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Stem Cell ReportsArticle

Human Neural Precursor Cells Promote Neurologic Recovery in a ViralModel of Multiple Sclerosis

Lu Chen, 1 ,7 Ronald Coleman, 2 ,7 Ronika Leang, 1 Ha Tran, 2 Alexandra Kopf, 1 Craig M. Walsh, 1Ilse Sears-Kraxberger, 3 Oswald Steward, 4 Wendy B. Macklin, 5 Jeanne F. Loring, 2 ,* and Thomas E. Lane 1 ,6 ,*1Department of Molecular Biology and Biochemistry, Sue and Bill Gross Stem Cell Center, Multiple Sclerosis Research Center, University of California,Irvine, Irvine, CA 92697, USA2Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA3Reeve-Irvine Research Center, University of California, Irvine, Irvine, CA 92697, USA4Reeve-Irvine Research Center, Departments of Anatomy & Neurobiology, Neurobiology & Behavior, and Neurosurgery, School of Medicine,University of California, Irvine, Irvine, CA 92697, USA5Department of Cell and Developmental Biology, School of Medicine, University of Colorado, Aurora, CO 80045, USA6Present address: Division of Microbiology & Immunology, Departmentof Pathology, School of Medicine, University of Utah, Salt Lake City, UT 84112, USA7Co-rst author*Correspondence: [email protected] (J.F.L.), [email protected] (T.E.L.)http://dx.doi.org/10.1016/j.stemcr.2014.04.005

This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/3.0/ ).

SUMMARY

Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonicstem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyondday 8posttransplantation. Improved motor skills were associated with a reduction in neuroinammation, decreased demyelination, andenhanced remyelination. Evidence indicates that the reduced neuroinammation is correlated with an increased number of CD4 +CD25 +FOXP3+ regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reducedT cell proliferation andincreased Treg numbers. The hNPCs acted, in part, through secretion of TGF- b 1 and TGF- b2. These ndings indi-cate that thetransient presence of hNPCs transplantedin an animal model of MS has powerful immunomodulatory effects andmediatesrecovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MStreatments.

INTRODUCTION

Multiple sclerosis (MS) is a chronic inammatory diseaseof the central nervous system (CNS) involving immunecell inltration into the central nervous system (CNS),which results in demyelination and axonal loss thatculminates in extensive neurological disability ( Steinman,1996 ). The demyelination is progressive over time;however, spontaneous, but transient, myelin repair canoccur during the course of the disease. Endogenous oligo-dendrocyte precursor cells (OPCs) are believed to beresponsible for spontaneous remyelination ( Lassmannet al., 1997 ), but it is unclear why these cells only actintermittently. An important unmet clinical need for MSpatients is an effective method to induce sustainedremyelination.

Cell transplantation therapy is a promising approach topromote remyelination in MS patients; human embryonicstem cells (hESCs) and induced pluripotent stem cells, aswell as fetal brain, are potential sources of therapeutic cells(Brustle et al., 1999; Mu ller et al., 2006 ). Studies in animalmodels have demonstrated the benets of cell therapy intreating mouse models of MS. For example, myelin genera-tion ( Buchet et al., 2011 ), accompanied by modulation of inammatory responses, followed CNS transplantation of

human neural precursor cells into animal models in

which myelin formation is defective or demyelination isinduced via immunization with encephalitogenic pep-tides. Another model, which we employed in this study,is based on ndings that persistent infection with a mouseneurotropic coronavirus correlates with chronic neuroin-ammation and immune-mediated demyelination ( Laneand Buchmeier, 1997 ).

Here, we demonstrate sustained neurologic recoveryout to 6 months following intraspinal transplantationof hESC-derived NPCs (hNPCs) into mice in whichinammatory-mediated demyelination was initiatedby persistent viral infection of the CNS. We observedclinical recovery associated with muted neuroin-ammation and decreased demyelination, along withemergence of CD4 +CD25 +FOXP3 + regulatory T cells(Tregs). Ablation of Tregs in hNPC-transplanted miceinhibited the improvement in motor skills and increasedneuroinammation and in vitro, hNPCs modulatedcytokine secretion and proliferation by antigen-sen-sitized T cells. Interestingly, the hNPCs were rapidlyrejected after transplantation into these immunocom-petent mice, indicating that the sustained neurologic re-covery was not dependent on stable engraftment of hNPCs.

Stem Cell Reports j Vol. 2 j 825837 j June 3, 2014 j 2014 The Authors 825
http://-/?-mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.stemcr.2014.04.005http://creativecommons.org/licenses/by-nc-nd/3.0/http://crossmark.crossref.org/dialog/?doi=10.1016/j.stemcr.2014.04.005&domain=pdfhttp://creativecommons.org/licenses/by-nc-nd/3.0/http://dx.doi.org/10.1016/j.stemcr.2014.04.005mailto:[email protected]:[email protected]://-/?-


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RESULTS

Intraspinal Injection of Human ESC-Derived NeuralPrecursor Cells Results in Clinical Improvement of

JHMV-Infected MiceHuman neural precursor cells (hNPCs) were derived fromWA09 hESCs using modications of published protocols(Trosset et al., 2006; Vogt et al., 2011 ). An important modi-cation wascell passagingto control cell density during the9-day-directed differentiation protocol, and the trans-planted cells had a uniform cellular morphology ( Fig-ure 1 A). Because there is considerable phenotypic diversityamong preparations of neural precursor cells ( Mu ller et al.,2008 ), we performed extensive microarray-based transcrip-tome analysis to dene a genomic phenotype for the cellsthat showed clinical activity. The microarray analysisrevealed a consistent prole of gene expression amongseparate populations of hNPCs differentiated by ourmethod ( Figure 1 B; Table S1 available online).

To evaluate the effects of hNPCs in promoting clinicalrecovery, we injected cells into thespinal cords of immuno-competent mice with established JHMV-induced clinicaldisease ( Carbajal et al., 2010 ), which was generated bythe injection of susceptible mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV); consistentwith previous studies, the mice showed viral persistencein white matter tracts and hind-limb paralysis, as well asdemyelination and neuroinammation ( Templeton andPerlman, 2007 ).

Injection of hESC-derived hNPCs, but not human bro-blasts, resulted in a reduction in the severity of clinicaldisease and improved motor skills ( Figure 1 C) that weresustained out to 6 months posttransplantation (pt) ( Fig-ure 1 D). Of 96 mice injected with hNPCs, 66 (73%)displayed a signicant (p < 0.05) improvement in motorskill recovery, whereas nine of the 63 mice (14%) trans-planted with the vehicle control improved. Living cellsand intraspinal injection were necessary for the clinicalrecovery; transplantation of dead cells or delivery of cellsvia intravenous or intraperitoneal injection did not resultin improved clinical outcome (data not shown).

Because transplantation of the hNPCs was performed inimmunocompetent mice, we expected the cells to berapidly rejected. To track the fate of the cells followinginjection, we transplanted hNPCs constitutively express-ing the Photinus pyralis luciferase gene and monitored theirpresence by daily luciferin injection and IVIS imaging.Luciferase signals within the spinal cord were highest atday 1 pt and declined below the level of detection by day8 pt. ( Figure 1 E). Imaging of transplanted cells revealedthat the hNPCs did not migrate extensively within thespinal cord and did not disseminate into peripheral tissues.To conrm the imaging data, spinal cords from hNPC-

transplanted mice were removed at dened times pt,serially sectioned 5 mm rostral and 5 mm caudal to theinjection site, and immunostained for a human-speciccytoplasmic antigen ( Uchida et al., 2012 ). As shown in

Figure 1 F, hNPCs were detected at days 1 and 4 pt, butstaining was very weak by day 7 pt. These ndings conrmthat transplanted hNPCs survived for approximately1 week after transplantation.

Neuroinammation and Demyelination Was Reducedin hNPC-Transplanted MiceTo investigate potential mechanisms underlying hNPC-mediated improvement in neurologic function, weexamined neuroinammation in hNPC-transplantedmice. Analysis of spinal cords from hNPC-transplantedmice at 21 days pt revealed a marked reduction in inam-matory cells present within spinal cords compared to thecontrols. Clinical recovery following hNPC transplanta-tion is associated with reduced neuroinammation in thespinal cord ( Figure 2 A). Immunostaining for activatedmacrophage/microglia and T cells ( Figures 2 B and 2C),critical in amplifying the severity of demyelination in JHMV-infected mice, revealed an overall reduction inimmunoreactive cells within the spinal cords that wassustained past 175 days pt. (data not shown). Quantica-tion of macrophage/microglia and T cell immunostainingindicated that hNPC transplantation resulted in a signi-cant (p < 0.01) reduction in both cell populationscompared to control mice ( Figure 2 D). Flow cytometry

analysis conrmed immunostaining by showing reducedinltration of CD4 + and CD8 + T cells into the CNS of hNPC-treated mice compared to controls ( Figures 3 A and3B). Accumulation of virus-specic CD4 + and CD8 +

T cells in the CNS of hNPC-treated mice was also deter-mined by tetramer staining ( Zhao et al., 2011 ). Inltrationof CD4 + T cells recognizing the immunodominant epitopewithin the viral matrix (M) glycoprotein spanning aminoacids 133147 (M133147) and CD8 + T cells specic forthe immunodominant epitope in the spike glycoproteinlocated on amino acids 510518 (S510518) were bothreduced in hNPC-transplanted mice ( Figures 3 A and 3B).In addition, macrophage (F4/80 +CD45 high ) inltrationwas reduced in hNPC-transplanted mice ( Figure 3 C). Anal-ysis of viral titers within the CNS of experimental miceindicated that control of viral replication within the spinalcords was not affected by hNPC transplantation; repli-cating virus was not detected ( Figure 3 D), and there wereno differences in transcript levels of viral RNA ( Figure 3 E).

Spinal cord sections from transplanted mice thatdisplayed improved motor function showed a dramaticreduction in the severity of demyelination when comparedto control mice ( Figure 4 A). Quantication of demyelin-ation indicated a signicant (p < 0.05) reduction in white

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Stem Cell ReportshNPCs Enhance Clinical Recovery of MS Model Mice


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matter damage in mice treated with hNPCs (1.9 0.4,n = 9) compared to control mice (3.1 0.1, n = 7) ( Fig-ure 4 B). EM analysis of spinal cord sections was performed

to determine whether remyelination was occurring inhNPC-transplanted mice. Assessment of the g-ratio, theratio of the inner axonal diameter to the total outer ber

Figure 1. Characteristics and Transplantation of hESC-Derived NPCs into JHMV-Infected Mice(A) Human neural precursor cells (hNPCs) after 9 days of directed differentiation. The cells are closely packed and have a distinctivemorphology, and the culture appears to be homogeneous.(B) Gene expression signature of hNPCs used for transplantation experiments. A heatmap shows the hierarchical clustering of 118 probesthat were signicantly differentially expressed (p < 0.001) in hNPCs. Five independent cultures of hNPCs were compared by global geneexpression analysis (Human HT-12 v. 4 Expression Beadchip), with four samples of human pluripotent stem cells (two hESC and two iPSClines) and a sample of human broblasts. The scale at right indicates the fold differences in gene expression, with yellow indicatingrelatively higher expression and blue indicating relatively lower expression.(C) Human broblasts or hNPCs were transplanted by intraspinal injection into JHMV-infected mice at day 14 postinfection. hNPC-transplanted mice improved (p < 0.05) motor skills compared to animals transplanted with either broblasts or vehicle alone (control).(D) Improved (p < 0.05) clinical recovery in hNPC-transplanted JHMV-infected mice wassustained outto 168days posttransplantation (pt)when compared to infected mice treated with vehicle alone. For experiments shown in (C) and (D), the data are the results of two in-dependent experiments with data shown as averages SEM.(E)Daily IVIS imaging of luciferase-labeledhNPCsrevealed that following intraspinal transplantation, cells were reduced to below thelevel of detection by day 8 posttransplantation; representative mice are shown. IVIS imaging was performed on vehicle-transplanted mice as acontrol.(F) Representative immunohistochemical staining for human cells (STEM121 antibody) at dened times pt conrms IVIS imaging dataand shows that following intraspinal transplantation, hNPCs are gradually eliminated, with very few cells remaining by day 7 post-transplant; 60 3 images are of framed areas in the 4 3 images.

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diameter, is a structural index of remyelination; lowerratios indicate more extensive myelination ( Liu et al.,2001; Moore et al., 2013 ). The calculated g-ratio of hNPC-transplanted mice (0.83 0.005, n = 533 axons) was signif-icantly (p < 0.001) lower than that of control mice (0.94 0.005,n = 541 axons)at 3 weeks pt ( Figure4 C), indicating agreater degree of myelination in the transplanted mice.

hNPC Treatment Increased the Frequency of CD4 + CD25 + FOXP3 + Treg Cells within the CNSTregs have been shown to dampen neuroinammationwithin the CNS of JHMV-infected mice ( Trandem et al.,2010 ). In our studies, hNPC-mediated clinical recoverywas associated with muted CNS inammation, suggestingthat the transplanted hNPCs may have promoted recoverythrough Treg-mediated mechanisms. Flow analysis of dissociated cells from spinal cords revealed an increasedfrequency of Tregs (CD4 +CD25 +FOXP3 +) in the spinalcords of hNPC-transplanted mice at day 10 pt ( Figures 5 Aand 5B). In order to clarify the role of Tregs in improvedclinical recovery, hNPCs transplanted mice were treatedat days 2, 0, and +2 pt with anti-CD25 monoclonal anti-

body, which efciently blocks Treg activity ( Kohm et al.,2006 ). Flow analysis of CD4 +CD25 +FOXP3 + T cells withindraining cervical lymph nodes of anti-CD25-treatedmice at day 9 pt conrmed efcient depletion of Tregs(>95%) (data not shown). This treatment inhibited func-tional improvement in hNPC-transplanted mice out today 20 pt ( Figure 5 C). Further, the anti-CD25 treatmentwas associated with increased neuroinammation inhNPC-transplanted animals when compared to trans-planted animals treated with control antibody ( Figures5D and 5E).

We determined whether hNPCs inuenced cytokineexpression and proliferation of activated T cells; previousstudies reported that transplanted hNPCs could modulateT-cell-mediated cytokine secretion ( Liu et al., 2013 ), andsecretion of proinammatory cytokines, including IFN- gand TNF- a , has been suggested to contribute to the severityof demyelination ( Pewe and Perlman, 2002; Sun et al.,1995 ). Stimulation of T cells isolated from hNPC-trans-planted mice with virus-specic CD4 + peptide resultedin reduced (p < 0.05) expression of proinammatorycytokines IFN- g and TNF- a , whereas expression of

Figure 2. hNPC-Mediated Clinical Recov-ery Is Associated with Dampened Neuro-inammation at Day 21 Pt(A) Clinical recovery following hNPC trans-plantation was associated with reducedneuroinammation indicated by H&Estaining of spinal cord sections (upper panel, control; lower panel, hNPC-treated).(B and C) Immunohistochemical stainingdemonstrated reduced (B; upper panel,control; lower panel, hNPC-treated) macro-phage inltration/microglia activation(IBA-1 reactivity, brown) and (C) CD3-pos-itive lymphocytes (pan-T cell marker, Alexa488/green staining) within spinal cords of hNPC-treated and control mice (upper panel, control; lower panel, hNPC-treated).(D) Quantication of IBA-1 and CD3-reac-

tive cells indicated a signicant reductionin macrophage/microglia and CD3-positivecells following hNPC transplantationcompared to control mice. Data are repre-sentative of at least two independent ex-periments with a minimum of three mice per group; data are presented as averages SEM. Mann-Whitney t tests were used todetermine the p values.




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and found that the TGF- b family of cytokines was differen-tially expressed (p < 1 3 10 6 ) in the hNPCs ( Figure 7 A).TGF-b2 transcripts were 250-fold higher in hNPCs, whereasTGF-b1 transcripts were 5-fold higher compared to hESCs(Figure7 A).Hepatocyte growthfactor (HGF) waspreviouslyshown to be associated with mesenchymal stem cell-medi-

ated recovery in an experimental autoimmune encephalo-myelitis (EAE) mouse model ( Baietal.,2012 ), butour resultsshowed that HGF transcripts were nearly undetectable inour hNPCs ( Figure 7 A). Consistent with the transcript anal-ysis, measurement of protein levels in culture mediumconditioned by hNPCs showed that there was an approxi-mate 40-fold higherlevelofTGF- b2 anda 3-foldhigher levelof TGF-b1 in hNPC-conditioned medium (CM) comparedto control medium ( Figure 7 B). Inclusion of neutralizingantibodies specic for TGF- b1 orTGF-b2 blocked theabilityof hNPCs to inhibit proliferation of virus-specic CD4 +

T cells, and inclusion of both antibodies had the largesteffect ( Figure 7 C). In addition, antibody targeting of TGF-b1 or TGF-b2, alone or in combination, reduced numbersof CD4 +CD25 +FOXP3 + T cells emerging in cocultures of hNPCs and T cells ( Figure 7 D), suggesting an importantrole for these cytokines in contributing to the immuno-modulatory effects of hNPCs.

DISCUSSION

There hasbeen growing interest in theuseof cell transplan-tation as therapy for neurological diseases since the rst

human trials using fetal neural precursors for Parkinsonsdisease were performed more than 20 years ago (reviewedin Barker et al., 2013 ). Currently, there are preclinicalstudies and clinical trials using broblastic adult mesen-chymal stem cells (reviewed in Neirinckx et al., 2013 ) andfetal-derived neural cells ( Uchida et al., 2012 ) for transplan-

tation to treat neurological disease. A recent clinical studyshowed that fetal neural cell transplants promoted myeli-nation in individuals aficted with Pelizaeus-Merzbacherdisease (PMD), a rare hypomyelinating disease ( Guptaet al., 2012 ).

Multiple sclerosis (MS) is a demyelinating disease that isan attractive target for cell therapy because of the lack of long-term therapeutic benet from current treatments.We investigateda cell-therapystrategy using human neuralprecursor cells derived from hESCs for their therapeuticpotential in a mouse model of demyelination induced byviral infection. Available evidence indicates that the causeof MS is multifactorial and includes genetic backgroundand environmental inuences ( Goodin, 2012; Pugliattiet al., 2008 ). Although no clear causal relationship betweenMS and viral infection has been rmly established, virusesare capable of persisting within the CNS and have beenimplicated in initiating or exacerbating MS symptoms(Gazouli et al., 2008 ).

We found that hNPC-mediated recovery in our modelwas associated with a marked reduction in neuroinamma-tion, characterized by reduced inltration of inammatoryT cells and macrophages within the spinal cords and emer-gence of regulatory T cells (Tregs). Importantly, recovery

A B

C

Figure 4. hNPC Treatment Reduced theSeverity of Demyelination(A) Representative luxol fast blue stain of spinal cord sections obtained from either hNPC-treated or control mice at 3 weeks pt.Dashed lines indicate areas of white matter damage.(B) Quantication of the severity of demy-elination at 3 weeks posttransplant in-dicates that white matter damage is signif-icantly decreased (p < 0.05) in hNPC-treatedversus control.(C) Representative EM images (12003 )showing increased numbers of remyelinatedaxons (red arrows) compared to demyeli-nated axons (blue arrows) in hNPC-trans-planted mice compared to control mice.Calculation of g-ratio, as a measurement of

structural and functional axonal remyelina-tion, revealed a signicantly (p < 0.001)lower g-ratio (indicative of remyelination)

in hNPC-treated mice (0.83 0.005, n = 533 axons) compared to control mice (0.94 0.005, n = 541 axons) at 3 weeks pt. For graphsshown in (B) and (C), data are representative of at least two independent experiments with a minimum of three mice per group; data arepresented as average SEM; the Mann-Whitney t test was used to determine the p values.




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occurred in spite of the rapid rejection of the hNPCsin these immunocompetent mice. In MS, as in otherneurodegenerative diseases, there is growing evidencethat long-term survival of transplanted cells is not requiredfor benecial effects. A recent study ( Bai et al., 2012 )reported that mesenchymal stem-cell-induced recovery inthe EAE mouse model and in lysolecithin-induced demye-lination was dependent upon release of HGF. Althoughwe have found that our hNPCs do not produce detectableHGF mRNA or protein, our ndings are similar with regardto immunomodulation, diminished spread of demye-lination, and remyelination. Additional studies also sup-port the immunomodulatory properties associated withtransplanted stem cells; in one study, intracerebroventri-cularly injected human glial cells rapidly died yet resultedin reduction of the clinical severity of EAE that correlatedwith inhibited proliferation of myelin-specic T cells(Kim et al., 2012b ). Similarly, human ESC-derived oligo-dendrocyte precursors did not survive past 8 daysfollowing intraventricular injection into mice with EAE,

yet animals displayed decreased neurologic disability, andthis was associated with increased numbers of regulatoryT cells within the CNS ( Kim et al., 2012a ). These reportsare consistent with growing evidence that transplantedstem cells rarely differentiate into cells of neural lineage,and their efcacy often appears to be through delivery of trophic factors ( Blurton-Jones et al., 2009; Mu ller et al.,2006 ) or by modulating inammation ( Neirinckx et al.,2013 ).

An important question that remains an area of ongoingwork is to determine whether the sustained presence of Tregs after hNPC rejection continues to dampen neuro-inammation long term. We believe this is possible becausehNPCs had already disappeared from the spinal cord at thetime we detected increased numbers of Tregs. Our resultssuggest that Tregs are a critical component of the recovery,because antibody-mediated ablation of Tregs reduced theclinical effects of hNPCs. We are interested in character-izing cytokine proles within both the CNS and drainingcervical lymph nodes, as recent studies argue that

Figure 5. hNPC-Mediated Recovery IsAssociated with Treg Emergence withinthe Spinal Cord(AE) Mice were infected intracranially withJMHV and transplanted with either hNPCs or vehicle alone at day 14 post-infection.(A)Examination of Tregs within the spinal cords by ow analysis (CD4+CD25+FOXP3+)revealed an increased frequency of Tregs inhNPC-transplanted animals at day 10 post-transplantation, a time at which animalsbegan to display improved motor skillscompared to control mice. A representativeow cytometric dot blot is shown. (B)Quantication of Treg numbers in spinal cords of mice indicated a signicant (p

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