a,d) and remyelination (–), and protects these cells from CNS inflammation- or chemical-induced damage.[14,34] In the present study, we showed that among three transduced molecules, overexpression of LINGO-1-Fc most effectively enhanced MBP intensity in vivo and induced oligodendrocyte maturation in vitro. The importance of blocking LINGO-1 signaling in the inhibition of astrogliosis is supported by significantly reduced expression levels of GFAP, Vimentin, and N-cadherin in LPS-activated astrocytes in vitro. Furthermore, adding LINGO-1-Fc significantly enhanced the therapeutic effects of NSCs transduced with IL-10 and/or NT-3, clearly demonstrating the nonredundant role of LINGO-1-Fc in the cocktail.

Given that persistent CNS inflammation and microglia activation are considered the main cause of continuous neural tissue damage and EAE chronicity,[44] treatment for neuroregeneration alone will be insufficient to ameliorate disease development, and it will need to be combined with an immunomodulation. Among immunomodulatory molecules used in EAE studies, IL-10, as the major product and common functional factor of regulatory T cells, tolerogenic DCs, and type 2 macrophages (M2), is considered highly potent in decreasing proinflammatory cytokine production.[30,45,46] While its systemic administration did not suppress EAE, IL-10 delivery by fibroblast cells into CNS inflamed foci effectively improved disease outcome.[47] We have shown that i.v. transplanted brain tissue-derived NSCs suppressed ongoing acute EAE and that their effects were significantly enhanced by transduction with IL-10.[6] In the present study, we observed that in acute EAE levels of proinflammatory cytokines were markedly lower in IL-10-NSC-treated mice than in sham-treated or NT-3/LINGO-1-Fc-NSC-treated EAE mice. Further, reduced pro-inflammatory cytokines and M1 response, together with enhanced M2 response, likely converted the CNS microenvironment in inflamed foci from hostile to supportive of neuroregeneration. Indeed, M2 cells are considered important for OPCs to mature into myelinating oligodendrocytes.[31] While IL-10 was a well-known anti-inflammatory molecular which ameliorate inflammation progressive in autoimmune disease, the clues from our results indicated IL-10 probably blockade demyelination by inhibited inflammatory cytokine expression and induced M2 macrophages/microglia response.

An ideal combination of multiple therapeutic reagents would enhance the effect of each of them. Interestingly, when IL-10 and NT-3 were combined, microglia activation was markedly inhibited compared with the group that was treated with IL-10 alone. Further, the combination of LINGO-1-Fc and NT-3 also markedly halted disease progression. Our in vitro data demonstrated that LINGO-1-Fc could significantly promote OPC differentiation into mature oligodendrocytes. However, we also found that NT-3 dramatically enhanced cell survival. A previous study showed that LINGO-1-Fc treatment promotes TrkB phosphorylation[14] and NT-3 combines with the Trk family receptor to activate its downstream pathway.[39] Treatment with NT-3 and LINGO-1-Fc may, therefore, enhance their effect to boost OPC differentiation and improve the treatment results of each of them compared with a single gene treatment. Moreover, inflammation and demyelination are the two major elements of pathogenesis and exert their effects in parallel in chronic EAE. Our results show that IL-10 not only decreases pro-inflammatory cytokine production, but also dramatically inhibits microglial activation. Surprisingly, NT-3 had no effect on microglial activation, but when combined with IL-10 it appeared to enhance the suppressive capacity of the latter. This enhancement might be attributable to the upregulation of IL-10 production by NT-3 (Supplementary Figure S5e), although the exact mechanism is not clear. The superior effect of cocktail over single gene or combinations of any two of these genes indicates that these three therapeutic genes in the cocktail enhance the effects of one another in reversing CNS autoimmune damage.

The safety issue of NSC transplantation has been a concern for clinical application. For example, coinjection of allogeneic adult NSCs with pancreatic islets resulted in NSC-derived tumor formation.[48] However, It has been found that NSCs, after transplantation, survive in the CNS for up to 15 months, without any sign of deleterious outcomes such as tumor formation, adverse immune responses or inappropriate anatomical accumulation.[49] Safety of lentiviral vector as a tool for gene therapy has been widely tested in clinical trials.[50,51] We in another series of experiments observed that all cocktail-NSC-injected EAE mice survived up to 300 days p.i. without toxicity or tumor formation (data not shown). We, therefore, anticipate that the current system is an effective and safe method for the future therapy. Phase 1 and 2 clinical trials using NSCs as therapies are ongoing to test their safety and practicability.[52]

Taken together, our data demonstrate that NSCs engineered to produce a “cocktail” of three therapeutic molecules effectively target the three major mechanisms underlying EAE chronicity and convert the hostile environment into one supportive of the neuroregenerative process, thus significantly promoting endogenous oligodendrocyte/neuron differentiation and axonal integrity. Further, in addition to being a delivery vehicle, NSCs have intrinsic capacity, albeit weak, for immunomodulation,[21,23] neural protection[53] and neural-repopulation,[16,19] functions that can be greatly enhanced by this therapeutic cocktail. Thus, this study provides a new approach to break the vicious cycle of inflammation, myelin damage and neuroregeneration blockade, and paves the way to a novel, inducible, and highly effective therapy for chronic CNS inflammatory demyelination, for which there is currently no effective therapy.

Materials and Methods

NSCs were generated from bone marrow of the femurs of C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME), 6–8 weeks of age, as described previously.[54] Cells were plated on poly-D-lysine/laminin (Sigma-Aldrich, St. Louis, MO) coated 24-well plate and cultured in serum-free Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (Invitrogen, Gaithersberg, MD) supplemented with 2% B27, 20 ng/ml epidermal growth factor, and 20 ng/ml basic fibroblast growth factor along with antibiotics. Cells were plated at a density of 1 × 106 cells/well, and media changed every four days. After 2 weeks, a proportion of individual cells proliferated to form distinct neurosphere. 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. Cells at 5th to 10th passages were used in the following in vitro and in vivo experiments.

Primary astrocytes, microglial cells and oligodendrocyte progenitor cells (OPCs) were isolated from newborn mouse brain, dissociated with Neural Tissue Dissociation Kit (Miltenyi Biotech, San Diego, CA) and purified with either anti-ASCA-2, anti-CD11b, or anti-A2B5 microbeads (Miltenyi Biotech) respectively. Astrocytes were cultured in DMEM/10% fetal bovine serum (FBS) cell culture medium, microglia in DMEM/10% FBS plus 5 ng/ml M-CSF (PeproTech, Rochy Hill, NJ), and OPCs in DMEM/F12 supplemented with 2% B27, 2 mmol/l Glutamax, 20 ng/ml basic fibroblast growth factor, and 20 ng/ml PDGF-AA (Invitrogen).

To generate vectors inducibly expressing multiple therapeutic genes, we used the pLVX-EF1a-Tet3G and pLVX-TRE3G-IRES vectors (Clontech, Moutain View, CA). In brief, we cloned and inserted the NT-3 and IL-10 cDNA into multiple cloning sites (MCSs) of the pLVX-TRE3G-IRES-puro vector. Primer sequences are listed in Supplementary Table S1. The puromycin gene was replaced by the hygromycin gene in the pLVX-TRE3G-IRES-puro vector, which was then named pLVX-TRE3G-IRES-hyg. Next, we inserted LINGO-1-Fc and copGFP cDNA into the MCS of pLVX-TRE3G-IRES-hyg. The Lenti-X HTX Packaging System (Clontech) was used to produce the lentivirus of Lenti-X Tet-On 3G, LV-TRE3G-NT-3-IRES-IL-10, and LV-TRE3G-LINGO-1-Fc-IRES-copGFP (Supplementary Figure S2a).

To establish the complete Tet-On Advanced System, we performed sequential transduction with the lentiviruses into NSCs following the user's manual for the Lenti-X Tet-On 3G inducible expression system. NSCs were first transduced with only the Lenti-X Tet-On 3G, followed by selection with G418 (500 µg/ml). Resistant clones were then screened for Tet-On expression and tested for inducibility. A favorable Tet-On-positive clone was then transduced with the LV-TRE3G-NT-3-IRES-IL-10 lentivirus. Doubly transduced cells were selected using puromycin (5 µg/ml), and the resulting puromycin-resistant clones were then screened for NT-3 and IL-10 inducibility. Afterwards, the neomycin and puromycin resistant clone was tranduced with the LV-TRE3G-LINGO-1-Fc-IRES-copGFP lentivirus and, finally, triply-transduced cells were selected using hygromycin (500 µg/ml) and the resulting hygromycin-resistant clones screened for LINGO-1-Fc and copGFP inducibility. NSC-transduced with Lenti-X Tet-On 3G, LVX-TRE3G-IRES-puro and LVX-TRE3G-IRES-copGFP-hyg were used as control NSCs.

The proliferation capacity of NSCs was tested by staining for Ki-67 in vitro. NSCs were seeded at a density of 2.0 × 104 cells/ml and incubated in NSC medium. After 24 hours, cells were fixed and stained using anti-Ki-67 antibody (Abcam, Cambridge, MA). The proliferation capacity was expressed as the percentage of Ki-67+-positive cells among GFP+ cells.

To evaluate the differentiation ability of engineered NSCs, dissociated single cells were plated on poly-D-lysine/laminin coated coverslip 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 mmol/l GlutaMax-I and 0.5 mmol/l cAMP. For astrocyte differentiation, DMEM was supplemented with 1% N-2, 2 mmol/l GlutaMax-I and 1% FBS. The oligodendrocyte differentiation medium requires Neurobasl medium supplemented with 2% B-27, 2 mmol/l GlutaMax-I and 20 ng/ml T3. Over 2 weeks, NSCs in differentiation media changed morphology and developed markers of neurons, astrocytes and oligodendrocytes as determined by immunocytochemistry staining. To determine the number of cells expressing a specific antigen, five areas of each coverslip were examined, and the percentage of positive cells labeled for a specific neural marker in the total number of DAPI+ cells was expressed as the mean value of specific neural differentiation.

Female C57BL/6 mice, 7–8 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, ME). All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and were carried out in accordance with the approved institutional guidelines and regulations. Mice were immunized at two sites on the back with 200 μg of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35-55) (Genescript, Piscataway, NJ) in 200 μl of emulsion containing 50% complete Freund's adjuvant with 5 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Lawrence, KS). All mice were i.p. injected with 200 ng pertussis toxin (Sigma-Aldrich) in Phosphate-buffered saline (PBS) on days 0 and 2 postimmunization (p.i.). Clinical EAE was scored daily in a blind manner, according to a 0–5 scale as described previously[55]: 0, no clinical signs; 0.5, stiff tail; 1, limp tail; 1.5, limp tail and wadding gait; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of another limb; 3, complete paralysis of both hind limbs; 4, moribound; and 5, death.

NSCs transduced with different vectors were i.v. injected as single-cell suspension (1.0 × 106 cells in 150 μl PBS/each mouse) via the tail vein at the onset (day 10 p.i.) or chronic stage (day 60 p.i.) of disease. Upon cell injection, doxycycline (Dox; Sigma-Aldrich) was administered subcutaneously (s.c.) at a dose of 100 mg/kg/day.[56] Mice injected with PBS only served as control. The group using unmodified NSCs was omitted given that showed therapeutic effects similar to GFP-transduced NSCs (data not shown). In addition, given that injection of Dox alone did not affect the clinical course of EAE,[26] a control group treated with Dox only was not included in the current study.

Mice were sacrificed at day 28 (for acute EAE) or day 120 (for chronic EAE) p.i. and transcardially perfused with PBS. Brain and lumbar spinal cords were harvested for pathological assessment and spleen for immunological assessment. CNS tissues were cut into 7 μm sections, fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (H&E) for assessment of inflammation and Luxol fast blue (LFB) for demyelination. Slides were assessed and scored in a blinded fashion for inflammation[57]: 0, none; 1, a few inflammatory cells; 2, organization of perivascular infiltrates; and 3, abundant perivascular cuffing with extension into the adjacent tissue. For demyelination quantification, total white matter was manually outlined, and area (%) of demyelination was calculated by Image-Pro Plus software.[58]

For immunohistochemistry, brain and spinal cord tissues were fixed using 4% paraformaldehyde for 1 day and then cryo-protected using 30% sucrose solution for 3 days. Fixed tissues were embedded in OCT compound (Tissue-Tek, Sakura Finetek, Japan) for frozen sections and then sectioned coronally at 12 μm. Transverse sections of brain and spinal cord were cut, and stained with different antibodies. Immunofluorescence controls were routinely generated by omitting primary antibodies. Results were visualized by fluorescent microscopy (Nikon Eclipse E600; Nikon, Melville, NY) or confocal microscopy (Zeiss LSM 510; Carl Zeiss, Thornwood, NY). For the quantifications of HE, FB, iNOS+Iba1+, Arg1+Iba1+, APP+, GFAP+ and MBP+, ten areas in the white matter at the lumbar spinal cord were selected (Supplementary Figure S3).

For toluidine blue staining, spinal cords were dissected out and incubated in 2% paraformaldehyde plus 2.5% glutaraldehyde overnight. Fixed tissues were then immersed in 2% osmium tetroxide, dehydrated through a graded acetone series, and embedded into EPON following procedures described previously.[34] Semi-thin sections of 1 μm thickness were stained with 1% toluidine blue and images captured by light microscopy. A minimum of 10 micrographs per mouse were obtained, and 5 mice per group were evaluated. All axons in the area of ventral spinal cord were counted. To quantify myelinated axons, a line-sampling method was used in accordance with our previous study.[34] G ratio, i.e., axon diameter divided by the entire myelinated fiber diameter, was determined using Image-Pro Plus software.[6,59]

Total RNA was extracted from cells or tissues using RNeasy Plus Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Reverse transcription was conducted using QuantiTect Reverse Transcription Kit (QIAGEN). Real-time PCR was performed using the QuantiFast SYBR Green PCR Kit (QIAGEN), and detection was performed using the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Neucleotide sequences of the primers were based on published cDNA sequences (Supplementary Table S2).

Spleen was mechanically dissociated through a 70 µm cell strainer (Falcon, Tewksbury, MA) and incubated with red blood cell lysis buffer (Miltenyi) for 1 minute. Harvested cells were washed with cold PBS before in vitro stimulation. To acquire CNS cells, spinal cords and brains were mechnically dissociated and incubated with Liberase (Roche, Nutley, NJ) for 30 min, passed through a 70 µm cell strainer and washed with cold PBS. Cells were then fractionated on a 70/30% Percoll (Sigma-Aldrich) gradient by centrifugation at 2,000 rpm for 20 minutes and MNCs were collected from the interface and washed with PBS.

Splenocytes at 1.0 × 106 cells/ml were cultured in triplicates in RPMI 1640 supplemented with 10% FBS in 24-well plates and stimulated with 25 μg/ml MOG35-55 for 72 hours. Supernatants were collected and assayed for IFN-γ, IL-17, GM-CSF, IL-5, and IL-10 by ELISA Kits (R&D System, Minneapolis, MN).

For surface-marker staining, cells were incubated with fluorochrome-conjugated Abs to CD4, CD8, CD11b, and CD11c (BD Biosciences, San Jose, CA) or isotype control Abs for 30 minutes on ice. For intracellular staining, splenocytes or CNS-infiltrating MNCs were stimulated with 25 and 10 μg/ml MOG peptide for 72 hours or overnight, respectively, followed by stimulation with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of GolgiPlug for 5 hours. Cells were surface-stained with mAbs against CD4 and CD8. Cells were then washed, fixed, and permeabilized with FIX&PERM solution (Invitrogen), and intracellular cytokines were stained with Abs against IL-17, IFN-γ, or GM-CSF (BD Biosciences). Flow cytometric analysis was performed on FACSAria (BD Biosciences) and data were analyzed with FlowJo software (Treestar, Ashland, OR).

Statistical analyses were performed using GraphPad Prism 6 software (GraphPad, La Jolla, CA). Data are presented as mean ± SD. When comparing multiple groups, data were analyzed by analysis of variance with Tukey's multiple comparisons test. A significance criterion of P < 0.05 was used for all statistical analysis.

Figure S1. Generation of bone marrow-derived NSCs in vitro. Figure S2. Vector construction and NSC transduction. Figure S3. Schematic diagram of the measured area of spinal cord white matter. Figure S4. Cocktail-NSCs are the most efficient in suppressing CNS inflammation. Figure S5. Cytokine production of MNCs from NSC-treated mice during acute EAE. Figure S6. Cocktail-NSCs reduce CNS inflammation and promote an M2 phenotype in macrophages/microglia. Figure S7. Cocktail-NSCs inhibit microglia and astrocyte activation in vitro. Figure S8. Neural cell differentiation of cocktail-NSCs in vivo. Figure S9. Cocktail-NSCs promoted primary OPC differentiation in vitro. Table S1. Primers used for pLV-TRE3G-NT-3-IRES-IL-10 and pLV-TRE3G-LINGO-1-Fc-IRES-copGFP vector construction. Table S2. Primers used for real-time quantitative RT-PCR analysis.