in vitro assessment of the direct effect of laquinimod on basic functions of human neural stem cells...

Journal of the Neurological Sciences xxx (2014) xxx–xxx

JNS-13358; No of Pages 9

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Journal of the Neurological Sciences

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In vitro assessment of the direct effect of laquinimod on basic functions ofhuman neural stem cells and oligodendrocyte progenitor cells

Eve E. Kelland a,⁎, Wendy Gilmore a, Liat Hayardeny c, Leslie P. Weiner a,b, Brett T. Lund a

a Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USAb Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USAc Pharmacology Unit, Global Innovative R&D, Teva Pharmaceutical Industries, Netanya, Israel

⁎ Corresponding author at: University of Southern CaMcKibben Hall Annex Room 246, Los Angeles, CA 90033fax: +1 323 442 3032.

E-mail address: [email protected] (E.E. Kelland).

http://dx.doi.org/10.1016/j.jns.2014.07.0580022-510X/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Kelland EE, et al, Inoligodendrocyte progenitor cells, J Neurol Sc

a b s t r a c t
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Article history:Received 13 January 2014Received in revised form 19 June 2014Accepted 28 July 2014Available online xxxx

Keywords:LaquinimodNeural stem cellOligodendrocyte progenitor cellMultiple sclerosis

Laquinimod is an orally active immunomodulatory small molecule that has shown clear clinical benefit in trialsfor relapsing–remitting multiple sclerosis and in experimental rodent models that emulate multiple sclerosis(MS). Studies in healthymice, and inmice with experimental autoimmune encephalomyelitis, have demonstrat-ed that laquinimod is capable of entering the central nervous system. It is therefore important to determine iflaquinimod is capable of a direct influence on basic functions of neural stem cells (NSC) or oligodendrocyteprogenitor cells (OPC)—cells critical for myelin repair in MS. In order to address this question, a series ofexperiments was conducted to determine the effect of exogenous laquinimod on viability, proliferation,migration and differentiation of human NSC and OPC in vitro. These data show, for the first time in cells ofhuman origin, that direct, short-term interaction between laquinimod and NSC or OPC, in an isolated in vitrosetting, is not detrimental to the basic cellular function of these cells.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is a chronic autoimmune and neurodegener-ative disease of the central nervous system (CNS). The disease affectsapproximately 400,000 individuals in the United States and about 2.5million worldwide, with evidence of increasing incidence over the last50 years [16,18,19,24,31]. Pathologically, MS is characterized by focalleukocyte infiltration into the CNS by multiple immune cell types, in-cluding autoreactive, myelin-specific T cells [28,38]. Targeted myelindestruction leads to demyelination, axonal loss and clinical neurologicalpresentation of the disease. In the earlier stages of MS there is evidenceof endogenous repair in the form of remyelination, which leads to axo-nal protection and restoration of nerve signaling [15,30]. The process ofremyelination involves the recruitment of neural stem cells (NSC) andoligodendrocyte progenitor cells (OPC) to lesion sites from either thesubventricular zone or neighboring parenchyma, possibly in responseto the release of chemoattractant molecules present in the active lesion[7–9,21,35,40]. Once in situ these cells undergo differentiation and

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remyelination occurs, but, as the disease progresses remyelinationfails for reasons that are still unclear. Possible mechanisms ofremyelination failure may involve overall neural cell dysfunction, per-sistent inhibitory signals or inappropriate cell migration [3,12,13,21].Despite the increasing availability of MS drug treatments in the last15 years, current FDA approved therapies are only moderately effectivein reducing relapse rate and stopping disease progression in the MSpopulation as a whole, and are not targeted at the direct protection orrepair of the CNS. While new therapies addressing this issue are ingreat demand it is important to confirm that existing and new immuno-modulatory agents, especially those capable of crossing the blood–brainbarrier, are not detrimental to the stem cell repair process.

Laquinimod, N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-3-quinoline-carboxamide, is an orally active immuno-modulatory small molecule that has been shown to prevent the onsetof disease, suppress established disease and reduce relapse rate in ex-perimental autoimmune encephalomyelitis (EAE; an established animalmodel of MS) [6,26,32,41]. Clinical studies in humans have also demon-strated efficacy in reducing active lesions and brain atrophy onMRI andslowing clinical disease progression [10,11]. Themechanismof action oflaquinimodmay involve a modest shift in immune function from a pro-inflammatory Th1 to an anti-inflammatory Th2/3, rather than a generalimmune suppression [17,33,37,39,41]. Alterations in immune cell traf-ficking and a reduction in acute axonal damagehave also been proposedas mechanisms of action [5,25,39]. Intriguingly, laquinimod may also

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have direct effects in the CNS. Using 14C-labeling in mice it was shownthat laquinimod, at a dose of 1–25 mg/kg, is capable of entering theCNS to ~7–8% in healthy and 13% in mice with EAE [39]. We thereforeestimate that the concentration of laquinimod in the CNS to be in the re-gion of 100 nM (0.1 μM). These data suggest that laquinimod is capableof directly affecting the functions of neural cells, especially NSC andOPC.To address this possibility, we conducted a series of experiments to testthe effect of direct exposure of laquinimod on NSC and OPC viability,proliferation, migration and differentiation, using human embryonicderived NSC and OPC as an in vitromodel system.

2. Experimental procedures

2.1. Laquinimod

Research grade laquinimod Na was provided by Teva Pharmaceuti-cal Industries (Netanya, Israel) and was prepared fresh for each experi-ment by reconstitution in distilled water at a stock concentration of100 mM. Laquinimod was diluted to appropriate working concentra-tions in complete NSC and OPC culture media.

2.2. Differentiation of hESC into NSC and OPC

Human NSC and OPC were derived from the human embryonicstem cell (hESC) line WA09 according to previously publishedmethods [21]. For NSC cultures, hESC-derived cell lines that metspecific NSC criteria (morphology, phenotype and ability to furtherdifferentiate to neural cell lineages), were maintained in neural-induction media (NIM) (DMEM/F12 supplemented with N2(Invitrogen), 2 μg/ml heparin (Sigma) and 0.5 mM sodium pyru-vate) containing 10 ng/ml basic fibroblast growth factor (bFGF,R&D Systems) and 20 ng/ml epidermal growth factor (EGF, R&DSystems) in tissue culture plates treated with growth factor reducedmatrigel (GFRM;1:30 dilution, BD Biosciences).

For OPC cultures, enriched populations of human OPC that met spe-cific OPC criteria, including phenotype, morphology, expression of mye-lin proteins and ability to becomemyelinating cells were maintained inITB media (a 50:50 mix of ITTSPP media [DMEM/F12 media supple-mented with 25 μg/ml human insulin, 50 μg/ml human apotransferrin,6.3 ng/ml progesterone, 10 μg/ml putrescine, 50 ng/ml sodium selenite,40 ng/ml triiodothyronine and 0.5 mM sodium pyruvate] and B27media [DMEM/F12 supplemented with 2X B27 (Invitrogen)]) contain-ing 20 ng/ml EGF on GFRM-coated tissue culture plates.

2.3. Measurement of cell viability

Cell viability and apoptosis were determined using fluoresceindiacetate and propidium iodide (FDA/PI) staining and by caspase activa-tion using CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein(Millipore) as previously described [20–22]. For FDA/PI quantificationNSC and OPC were seeded at a concentration of 15,000 cells/well toGFRM-coated 48-well plates. For CaspaTag assay cells were seeded at50,000 cells/well in 12-well plates treated with GFRM. Twenty-fourhours later cultures were dosed, in duplicate, with increasing logdoses of laquinimod-Na or the positive cell death control staurosporine(500 nM, Sigma). Cells were incubated for 24 h, 48 h and 7 days at 37 °C5% CO2, with media replenishment every 3 days. Following FDA/PIstaining cell counts (living vs. dead cells) were determined using aNikon Eclipse 2000 inverted fluorescence microscope from three ran-dom fields of view per well. For CaspaTag assay, caspase and PI stainingwas quantified using a BD FACS Caliber Flow Cytometer using instru-ment settings and fluorochrome compensations defined in preliminaryexperiments.

Please cite this article as: Kelland EE, et al, In vitro assessment of the directoligodendrocyte progenitor cells, J Neurol Sci (2014), http://dx.doi.org/10

2.4. Measurement of cell proliferation

Proliferation wasmeasured using [3H] thymidine incorporation intocells harvested by an automated cell harvester (Tomtec Harvester 96Mach III M). Cells were seeded onto a 96-well plate at 15,000 cells/well (NSC) and 25,000/well (OPC) for 24h time point and at 8000cells/well for 4 day time point. Twenty-four hours later cells were treat-ed with increasing concentrations of laquinimod-Na. For NSC and OPCcultures, proliferation media containing either 20 ng/ml EGF and10 ng/ml bFGF, 20 ng/ml EGF or 10 ng/ml PDGF, 10 ng/ml bFGF and20 ng/ml EGF were used as a positive control. For 24h proliferation0.2 μCi [3H] thymidine/well, and for 4 day assay, 1 μCi [3H] thymidine/well was added to the adhered cells immediately following laquinimoddosing. Radioactivity (CPM) was counted using a Wallac Trilux 1450Microbeta liquid scintillation counter and data expressed as percentagechange from control (basal media only).

2.5. Chemotaxis of NSC and OPC

The migratory capacity of NSC and OPC-enriched cultures wasassessed using Boyden chambers (8 μm, Corning Costar) as previouslydescribed [21]. Briefly, 60,000 cellswere seeded to inserts andmigrationtowards lower chambers containing varying concentrations oflaquinimod or the control chemoattractants CXCL12 or PDGF-AA wasmeasured. To determine if direct interaction of cells with laquinimodpromoted or hindered cell migration to positive stimuli (such asCXCL12 or PDGF-AA), cells were incubated with varying concentrationsof laquinimod for 30min prior to adding to the upper chamber inserts ofthe migration chamber. Cell counts were measured using a phase con-trast microscope from five random fields of view per treatment.Appropriate controls with no chemokine, irrelevant soluble factor, nomigrated cells and migrated cells were carried out within each experi-ment. Results were calculated as a percentage of the control (mediaonly) and the validity of the results determined by performing threeindependent experiments.

2.6. Laquinimod differentiation studies

2.6.1. NSC neuronal differentiationDifferentiation of NSC to immature neurons (as measured by

doublecortin (DCX) expression and morphology change) was achievedby culturing NSC on poly-D-lysine/laminin coated tissue culture platesin NIM in the absence of growth factors for at least 7 days. To enhancedifferentiation, NSC were treated with BDNF and cAMP in NIM mediaas described by Zhang et al. [42]. Cells were treated with laquinimodat day 1 of the differentiation protocol and media, with and withoutlaquinimod, were replenished every 2 days. DCX positive cells andtotal number of DAPI positive cells per field of view were countedfrom at least three random fields of view per well and the experimentwas repeated at least three times. Differentiated cells are expressed aspercentage DCX/nestin positive or as DCX only positive.

2.6.2. OPC differentiationFor differentiation studies, enriched OPC populations were

cultured in ITB media without EGF for 24 h and subsequentlydosed with laquinimod for 10 days, with media changes every2 days. Cultures were either lysed for quantification of oligodendro-cyte proteins CNPase or myelin basic protein (MBP) byWestern blot,or analyzed directly by immunofluorescence analyses using the im-mature oligodendrocyte marker O4. Cell counts (O4-positive vs.dapi-positive nuclei) were measured using a 20× objective on aNikon Eclipse 2000 inverted fluorescence microscope from threerandom fields of view per well.



3E.E. Kelland et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx

2.7. Bioactivity of laquinimod

Primary cultures of human brain progenitor-derived astrocyteswere obtained as a cryopreserved stock of 106 cells/ml (Life Technol-ogies: N7805-100). Astrocytes were thawed and cultured at 2 × 106

cells/cm2 on growth factor reduced matrigel (GFRM)-coated tissueculture plates in DMEM containing N-2 supplement and 10% fetalbovine serum. Following initial culture human astrocytes were plat-ed at a density of 40,000 cells/well on GFRM-coated 48-well tissueculture plates. Twenty-four hours later cells were pre-incubatedwith 0, 100 nM and 1 μM laquinimod for 2 h and stimulated withor without 10 ng/ml IL-1β or 10 ng/ml IL-1β and 10 ng/ml IFNγ for18 h. Supernatants were collected for measurement of the concen-trations of IL-6, IP-10 (CXCL10) and RANTES (CCL5) as previouslydescribed [23] using a commercially available cytometric beadarray (CBA) according to manufacturer's recommendations (BDPharmingen, San Diego, CA). Cells were fixed with paraformalde-hyde, blocked in 20% goat serum and analyzed for expression ofglial acidic fibrillary protein (GFAP; Sigma) and vimentin (Millipore)using standard immunocytochemical techniques.

2.8. Statistical analysis

Continuous variables, such as data obtained with different typesof cells (NSC, immature neurons, OPC or oligodendrocytes) or fol-lowing different treatments of the various cell populations werecompared using Student's two-tailed t-tests for normally distributeddata, or the Mann–Whitney rank sum test if the data did not satisfythe assumptions for normally distributed data. Two-sided p valueswere calculated on raw data and a p value of 0.05 or less was consid-ered significant; increasing significance of data was notated infigures as *p b 0.05, **p b 0.01, ***p b 0.001 versus respective testgroup.

3. Results

Regeneration and repair in the CNS in MS is likely to be the result ofNSC and OPC recruitment, proliferation and differentiation to, and at,sites of demyelination. Studies have demonstrated that laquinimod isable to enter the CNS [39] and recent clinical trial data indicate potentialfor a neuroprotective role in the CNS following treatment withlaquinimod [10,36]. We therefore conducted a series of experimentsto assess the effects of in vitro exposure to laquinimod on basic biologi-cal activities of NSC and OPC. For these studies we used human embry-onic stem cell derived NSC and OPC cultures as an in vitromodel system,as described by Kelland et al. [21]. In our hands, NSC cultures expressedtheNSCmarkers nestin (97.4± 0.9%) andmusashi-1 (91.7± 5.3%), theimmature neuronal marker doublecortin (DCX) (9.5 ± 2.7%) and failedto express markers for the astrocyte lineage or oligodendrocyte lineage(data not shown). Following removal of growth factors for 7 days thesecells demonstrated increased expression of the immature neuronalmarker DCX, indicating differentiation to the neuronal pathway. OPCcultures expressed OPC markers NG2 (70.3 ± 6.0%), A2B5 (52.3 ±5.5%) and PDGFRα (64.0 ± 6.5%). OPC also expressed the immature ol-igodendrocyte marker O4 (7.1 ± 1.5%) and following removal of EGFfrom the culture media for at least 7 days demonstrated increased O4expression. All of these data were consistent with our previously pub-lished observations [21].

3.1. Short-term exposure to laquinimod is not detrimental to NSC and OPCviability in vitro

For the first series of laquinimod studies we addressed the issueof cell viability. Exposing human embryonic stem cell derived NSCand OPC cultures to increasing concentrations of laquinimod for24h, 48h and 7 days was not detrimental to the cells (Fig. 1).


Fig. 1A shows that following short term exposure of NSC tolaquinimod, only sporadic, minor changes in viability were ob-served, with no dose-specific response (24 h: control 91.0 ± 2.9%vs. LAQ 10 nM 84.1 ± 2.1% cells viable, p b 0.05). Treatment of NSCwith the positive control cell death inducer, staurosporine (500nM) for 24h (Fig. 1Ai) or 48h (data not shown) resulted in significant(p b 0.01) cell death (24 h: 41.6 ± 7.1% and 48 h: 48.8 ± 0.3% cellsviable), indicating that cell death pathways are intact in these cells.Following 7 days of laquinimod treatment there was a small butsignificant reduction in propidium iodide (PI) staining at both10 μM (4.11% decrease from control; p b 0.005) and 100 μM con-centrations (6.16% decrease from control, p b 0.005) (Fig. 1Bi).Laquinimod treated OPC demonstrated no significant changes incell viability at any of the time points or concentrations tested(Fig. 1C and D). Furthermore, treatment with laquinimod up to100 μM did not induce any obvious morphological changes fromcontrol treated cells in either NSC or OPC cultures in vitro (Fig. 1:Aii & iii and Cii & iii).

3.2. Treatment of OPC with laquinimod results in minor changes incell proliferation

Wenext addressedwhether treatmentwith laquinimod affected cellproliferation in NSC or OPC cultures (Fig. 2). Cells were treated withlaquinimod in various culture conditions for 24 h or 4 days and cell pro-liferation measured by [3H] thymidine incorporation. For both timepoints NSC were cultured either in increasing concentrations oflaquinimod in NIM containing no growth factors (basal media) or in-creasing concentrations of laquinimod in NIM containing the growthfactors EGF (20 ng/ml) and bFGF (10 ng/ml) (proliferation media). Forthe 4 day time point NSC were also cultured with sub-maximal concen-trations of EGF and bFGF in order to determine if treatment withlaquinimod could enhance proliferation in non-optimized conditions.Culture of NSC in the presence of 20 ng/ml EGF and 10 ng/ml bFGF (pro-liferationmedia and positive control) resulted in a significant (p b 0.05)increase in thymidine incorporation compared to NSC treated withbasal media at both the 24 h (Fig. 2A: 175.3 ± 32.0% vs. 100% control)and 4 day (Fig. 2B: 658.9 ± 52.3% vs. 100% control) time points. Addi-tion of laquinimod to either growth condition did not significantly affectNSC proliferation in vitro.

The effect of laquinimod on OPC proliferation was also studiedafter 24 h or 4 days of culture. For both time points OPC were cul-tured in either: 1) increasing concentrations of laquinimod in ITBmedia containing no growth factors (basal media), 2) increasingconcentrations of laquinimod in ITB media containing the growthfactor EGF (20 ng/ml) or 3) increasing concentrations of laquinimodin ITB media containing PDGF-AA (10 ng/ml), bFGF (10 ng/ml)and EGF (20 ng/ml). There was a significant (p = 0.019) dose-dependent effect of laquinimod on OPC proliferation in basal media(absence of growth factors) for 24 h (Fig. 2C, clear boxes). 10 nMlaquinimod treatment for 24 h in basal media resulted in asignificant increase (p b 0.05) in thymidine incorporation, 100 nMshowed no effect, and at the higher 1 μM dose (10-fold higherthan is estimated to enter the CNS) there was a small, but significant(p b 0.05) decrease in thymidine incorporation. Treatment with thelower dose of laquinimod for 4 days in culture also showed similarincreased proliferation (Fig. 2D, clear boxes), however the dose–re-sponse observed with 24 h of treatment did not continue through4 days of laquinimod treatment. Culture of OPC in the presence of ei-ther formulation of growth factors (which both independently serveas positive controls in this assay) resulted in significantly increasedproliferation after both 24 h (Fig. 2C, gray boxes) and 4 days(Fig. 2D, gray and black boxes). The addition of laquinimod to thegrowth factor formulations neither enhanced nor inhibited thebasal level of OPC proliferation at any of the doses tested.



Fig. 1. Effect of laquinimod onNSC andOPC cell viability in vitro. NSC andOPCwere incubatedwith increasing concentrations of laquinimod (x-axes). Cell viabilitywas quantitated after 24h with FDA/PI staining (A and C) and apoptosis was measured at 7 days using the CaspaTag assay (B and D). For CaspaTag assay, NSC or OPC were either dually stained for caspase acti-vation and propidium iodide (data not shown) or propidium iodide only (Bi and Di). NSC or OPC were also treated for 24 h with the cell death inducer staurosporine (STS, 500 nM) as apositive control for cell death in culture. Side panels (Aii and iii) and (Cii and iii) show photomicrographs of typical FDA/PI staining of control and 100 μM laquinimod treated cells anddemonstrate normal cell morphology. Data are expressed as the percentage mean ± S.E.M. FDA/PI positive or propidium iodide positive from at least three independentexperiments. Statistically significant differences (paired t-test) compared to control samples (medium only) are identified by asterisks, and level of significance represented as follows:*p b 0.05, **p b 0.01, or ***p b 0.001.


3.3. Laquinimod is a weak chemoattractant for NSC and OPC and does notaffect migratory responses to known chemoattractants in vitro

In this series of experiments the effect of laquinimod on NSC andOPC migratory responses was determined using the following para-digms: 1) chemoattractant potential of laquinimod and 2) effect ofpre-treatment of NSC and OPCwith laquinimod onmigratory responsesto known chemoattractants. After 24 h, NSC demonstrated a small, butsignificant increase (132.92 ± 22.21% vs. 100% control, p b 0.05) in mi-gration in response to 10 μM laquinimod (lower chamber, Fig. 3A).Human NSC express the chemokine receptors CXCR4 and CXCR1,therefore as a positive control, we exposed cells to the cognatechemoattractant ligands CXCL12 (SDF-1α) and CXCL8 (IL-8). As expect-ed, exposure of NSC to CXCL12 (200 ng/ml) and CXCL8 (500 ng/ml) re-sulted in significant (p b 0.001) NSC chemotaxis (Fig. 3A). NSC failed tomigrate towards culture media containing the proliferative mitogensEGF and bFGF. Co-treatment of laquinimod with sub-maximalchemoattractant concentrations of CXCL12 (25 ng/ml and 100 ng/ml re-spectively) did not enhance NSCmigration (data not shown). Addition-ally pre-treatment of NSCwith increasing concentrations of laquinimod(upper chamber) did not affect their migratory response to CXCL12(lower chamber) (Fig. 3B).

The effect of laquinimod on OPC migration was also assessed. After24 h OPC demonstrated a small, but significant increase (113.86 ±5.73% vs. 100% control, p b 0.05) in migration in response to 100 nMlaquinimod (lower chamber) (Fig. 3C). The positive controlchemoattractants PDGF-AA (100 ng/ml) and CXCL8 (500 ng/ml)resulted in the expected significant OPC chemotaxis (Fig. 3C) [21].Pre-treatment of OPC with increasing concentrations of laquinimod(upper chamber) did not affect OPC migratory responses to PDGF-AA(Fig. 3D).


3.4. Laquinimod does not influence NSC early neuronal differentiation

Since NSC are an essential component of repair in the CNS wewished to determine if laquinimod influences NSC neuronal differentia-tion in vitro. For these experiments NSC were cultured in either 1) NIMcontaining 20 ng/ml EGF and 10 ng/ml bFGF (favoring proliferative con-ditions), 2)NIMcontaining no growth factors (favoringdefault differen-tiation conditions), 3) NIM containing BDNF (10 ng/ml) and cAMP(100 ng/ml) (favoring neuronal differentiation conditions) [42] or 4)NIM containing BDNF, cAMP and bFGF (favoring proliferation and neu-ronal differentiation conditions). For all of the culture conditions NSCwere treated with increasing concentrations of laquinimod for 7 days(with media changes every other day). Differentiation was quantifiedusing cell morphology and immunocytochemistry to detect the NSCmarker nestin and the immature neuronal cell marker DCX. We notedthat NSC differentiation to the neuronal lineage is accompanied by in-creased DCX expression and altered cell morphology in the absence oflaquinimod (Fig. 4: Ai andDii). As theNSC differentiates, itsmorphologychanges from a fibroblast-like appearance to a bi-polar elongated cellthat aligns and orientates to neighboring elongated cells such that uni-form polar alignment and bundling of cell nuclei is observed, as demon-strated in Fig. 4Dii: ‘Control’. The change in morphology to a bi-polarelongated cell is always accompanied by DCX expression (NSC with afibroblast-like appearance do not express DCX) and dual staining fornestin. As the cell further differentiates and matures, nestin expressionis lost. Therefore for this series of experiments, data are expressed aspercentage population of cells expressing either 1) nestin only to denotethe NSC population (Fig. 4B), 2) DCX and nestin dual staining to denoteNSC that have begun to differentiate to cells of a neuronal lineage(Fig. 4Ai) and 3) DCX only (N50% loss in nestin expression) to denotefurther maturation of these cells (Fig. 4Aii). In NSC cultured in



Fig. 2. Effect of laquinimod on NSC and OPC proliferation in vitro. The effect of laquinimod on NSC (A and B) or OPC (C and D) [3H] thymidine incorporation was determined after 24 h ofculture or after 4 days of culture. (A and B) For both time points NSCwere treatedwith increasing concentrations of laquinimod (x-axes) inNIMmedia containing no growth factors (clearboxes), or 20 ng/ml EGF and 10 ng/ml bFGF (black boxes) which served as a positive proliferation control. For 4 day cultures NSC were also cultured in increasing concentrations oflaquinimod in NIM media containing sub-optimal concentrations of EGF and bFGF respectively (dark gray boxes). (C and D) For both time points, OPC were treated with increasingconcentrations of laquinimod whilst cultured either in: 1) ITB media containing no growth factors (clear boxes); 2) ITB media containing 20 ng/ml EGF (gray boxes) or 3) ITB mediacontaining 10 ng/ml PDGF-AA, 10 ng/ml bFGF and 20 ng/ml EGF (black boxes). OPC optimal growth conditions were observed with ITB media containing 20 ng/ml EGF at 24 h and4 days and served as a positive proliferation control. Data are expressed as the percentage change from control mean ± S.E.M. from at least three independent experiments. Statisticallysignificant differences (paired t-test) are identified by asterisks, compared groups identified by connecting line, and level of significance represented as follows: *p b 0.05.


proliferating conditions (EGF and bFGF) for 7 days approximately 10% ofthe population of cells express DCX (Fig. 4Ai). In these culture condi-tions, DCX-expressing cells, though bi-polar in morphology, have ascattered distribution in the field of view (Fig. 4Di). Removal of growthfactors for 7 days resulted in a significant increase in expression of DCX(43.3 ± 3.8% population, p b 0.01) with cells displaying bi-polar elon-gated morphology and uniform cell alignment in the field of view(Fig. 4: Ai and Dii). Similar trends were also observed when BDNF andcAMPwere added to the cultures (33.5±2.7%DCX+population). Inter-estingly, addition of BDNF and cAMP significantly enhanced (p b 0.01)cell maturation (DCX+ only) as compared to removal of growth factors(Fig. 4Aii: 11.1 ± 0.7% BDNF/cAMP vs. 6.8 ± 1.6% no growth factors).The presence of 10 ng/ml bFGF in the proliferation/differentiation(bFGF, BDNF and cAMP) culture media kept NSC in a proliferativestate with only 9.7± 1.7% cells expressing DCX. Addition of laquinimodin all of these culture conditions for 7 days did not affect any stage ofearly NSC differentiation or maturation in vitro. Moreover laquinimodtreatment did not influence cell morphology and cell alignment.

3.5. OPC differentiation is not affected by laquinimod treatment

Following a demyelinating injury OPC are recruited to lesion siteseither from the subventricular zone or neighboring parenchyma, andare able to differentiate and remyelinate damaged axons. An essentialstep in this remyelination is maturation of the OPC to oligodendrocytes.Therefore it is important to determine if laquinimod influences OPCdifferentiation. To this end human OPC were treated with varying


concentrations of laquinimod for 10 days in the absence of proliferativegrowth factors (with media changes every other day). OPC differentia-tion was quantified using immunocytochemistry to detect expressionof the oligodendrocyte marker O4, and by Western blot analysis of theoligodendrocyte specific proteins CNPase and myelin basic protein(MBP). OPC cultured in ITB media in the absence of growth factors for10 days demonstrated a significant (p b 0.05) increase in expressionof O4 (18.2± 2.5%) comparedwith control cells at day 1 of the differen-tiation protocol (7.1 ± 1.5%). Treatment of OPC with laquinimod for 10days did not significantly alter the number of cells expressing O4(Fig. 5A and B). Changes in the expression of the OPC marker NG2 andoligodendrocyte markers CNPase and MBP in Western blots revealedsubtle changes (from time-point matched control) in NG2 expressionfollowing treatmentwith 10 nM and 1 μM laquinimod (Fig. 5C). CNPaseand MBP expression did not change between time-point matchedcontrol and laquinimod treated cultures.

3.6. Bioactivity of laquinimod: human astrocytes show decreased pro-inflammatory cytokine secretion upon laquinimod treatment

Since laquinimod had no effect on NSC and OPC viability, prolifera-tion, response to chemotactic factors or differentiation in vitro, it is im-portant to demonstrate that the laquinimod preparations we used areindeed capable of producing a biological effect on cells of the CNS line-age. Bruck et al. [4] recently demonstrated a direct effect of laquinimodon astrocyte cultures, thereby providing a viable model for confirmingbiological activity in the laquinimod preparations used in this study.



Fig. 3. Effect of laquinimod on NSC and OPC migration in vitro. Migration of NSC (A) and OPC (C) in response to laquinimod and inhibition of chemotactic migration of NSC (B) and OPC(D) by laquinimod. (A) NSC chemotaxis in response to increasing concentrations of laquinimod for 24 h. For all conditionsmedia was NIM culture media without growth factors. Positivecontrolswere CXCL12 (200 ng/ml) and CXCL8 (500 ng/ml). (B) Inhibition of NSCmigration by laquinimod; NSCwere pre-treatedwith increasing concentrations of laquinimod for 30minprior to exposure to 200 ng/ml CXCL12 for 24 h. (C) OPC chemotaxis in response to increasing concentrations of laquinimod for 24 h. For all conditions ITB media containing no growthfactors was used. Positive controls were PDGF-AA (100 ng/ml) and CXCL8 (500 ng/ml). (D) Inhibition of OPC migration by laquinimod; OPC were pre-treated with increasingconcentrations of laquinimod for 30 min prior to exposure to 100 ng/ml PDGF-AA for 24 h. Data are expressed as the percentage change from control (media) mean ± S.E.M. from atleast three independent experiments. Statistically significant differences (paired t-test) are identified by asterisks, compared groups identified by connecting line, and level of significancerepresented as follows: *p b 0.05 or ***p b 0.001.


Thus, pre-treatment of astrocytes with laquinimod prior to stimulationwith 10 ng/ml IL-1β significantly (p b 0.05) reduced levels of IL-6 andIP-10 in the culture supernatants compared to IL-1β stimulation alone(Fig. 6A and B). In contrast, pre-treatment of human astrocytes withlaquinimod prior to stimulation with 10 ng/ml IL-1β and IFNγ signifi-cantly (p b 0.05) increased the levels of RANTES in the culture superna-tants compared to IL-1β and IFNγ stimulation alone (Fig. 6C).Immunocytochemical staining revealed decreased ‘reactive’ astrocytemorphology in IL-1β stimulated astrocytes pre-treatedwith laquinimod(Fig. 6D–G). These findings are consistent with those of Bruck et al. [4]and support not only that the laquinimod preparations we used are bi-ologically active, but also that laquinimod has no effect on the NSC andOPC functions tested in this study.

4. Discussion

Approved therapies for the treatment of MS that can readily pene-trate the CNS have the potential to directly or indirectly modulate spe-cific brain cell functions. Since laquinimod is capable of entering theCNS, there is potential for direct interaction with neural cells, especially


NSC and OPC that are activated by CNS damage and are essential to therepair process. In the adult CNS, NSC and OPC have been demonstratedto be recruited to, and be present in and around, activeMS lesions [9,35,40]. It is assumed that they undergo differentiation, remyelinate an ex-posed axon andpermit efficient nerve conduction, aswell as provide ax-onal protection and as a consequence, prevent further damage anddisease progression in MS. Using human embryonic stem cell derivedNSC and OPC cultures, as a model system, we tested the direct effectof laquinimod treatment on NSC and OPC viability, proliferation, migra-tion and differentiation in vitro. We have previously demonstrated thatthese human cells are capable of differentiating into cells of oligoden-drocyte, neuronal and astrocyte lineages and that when transplantedinto the corpus callosum of mice deficient in myelin basic protein dem-onstrate functionalmyelin formation [21]. The data clearly show that di-rect exposure to laquinimod, over a concentration range of 10 nM to 1μM,was not detrimental to basic functions of these cells. The lack of a di-rect effect of laquinimod on primary murine OPC cultures was also re-cently demonstrated [4]. To our knowledge, laquinimod has not beentested in NSC or OPC of human origin. We also confirmed thatlaquinimod was bioactive in our hands by duplicating the findings of



Fig. 4. Effect of laquinimod on NSC neuronal differentiation in vitro. NSC were cultured under the four different conditions identified at the head of each figurewith increasing concentra-tions of laquinimod (x-axes) for 7 days. Differentiationwas quantifiedby immunocytochemistry using theneural stemcellmarker nestin (greenfluorescence) and the immature neuronalcellmarker doublecortin (DCX, redfluorescence); cell nucleiwere stainedwithDAPI (bluefluorescence). (A) (i) Percentage of DCX and nestin dual positive cells and (ii) percentage of DCXpositive cells only. (B) Percentage of nestin positive cells. (C) Average number DAPI positive nuclei per field of view. (D) Representative images of NSC cultured in the presence oflaquinimod for 7 days, ×200magnification. Note that DCX expression is restricted to cells with elongatedmorphology denoting neuronal differentiation; some of these cells retain nestinexpression that is subsequently lost upon further maturation. Data are expressed as the mean percentage population positive for specific lineage marker ± S.E.M. from at least threerandom fields of view and from three independent experiments. Statistically significant differences (paired t-test) are identified by asterisks, compared groups identified by connectingline, and level of significance represented as follows: **p b 0.01.


Bruck et al. [4]. Therefore, this lack of a direct biological effect on cells ofthe NSC and OPC lineage is relevant to the mechanism of action of thisdrug. Direct mechanisms of action of laquinimod in the CNS appearmore likely to be via modulation of astrocyte activity, potentially causinga shift in favor of anti-inflammatorymechanisms to create amorepermis-sive environment for repair [1,4,17]. If this is the case then general sup-pression of inflammation in CNS autoimmune demyelinating disordersmay not be an optimal approach to successful long-term therapy, espe-cially in view of reports that ‘inflammation’ is required for remyelinationand repair in demyelinating disorders [2,14,27,29,34]. The possibility thatlaquinimod promotes ‘beneficial’ inflammation to preserve NSC and OPC


function and promote stem cell recruitment and repair in lesions thathave not yet succumbed to axonal degradation is compelling, as is thepossibility that laquinimod may offer direct protection of NSC and OPCfrom immune insult and is worthy of further investigation.

Conflict of interest

The authors disclose no conflict of interest. Dr. Liat Hayardeny isemployed by Teva Pharmaceutical industries. Drs. Weiner and Lundserved as consultants for Teva in 2012 and 2013 for which they receivedhonoraria.



Fig. 5. Effect of laquinimod on OPC differentiation in vitro. (A) Effect of treatment with increasing concentrations of laquinimod on O4 expression in OPC cultured in ITB media in theabsence of growth factors for 10 days. Data are expressed as percentagemean± S.E.M. (O4-positive vs. dapi nuclei) from at least three independent experiments. Statistically significantdifferences (paired t-test) are identified by asterisks, compared groups identified by connecting line, and level of significance represented as follows: *p b 0.05. (B) Typical photomicro-graphs of laquinimod treated OPCwith O4 staining (red) and DAPI stained nuclei (blue). (C) RepresentativeWestern blot analyses of protein expression by OPC cultures in the presence oflaquinimod for 10 days. OPC populations are measured by NG2 expression, whereas oligodendrocyte populations are measured by CNPase andMBP expression;α-tubulin was used as aprotein loading control.

Fig. 6. Pre-treatment with laquinimod alters secretion of critical immune effector solutes by stimulated human astrocytes in vitro. (A–C) Human astrocytes were pre-treatedwith varyingconcentrations of laquinimod prior to stimulationwith either 10ng/ml IL-1β or 10ng/ml IL-1β plus 10ng/ml IFNγ, and culture supernatants collected 18 h later. The concentrations of IL-6,IP-10 and RANTES were measured using cytometric bead array. Data are expressed as concentration (pg/ml) ± S.E.M of at least three different cell preparations. (D–G) Representativephotomicrographs of laquinimod treated astrocytes displaying altered morphology. Astrocytes in control (media only) conditions (D) and stimulated with IL-1β for 18 h with nolaquinimodpre-treatment (vehiclemedia control) (E); 100 nM laquinimodpre-treatment (F) and 1 μM laquinimodpre-treatment (G). Statistically significant differences between groupsare indicated by adjoining line, * indicates a p-value of b0.05.


Please cite this article as: Kelland EE, et al, In vitro assessment of the direct effect of laquinimod on basic functions of human neural stem cells andoligodendrocyte progenitor cells, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.07.058



Acknowledgments

The authors would like to thank Peili Li for her technical assistanceand colleagues from the USC Comprehensive MS Care Center for theirinsight and helpful discussions during the course of this study. Thisstudy was supported by an investigator initiated grant from Teva Phar-maceutical Industries.

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