Journ

alof

Cell

Scie

nce

Macrophage migration inhibitory factor (MIF)promotes cell survival and proliferation of neuralstem/progenitor cells

Shigeki Ohta1, Aya Misawa1, Raita Fukaya2, Satoshi Inoue3, Yonehiro Kanemura4,5, Hideyuki Okano6,Yutaka Kawakami1,*,` and Masahiro Toda2,*,`

1Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku,160-8582, Japan2Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan3Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan4Division of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka,Chuo-ku, Osaka 540-0006, Japan5Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan6Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

*These authors contributed equally to this work`Authors for correspondence (yutakawa@sc.itc.keio.ac.jp; todam@sc.itc.keio.ac.jp)

Accepted 23 February 2012Journal of Cell Science 125, 3210–3220� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.102210

SummaryIn a previous study, we showed that murine dendritic cells (DCs) can increase the number of neural stem/progenitor cells (NSPCs) in vitroand in vivo. In the present study, we identified macrophage migration inhibitory factor (MIF) as a novel factor that can support the

proliferation and/or survival of NSPCs in vitro. MIF is secreted by DCs and NSPCs, and its function in the normal brain remains largelyunknown. It was previously shown that in macrophages, MIF binds to a CD74–CD44 complex. In the present study, we observed theexpression of MIF receptors in mouse ganglionic-eminence-derived neurospheres using flow cytometry in vitro. We also found CD74

expression in the ganglionic eminence of E14 mouse brains, suggesting that MIF plays a physiological role in vivo. MIF increased thenumber of primary and secondary neurospheres. By contrast, retrovirally expressed MIF shRNA and MIF inhibitor (ISO-1) suppressedprimary and secondary neurosphere formation, as well as cell proliferation. In the neurospheres, MIF knockdown by shRNA increased

caspase 3/7 activity, and MIF increased the phosphorylation of Akt, Erk, AMPK and Stat3 (Ser727), as well as expression of Hes3 and Egfr,the products of which are known to support cell survival, proliferation and/or maintenance of NSPCs. MIF also acted as a chemoattractantfor NSPCs. These results show that MIF can induce NSPC proliferation and maintenance by multiple signaling pathways actingsynergistically, and it may be a potential therapeutic factor, capable of activating NSPC, for the treatment of degenerative brain disorders.

Key words: CD74, MIF, Dendritic cells, Neural stem/progenitor cells

IntroductionTo date, one promising approach for the treatment of

neurodegenerative diseases such as Parkinson’s disease, e.g.

amyotrophic lateral sclerosis, stroke, and spinal cord injury (SCI),

is neural stem/progenitor cell (NSPC)-based therapy, which

involves neural regeneration through NSPC transplantation and

strategies for the activation of endogenous NSPCs. It is not yet clear

which of these therapeutic approaches is the most effective (Okano,

2002; Okano et al., 2007; Okano and Sawamoto, 2008; Aboody

et al., 2011). NSPCs are multipotent precursors present in both

the embryonic and adult brains, which are capable of undergoing

self renewal as well as of generating neurons, astrocytes, and

oligodendrocytes (Reynolds and Weiss, 1992; Gage, 2000), thereby

contributing to neurogenesis (van der Kooy and Weiss, 2000;

Temple, 2001). A number of factors have been shown to participate

in the regulation of embryonic and adult NSPCs (Shimazaki, 2003;

Zhao et al., 2008), yet the identification of molecules capable of

regulating NSPCs is required to better understand neural stem cell

biology and its therapeutic potential.

In a previous study, we showed that the implantation of mouse

splenic conventional dendritic cells (cDCs) into an injured site

leads to functional recovery in a mouse SCI model (Mikami et al.,

2004). To examine the effects of DCs on SCI, we characterized

DCs derived from bone marrows of common marmosets (Ohta

et al., 2008), and observed functional recovery from SCI by DC

transplantation (Yaguchi et al., 2009) in our established SCI model

(Iwanami et al., 2005), suggesting that DCs may be used for SCI

therapy. In the mouse SCI model, cDCs have been shown to induce

de novo neurogenesis in vivo and to increase NSPC proliferation in

vitro and in vivo, although details of the mechanism underlying

this induction remain unknown. Thus, the molecular mechanism

by which cDCs are capable of inducing the proliferation and/or

survival of NSPCs in the SCI model remained to be elucidated.

Based on these observations, we sought to identify molecules

produced by cDCs that can increase the proliferation and/or

survival of NSPCs in vitro. Using a lentivirus-mediated functional

cloning system, we identified a candidate gene, CD74, which

is a non-polymorphic type II integral membrane protein that is

3210 Research Article

Journ

alof

Cell

Scie

nce

expressed on antigen presenting cells including DCs, B cells

and macrophages. CD74 was initially considered a major

histocompatability complex class II chaperone (Stumptner-

Cuvelette and Benaroch, 2002). Nonetheless, macrophage

migration inhibitory factor (MIF) was later demonstrated to be a

ligand of CD74 (Leng et al., 2003), which can generate a complex

with CD44 and act as a receptor for MIF (Shi et al., 2006). To date,

MIF has also been identified as a non-cognate ligand for CXCR2

and CXCR4 (Schwartz et al., 2009). MIF is known to be a pro-

inflammatory factor in many diseases including atherosclerosis

and rheumatoid arthritis (Morand et al., 2006). Additionally, MIF

has been shown to induce cell proliferation in macrophages (Shi

et al., 2006), B lymphocytes, and prostate cancer cells (Meyer-

Siegler et al., 2006; Starlets et al., 2006). However, the beneficial

role of MIF through AMP-activated protein kinase (AMPK)

activation in an ischemia model was recently reported (Miller et al.,

2008), indicating that MIF may have multiple unknown functions

in various diseases. In the central nervous system (CNS), MIF

expression has been reported in the rat forebrain ventricular zone

(Suzuki et al., 1999), yet the function of MIF in the CNS and in

NSPSCs has yet to be elucidated. We therefore decided to

investigate the roles of MIF in NSPCs. In the present study, we

observed MIF secretion by NSPCs and cDCs. MIF could promote

proliferation and/or survival of NSPCs. In addition, MIF secreted

by cDCs partially supports the stimulation of NSPC proliferation

and/or survival. This is the first report that shows the functions of

MIF–CD74 signaling in NSPCs.

ResultsMolecular cloning of NSPC growth factors derived from

dendritic cells

To identify mouse cDC-derived molecules that can induce NSPC

proliferation, we first constructed a novel lentiviral expression

vector, EFII-BSTXI-SV40-hrGFP, which contains a BSTXI

cloning site and encodes GFP driven by a SV40 promoter

(supplementary material Fig. S1A). A full-length cDNA library

was generated from mouse splenic cDCs using an oligo-capping

cDNA synthesis method (Maruyama and Sugano, 1994). The

full-length cDNAs were cloned into the BSTXI site of EFII-

BSTXI-SV40-hrGFP and the plasmids were amplified once in

bacterial cells. Ninety-six randomly selected clones generated

from the cDNA libraries were subjected to sequence analysis, and

92.4% of the clones containing the full-length cDNA were

identified. The lentiviral cDNA expression plasmid library

vectors were subjected to lentivirus production. Next, NSPCs

cultured from mouse E14.5 GE were infected with the lentivirus-

expressing cDC-derived cDNA. GFP-positive single cells

dissociated from neurospheres were seeded onto 96-well plates

at a low density (1 cell/ml). After 10 days of culture, some highly

proliferative neurospheres were observed. Those single spheres

were picked up, expanded, and subjected to a second round of

screening based on their ability to form neurospheres compared

to NSPCs infected with empty control vectors. Finally, genomic

DNA PCR was performed to verify the cDNA inserted into

the neurospheres (supplementary material Fig. S1B). DNA

sequencing analysis revealed that two of the four neurosphere

clones that were selected in the secondary screening encoded the

same CD74 cDNA (NCBI accession number, AK154096.1), and

the other two clones did not contain the full-length cDNA. Thus,

we did not perform further analyses on the latter two clones.

CD74 can induce cell proliferation and/or promote survival

of NSPCs

To examine whether CD74 can induce the proliferation and/or

promote the survival of NSPCs, CD74 was retrovirally expressed

in NSPCs. Cell proliferation assay were performed on these cells

using a Cell Titer-Glo Luminescent Cell Viability Assay kit.

CD74-overexpressing NSPCs showed a significant increase in cell

proliferation compared to NSPCs expressing the control virus at 4

days post-infection (1.360.02 fold of control, P,1025; n53;

Fig. 1A). This result was confirmed by cell number quantification

using Trypan Blue staining at 5 days post-infection (1.4760.26

fold of control, P,0.05; n53). Next, we performed a secondary

neurosphere assay using the same CD74-expressing retrovirus and

compared the results to cells infected with the control virus (pMX-

Ig). At a low seeding density condition (1 cell/ml), a greater

number of secondary neurospheres was generated from CD74-

overexpressing primary neurospheres, indicating the accuracy of

the expression cloning strategy (2.860.75 fold of control,

P50.001; n58; Fig. 1B). These results show that activation of

CD74 signaling in neurospheres can support the proliferation and/

or survival of NSPCs without other stimulation in vitro.

MIF and MIF receptor expression in NSPCs

Although CD74 overexpression in NSPCs supported cell

proliferation and/or survival, the detailed mechanisms

underlying these effects were unclear. CD74 was originally

identified as a MHC Class II-associated molecule in antigen-

presenting cells (APCs) (Stumptner-Cuvelette and Benaroch,

2002). To date, many studies have reported that MIF is a CD74

ligand that can activate various signaling pathways in some cells

including tumor cells (Bucala and Donnelly, 2007). Therefore,

we considered the possibility that MIF expressed endogenously

in NSPCs can support the proliferation of NSPCs through CD74,

which is expressed in NSPCs. If this were the case, CD74

overexpression in NSPCs could induce the proliferation and/or

survival of NSPCs without the addition of exogenous MIF,

consistent with the results above. In addition, the examination of

Fig. 1. CD74 overexpression increases the viability and self-renewal

ability of NSPCs in vitro. (A) Neurospheres generated from E14.5 mouse GE

were infected with retroviruses encoding GFP together with CD74 (pMX-

CD74) or GFP (pMX-Ig). Neurosphere viability was assessed for 4 days post-

infection (days in vitro; DIV) using a CellTiter Glo Luminescent Cell

Viability assay. (B) Neurospheres infected with either pMX-Ig or pMX-CD74

for 5 DIV were dissociated into single cells, then GFP-positive cells were

plated at a density of 1 cell/ml in 96-well plates (200 ml/well) and the number

of newly generated secondary (2 ) neurospheres was counted 10 days later.

All data are means 6 s.d. Similar results were obtained in three independent

experiments. **P,0.01 versus control; Student’s t-test.

Identification of MIF as a newly characterized growth factor of NSPCs 3211

Journ

alof

Cell

Scie

nce

soluble factors that can support NSPC proliferation and/orsurvival is desirable, especially when considering the

therapeutic potential of MIF. We originally sought to findunknown factors secreted by cDCs that can support theproliferation and/or survival of NSPCs. Thus, we focused onstudying MIF in NSPCs and first examined the amount of MIF

secreted in the supernatants of splenic cDCs and of mouse GE-and spinal cord-derived neurospheres, which are culturedovernight, using ELISA. We found that both neurospheres

derived from different embryonic tissues and cDCs secretedMIF, although cDCs secreted a higher amount of MIF comparedto the neurospheres (Fig. 2A). We also observed MIF secretion in

human NSPCs (1.8760.1 ng/ml for NSPCs cultured overnight).In addition, we performed histological analyses. E14.5 GEs wereobserved using immunohistochemical techniques, and some cellsexpressed Nestin (a marker of NSPCs and hair-follicle bulge stem

cells) (Amoh et al., 2005) and CD74 in the ventricular lining(Fig. 2B). We also observed MIF expression in the GE usingimmunohistochemistry and RT-PCR (Fig. 2C; supplementary

material Fig. S2), which is consistent with a previous report thatMIF is expressed in the ventricular zone of the fetal rat brain(Suzuki et al., 1999). In addition, CD74 expression in cultured

neurospheres generated from E14.5 GE was also shown byimmunocytochemical staining of the NSPC marker, Nestin(Fig. 2D). The expression of other MIF receptors (CD44,

CXCR2, CXCR4 in addition to CD74) in neurospheresgenerated from E14.5 GE was examined using flow cytometry(Fig. 2E). The gene expression of MIF and its receptors in theGE, GE-derived neurospheres and cDCs was confirmed by RT-

PCR analysis (supplementary material Fig. 2B).

MIF contributes to the proliferation of NSPCs

To characterize the effects of MIF on NSPCs, we examinedchanges in cell NSPC proliferation with MIF treatment. Theaddition of MIF to single cells dissociated from neurospheres

slightly increased cell viability at day 4. This increase was smallbut statistically significant (100 ng/ml, 1.160.05 fold of control,P50.001, n54; 400 ng/ml, 1.160.06 fold of control, P50.001,n54; Fig. 3A). We further examined the effects of MIF on NSPC

proliferation by performing BrdU chase experiments in NSPCswith MIF treatment. Immunocytochemical analysis showed anincrease in BrdU incorporation (1.2460.12 fold of control,

P50.034; n53; Fig. 3B), supporting the proliferation assayresults. Next, we examined the effects of a MIF-specificinhibitor, ISO-1, on NSPCs and observed that ISO-1 treatment

(100 mM) decreased cell viability (0.5460.05 fold of control,P50.05; n53; Fig. 3C). In addition, the gene silencing effect ofMIF on NSPC proliferation was tested using siRNA. First, weused two siRNAs to knock down MIF gene expression in NSPCs.

A decrease in gene expression was observed in the transienttransfection system, accompanied by a decrease in cell viabilityfollowing treatment with both siRNAs (supplementary material

Fig. S3). Since efficient gene silencing of MIF in NSPCs wasexpected, a retrovirally expressed short hairpin RNA (shRNA)-MIF vector was constructed and NSPCs were infected with the

virus. Six days after infection with an shRNA-MIF-expressingvirus, cell viability decreased (0.6860.06 fold of control,P50.015; n53; Fig. 3C), supporting the results of the transient

gene knockdown experiment. Cell cycle analysis also showedthat gene silencing of MIF in NSPCs decreased the S-phasepopulation, which was in accordance with the increase in BrdU

incorporation observed with MIF treatment (Fig. 3D). Taken

together, MIF contributes to NSPC proliferation, although the

effect of MIF knockdown was greater than that of exogenous

MIF addition. Finally, the effects of MIF treatment on NSPC

apoptosis were examined. Gene silencing of MIF led to an

increase in caspase 3/7 activity in shRNA-MIF-infected NSPCs

compared to controls 5 days after infection (1.460.07 fold of

control, P50.004; n54; Fig. 3E). Thus, MIF plays an important

role in NSPC proliferation and survival.

MIF increases the self-renewal of NSPCs

To identify changes in NSPC’s self-renewal capacity following

MIF treatment, we performed a neurosphere-formation assay, the

most commonly used method to measure the capacity of NSPC

self-renewal. We first added MIF exogenously to single NSPCs

plated at a low cell density (1 cell/ml) on 96-well plates and then

Fig. 2. Expression of MIF and CD74 in NSPCs. (A) MIF protein was

secreted into the medium of mouse GE-derived or SPC (spinal cord)-derived

NSP (neurosphere), and splenic cDCs (DCs), which were cultured using NSP

medium (Med). The same number of each cell type (105 cells/well) was

cultured in culture medium for 24 hrs before the supernatants were subjected

to ELISA analysis. (B) The expression of Nestin (NSPC marker) and CD74 in

E14.5 GE. A magnified image of the boxed area is shown in B9. Scale bars:

100 mm (B), 10 mm (B9). (C) MIF expression in E14.5 GE. A magnified

image of the boxed area is shown in C9. Scale bars: 100 mm (C), 10 mm (C9).

(D) Nestin and CD74 expression on the cell surface of E14.5 GE-derived

neurospheres. Scale bar: 50 mm. (E) Representative flow cytometory profiles

of the expression of MIF receptors (CD74, CD44, CXCR2, CXCR4) on the

cell surface of E14.5 GE-derived neurospheres. Neurospheres were stained

with fluorescent-dye-conjugated antibodies and analyzed by flow cytometry.

Journal of Cell Science 125 (13)3212

Journ

alof

Cell

Scie

nce

evaluated the number of primary neurospheres that were

generated. MIF increased the number of primary neurospheres in

a dose-dependent manner (200 ng/ml, 2.260.87 fold of control,

P50.02, n58; 400 ng/ml, 3.461.6 fold of control, P50.006, n58;

Fig. 4A). This increase in primary neurosphere formation

following exogenous MIF addition was attenuated by both anti-

CD74 neutralizing antibody and ISO-1 treatment (supplementary

material Fig. S4). Next, we performed primary sphere formation

assays at a cell density of 4 cells/ml using three different

conditions. First, we applied anti-CD74 neutralizing antibody in

conditions conducive to primary sphere formation to block the

interaction between endogenously expressed MIF and CD74. A

decrease in the number of primary spheres was observed

(0.3860.09 fold of control, P50.006; n55; Fig. 4B). In addition

to CD74, we also blocked CXCR4 and CXCR2 using neutralizing

antibodies, and observed that the blockade of CD74 alone showed

a significant decrease in the number of primary neurospheres

(supplementary material Fig. S5). In the second condition, ISO-1

was added to the same culture condition, and this treatment also

resulted in a decrease in the number of primary neurospheres

(0.260.15 fold of control, P,1025; n58; Fig. 4C). Finally, we

examined the effects of MIF gene silencing using shRNA-MIF and

observed a decrease in primary neurosphere formation (0.0560.01

fold of control, P50.01; n58; Fig. 4D). Taken together, these data

demonstrated that the MIF-CD74 ligand–receptor system can

contribute to primary neurosphere formation, suggesting that MIF

may support the proliferation and/or survival of NSPCs in vitro. To

study the effects of MIF treatment on the self-renewal ability of

NSPCs, a secondary neurosphere assay was developed. Exogenous

addition of MIF increased the number of secondary neurospheres

(2.060.59 fold of control, P50.003; n58; Fig. 4E). In addition,

ISO-1 and shRNA-MIF treatment also attenuated the formation of

secondary neurospheres compared to controls (ISO-1, 0.6560.11

fold of control, P,1025; n58; shMIF, 0.5060.11 fold of control,

P,1025; n58; Fig. 4F,G). Thus, MIF may contribute to the self-

renewal ability of NSPCs in vitro. As previously mentioned, one of

the aims of this study was to identify molecules secreted by cDCs

that support the proliferation of NSPCs. Thus, anti-CD74

Fig. 3. MIF contributes to the proliferation of

NSPCs. (A) MIF increases cell viability in a dose-

dependent manner. Single cells dissociated from

neurospheres were cultured with the indicated

amount of MIF in 96-well plates (5000 cells/well).

After 5 days of culture, cell viability was measured

using a Cell Titer-Glo assay kit. (B) MIF increased

BrdU incorporation in NSPCs. E14.5 GE-derived

NSPCs were treated with MIF (400 ng/ml) and

after 3 days in culture were pulsed for 24 hrs with

10 mM BrdU. Cells were fixed, permeabilized and

stained with anti-BrdU antibody. Data are

presented as the percentage of BrdU incorporation

into the NSPCs. (C) ISO-1 treatment (100 mM)

attenuates proliferation of NSPCs (left panel). MIF

targeting via retroviral shRNA significantly

reduced NSPC growth compared to Luciferase-

shRNA (shRNA-Luc; control, right panel), as

assessed using a Cell Titer-Glo assay kit. (D) Flow

cytometric analysis of NSPCs transduced with a

retrovirus expressing luciferase shRNA or MIF

shRNA. MIF knockdown was associated with a

decline in the percentage of cells in S phase after 5

days of shRNA infection. (E) MIF knockdown by

retrovirally expressed shRNA demonstrated an

increase in caspase 3/7 activity in NSPCs 3 days

after infection. Data in A–C,E are means 6 s.d.

(n53), and similar results were obtained in three

independent experiments. *P,0.05, **P,0.01

versus control; Student’s t-test.

Identification of MIF as a newly characterized growth factor of NSPCs 3213

Journ

alof

Cell

Scie

nce

neutralizing antibody was added to NSPCs and cDCs in a co-

culture system established in a previous study (Mikami et al.,

2004). The increase in primary neurosphere formation by cDCs

was partially attenuated by antibody treatment, indicating that the

MIF–CD74 pathway may contribute to DC-induced NSPC

activations (supplementary material Fig. S6).

MIF does not change the differentiation potential of NSPCs

Next, we examined the effects of MIF on the differentiation

potential of NSPCs in vitro. E14.5 GE-derived neurospheres were

cultured with exogenous MIF (400 ng/ml) for 5 days and then

differentiated into three neural lineages (neurons, astrocytes, and

oligodendrocytes) in the absence of growth factors. There was

no significant difference in the cell differentiation potential of

NSPCs by MIF treatment compared to controls (Fig. 5). Thus,

MIF did not change the cell fate of NSPCs in vitro, although it

did induce the self-renewal ability of NSPCs.

Signaling analyses of MIF in NSPCs

MIF has been reported to activate many signaling pathways

including the Erk and Akt pathways in many cells (Meyer-Siegler

et al., 2006; Shi et al., 2006; Lue et al., 2007). In the present

study, we examined whether MIF could induce the activation of

Fig. 4. MIF increases primary and secondary neurosphere formation. (A) Single dissociated cells of neurospheres were plated into a 96-well plate using a cell

sorter and cultured in the presence of both EGF and FGF2, with or without MIF. MIF increased the number of primary neurospheres in a dose-dependent manner.

(B–D) Single dissociated cells of neurospheres were plated into a 96-well plate and cultured in the presence of both EGF and FGF2 with either IgG or anti-CD74

(B), DMSO or MIF inhibitor (ISO-1, 100 mM; C), or shRNA-Luc or shRNA-MIF retrovirus (D). (E) Neurospheres were grown for 5 days in the presence of both

EGF and FGF2, with or without MIF (400 ng/ml), and the generated spheres were dissociated into single cells, then plated into a 96-well plate using a cell sorter

and cultured in the presence of both EGF and FGF2. (F) Primary neurospheres were cultured with either DMSO or ISO-1 (100 mM) for 5 DIV, then dissociated

cells were cultured in 96-well plates in the presence of EGF and FGF2, and the number of newly generated secondary neurospheres was counted. (G) Targeting

MIF via retrovirus shRNA significantly attenuated the efficiency of secondary neurosphere formation. Neurospheres treated with either Luc-shRNA or MIF

shRNA for 5 DIV were plated into a 96-well plate and cultured in the presence of EGF and FGF2. The number of primary and secondary neurospheres was

counted between days 10 and 14 of culture. All neurosphere formation assays were performed at a low cell density (A,E: 1 cell/ml; B–D,F, G: 4 cells/ml). Data are

means 6 s.d., and similar results were obtained in three independent experiments. *P,0.05, **P,0.01 versus control; Student’s t-test.

Fig. 5. MIF does not alter cell lineage commitment of NSPCs.

Neurospheres grown in the presence of either EGF and FGF2 or EGF and

FGF2 plus MIF (400 ng/ml) for 5 days were allowed to dissociate. They were

then plated onto poly-L-ornithine-coated coverslips at a density of 26105

cells/cm2 and cultured for another 5 days without growth factors and MIF.

Fully differentiated cells were fixed and subjected to immunocytochemical

analyses. No significant changes were observed in the percentages of DAPI-

positive cells that were labeled with a neural marker (b-III tubulin), an

astrocyte marker (GFAP) or an oligodendrocyte marker (MBP). Data are

averages of three independent experiments. Values are means 6 s.d.;

Student’s t-test.

Journal of Cell Science 125 (13)3214

Journ

alof

Cell

Scie

nce

some pathways that have been reported to be important for cell

proliferation and/or survival, as well as for the maintenance of

self-renewal ability and stem cell properties of NSPCs. MIF

activated Erk, a well known cell proliferation marker and Akt,

which contributes to NSPC survival (Otaegi et al., 2006; Kalluri

et al., 2007), as shown by a higher Erk and Akt phosphorylation

status after 30 minutes of MIF treatment, although Erk activity

was attenuated at 60 minutes (Fig. 6A). In addition, STAT3

(Ser727) phosphorylation has been reported to be important

for the maintenance of the stem cell properties of NSPC

(Androutsellis-Theotokis et al., 2006; Nagao et al., 2007).

Accordingly, MIF increased STAT3 (Ser727) phosphorylation,

although no significant increase of STAT3 (Tyr705)

phosphorylation was observed following MIF treatment

(Fig. 6B). In contrast, gene silencing of MIF caused the down

regulation of Bcl-2 and Bcl-xl, which are known as anti-apoptotic

factors. This result is in accordance with the previous result that

MIF knockdown in NSPCs increases caspase 3/7 activity, as

shown in (Fig. 3E). It has been reported that AMPK is stimulated

by MIF in rat cardiomyocytes under hypoxic conditions with an

increase in the number of GLUT4 transporters on the cell surface

(Miller et al., 2008). Additionally, AMPK agonist (AICAR) is

known to increase the cell surface expression of GLUT1 in rat

ventricular papillary muscle cells (Li et al., 2004). In our study,

MIF treatment increased AMPKa/b phosphorylation (Fig. 6D)

and upregulation of the Glut1 gene was also observed (1.8360.46

increase of control; P50.001; n53; supplementary material Fig.

S7A) along with an increase in GLUT1 translocation to the cell

surface (supplementary material Fig. S7B). These results indicate

that MIF may support NSPC survival through glucose

metabolism. EGFR and VEGF-A are also important factors that

support NSPC stem cell properties (Wada et al., 2006; Aguirre

et al., 2010). Thus, we confirmed changes in Egfr and Vegf-A

gene expression levels with MIF treatment. Egfr upregulation

following MIF treatment was confirmed by qRT-PCR analysis

(2.1360.23 fold of control, P50.002; n53; Fig. 6E). Vegf-A

gene expression also statistically increased by MIF treatment

(1.3760.23 fold of control, P50.05; Fig. 6F), which is consistent

with the study in fibroblasts and hepatocellular carcinoma cells

(Ren et al., 2003; Kim et al., 2007). In addition, we assessed

whether MIF induces gene expression of Hes3, which is reported

to be activated by Stat3-pSer727 in NSPCs (Androutsellis-

Theotokis et al., 2006). As expected, Hes3 gene expression was

upregulated by MIF (2.0960.2 fold of control, P50.001; n53;

Fig. 6G), although changes in Hes1 gene expression could not be

observed through MIF treatment (1.0560.1 fold of control, n53)

under the conditions of this assay.

Effect of MIF on cell migration

MIF has been shown to play a role in the migration of cells

including monocytes. To extend these findings to NSPCs, we

performed a cell migration assay in vitro using a xCELLigence

SP system. MIF was added to the bottom chamber and single

dissociated NSPCs were seeded onto the cell insert of the upper

Fig. 6. MIF signaling in NSPCs. (A) MIF (400 ng/ml) induced Akt and ERK phosphorylation in NSPCs. (B) MIF (400 ng/ml) induced phosphorylation of

STAT3 at Ser727, which is important for maintenance of stem cell properties in a time-dependent manner in NSPCs. (C) Targeting MIF via retroviral shRNA

decreased the expression of Bcl-2 and Bcl-xl, which are known as anti-apoptotic factors in NSPCs. (D) MIF (400 ng/ml)-induced phosphorylation of

AMPKa at Thr172 and AMPKb at Ser108 in a time-dependent manner in NSPCs. (E–G) MIF increases the expression of Egfr (E), Vegf-A (F) and Hes3 (G) in

NSPCs 24 hrs after MIF treatment. Each mRNA expression level was normalized against GAPDH mRNA levels, then expressed relative to the normalized value

of controls. For the graphs, the data were compiled from three independent experiments. Data are averages of three independent experiments. Values are means 6

s.d.. *P,0.05, **P,0.01 versus control; Student’s t-test.

Identification of MIF as a newly characterized growth factor of NSPCs 3215

Journ

alof

Cell

Scie

nce

chamber. The number of cells that migrated through the

membrane of the cell insert was defined as the cell migration

index. MIF-treated NSPCs displayed increased migration

compared to controls. In addition, we performed MIF receptor

blockade experiments using CD74, CXCR4, and CXCR2

neutralizing antibodies in this system. CD74 blockade most

effectively inhibited MIF-mediated migration among antibodies

(Fig. 7A). To further confirm the results, in vitro slice cultures

were performed. Lentivirally labeled GFP-positive neurospheres

of similar size were plated on the ganglionic eminence (GE) of

E14.5 mouse brain slices and migration of GFP-positive cells onthe brain slices was observed. Ubiquitous MIF expression was

observed by immunohistochemical analysis in the GE of E14.5mouse brains (Fig. 2C) and MIF gene expression in the GE ofE14.5 mouse brains was also confirmed by RT-PCR analysis(supplementary material Fig. S2), suggesting that MIF can act as

a chemoattractant for NSPCs in vivo. MIF gene expression in theGE of E14.5 mouse brains was also confirmed by RT-PCRanalysis (supplementary material Fig. S2). Thus, ISO-1 was

added to the culture medium to block the effects of MIF on NSPCmigration. ISO-1 treatment led to a significant reduction in cellmigration compared to controls at zone II, as shown in Fig. 7D

(0.3760.06 fold of control, P50.05; n58; Fig. 7B–E). Inaddition, we blocked SDF1 using SDF1-neutralizing antibodyin the same culture conditions, and we observed a significantreduction of NSPC migration by SDF1 blockade (supplementary

material Fig. S8). Taken together, these results indicate MIFinvolvement in NSPC migration.

DiscussionIn the present study, we identified MIF as a factor secreted bycDCs and NSPCs that can act on its own to support the

proliferation and/or survival, as well as the self-renewal ofNSPCs in vitro. MIF receptors were expressed in the NSPCs,suggesting that MIF can maintain NSPCs through autocrine and/or paracrine mechanisms. MIF also showed chemokine-like

characteristics. These findings indicate that MIF may be effectivefor the treatment of neurodegenerative diseases.

Many factors can contribute to the proliferation and/or survival

of NSPCs, including EGF, FGF2, VEGF, CNTF/LIF, PACAP,and PEDF (Reynolds and Weiss, 1992; Kuhn et al., 1997;Chojnacki et al., 2003; Ohta et al., 2006; Wada et al., 2006;

Andreu-Agullo et al., 2009). In this present study, we identifiedMIF as a proliferation and/or survival factor for NSPCs derivedfrom the GE of E14.5 mice. MIF has been reported to activate

ERK (Shi et al., 2006), and this activation was observed inNSPCs. In addition, gene silencing of MIF in NSPCs decreasedBcl2 and Bcl-xl expression and activated caspase 3/7,demonstrating that MIF can act as a survival factor NSPCs.

Moreover, the Akt signaling pathway is also known to beimportant for cell proliferation and survival of NSPCs (Otaegiet al., 2006; Kalluri et al., 2007). Consistent with these previous

studies, we showed that MIF treatment activated Akt and led toNSPC proliferation and/or survival. MIF treatment also led to anincrease in the number of secondary neurospheres, indicating its

ability to increase NSPC self-renewal. EGF and Notch signalingpathways are known to be essential for the maintenance of NSPCprogeny (Doetsch et al., 2002; Mizutani et al., 2007; Aguirreet al., 2010). We observed an increase in Stat3-pS727

phosphorylation, which is downstream of the Notch signalingcascade and is important for the survival and/or self-renewal ofNSPCs (Androutsellis-Theotokis et al., 2006; Nagao et al., 2007).

A previous study showed increased Hes3 expressionaccompanied by Akt and Stat3-pS727 phosphorylation throughNotch activation in NSPCs (Androutsellis-Theotokis et al., 2006),

which is consistent with our findings. Furthermore, we observedincreases in gene expression of Egfr in NSPCs, indicating thatMIF can stimulate EGF signaling pathways, as well as pathways

downstream of Notch to induce the self-renewal capacity ofNSPCs. In addition, MIF was found to be secreted in the humanembryonic-brain-derived-NSPC culture supernatant. This result

Fig. 7. The effects of MIF on NSPC migration. (A) Migration kinetics of

NSPCs. NSP medium containing 10% FCS, with either BSA or MIF, was

added to the bottom chamber of an xCELLigence SP system (Roche

Diagnostics), a real-time migration monitoring device. NSPCs (20,000 cells/

well) were seeded onto the upper chamber in NSP medium and the number of

cells that migrated to the bottom chamber was counted. In the MIF receptor

blockade experiment, anti-CD74, CXCR4 and CXCR2 neutralizing antibodies

were added to the upper chamber at a concentration of 20 mg/ml. MIF-

mediated cell migration was prevented by the addition of anti-CD74 antibody.

The number of cells that migrated through the membrane of the cell insert

after 24 hrs of culture was defined as the cell migration index. Data are means

6 s.d. of four experiments. *P,0.05, **P,0.01; Student’s t-test. (B–E) MIF

inhibitor (ISO-1) inhibited the migratory potential of neural stem cell colonies

derived from the GE. A lentivirally GFP-labeled neurosphere derived from an

E14.5 GE was deposited on an E14.5 brain slice (B). After 2 days of co-

culture, cell migration out of neurospheres (200 mm diameter) treated with

ISO-1 (100 mM) (C) or DMSO (D) was observed using fluorescent

microscopy. The number of cells that migrated from neurosphere colonies to

zones I and II in D was quantified based on fluorescence intensity, and

defined as migration cell index in E. Scale bar: 200 mm. Data are

means 6 s.d. (n58). *P,0.05; Student’s t-test.

Journal of Cell Science 125 (13)3216

Journ

alof

Cell

Scie

nce

indicates that MIF may exert the same function on human NSPCsas seen in murine NSPCs.

AMPK is an important regulator of both glycolysis and glucose

uptake in NSPCs (Rafalski and Brunet, 2011). AMPK was shownto play an important role in brain development, as indicated byAMPKb1 knockout mice, which showed defects in cell cycle

regulation of NSPCs (Dasgupta and Milbrandt, 2009). In othercell types such as neurons and an immortalized cerebellar cellline, AMPK activation showed a protective effect against glucose

deprivation and oxidative stress (Culmsee et al., 2001; Park et al.,2009). Additionally, in the ischemic heart model, MIF showed aprotective effect accompanied by activation of AMPK signaling

and glucose uptake (Miller et al., 2008). Intriguingly, theinduction of Glut1 mRNA was enhanced in hypoxic MIF-overexpressing breast cancer cells (Oda et al., 2008), whichcoincided with our result that MIF can induce Glut1 gene

expression in NSPCs. Thus, MIF may protect NSPCs against celldeath through AMPK activation, which correlates with energymetabolism. In future studies, the protective effect of MIF on

NSPCs should be examined based on energy metabolism, whichis known as a key NSPC regulatory system (Rafalski and Brunet,2011).

MIF has been reported to enhance the migration ofhepatocellular carcinoma cells, melanoma cells, and monocytes(Shimizu et al., 1999; Ren et al., 2003). In addition, the inhibitionof MIF binding to CD74 decreases prostate cancer cell invasion

(Meyer-Siegler et al., 2006). SDF1, a CXCR4 ligand, has beenshown to increase the motility of type A cells in thesubventricular zone (SVZ) of the adult mouse (Kokovay et al.,

2010) and to promote NSPC transmigration (Imitola et al., 2004).In fact, we observed that SDF1 blockade led to a significantdecrease of NSPC migration in the brain slice culture system. In

our study, MIF showed an effect similar to that of SDF1 throughthe enhancement of NSPC migration in vitro. Furthermore, NSPCmigration on cultured brain slices was blocked by a MIF-specific

inhibitor. Thus, it would be interesting to compare thechemoattractant abilities of MIF and SDF1 in an in vivo modelusing knock out mice. Taken together, MIF may contribute toNSPC migration in the developmental brain and in other

inflammatory conditions seen in stroke and neurodegenerativediseases, as discussed below.

MIF has been shown to have pro-inflammatory effects and to

modulate the immune system in diseases such as septic shock,rheumatoid arthritis, and atherogenesis (Bernhagen et al., 1993;Morand et al., 2006). Inflammation is associated with stroke and

many neurodegenerative disorders, which include Alzheimer’sdisease, multiple sclerosis, and Parkinson’s disease (Glass et al.,2010). MIF expression in the brain cytosol and cerebrospinalfluid is higher in Alzheimer’s disease patients compared to age-

matched controls (Bacher et al., 2010). Increased expression ofCD74 in neurofibrillary tangles in Alzheimer’s disease (Bryanet al., 2008) has been similarly reported, although the detailed

function of the MIF–CD74 system in Alzheimer’s disease relatedinflammation remains to be elucidated. Given the complexity andthe multiple functions of inflammatory factors, which include

cytokines, it is still difficult to understand the roles that thesefactors play in physiological conditions. Indeed, in some cases,inflammatory responses are beneficial (Wyss-Coray and Mucke,

2002). The effect of inflammatory factors on NSPCs has beenstudied and a variety of responses have been discovered (Ziv andSchwartz, 2008; Carpentier and Palmer, 2009; Russo et al.,

2011). TNF-a, IL-1b, and SDF1 (CXCL12) are capable of

increasing NSPC proliferation (Whitney et al., 2009; Wu et al.,

2009). In this study, we showed that MIF can increase the

proliferation and/or survival of NSPCs, as was seen with SDF1. It

is noteworthy that a new role of NSPCs as a potent inducer of

anti-inflammatory responses has been reported (Einstein et al.,

2003), indicating that the increase in NSPCs number by MIF in

neurodegenerative diseases may be a useful therapeutic strategy

that is based on immunoregulation. These results define new

models of MIF action and establish a link between NSPC

maintenance and molecular pathways that are central to

inflammation.

In a previous study, we showed functional recovery in a mouse

SCI model through the administration of splenic cDCs, which

activate endogenous NSPCs. In addition, cDCs increased the

number of NSPCs in vitro (Mikami et al., 2004). In this study, we

showed that the MIF–CD74 system can contribute in part to cDC

activity, which increases the number of NSPCs in vitro, implying

that MIF can support functional recovery from SCI through either

NSPC activation as seen in cDCs or the recruitment of NSPCs

to the injured site, which displays chemoattractant abilities.

Although many therapeutic approaches for SCI treatment based

on regenerative medicine have been designed, including cell

therapy using NSPCs, oligodendrocyte progenitors, and

multipotent hair follicle stem cells (Thuret et al., 2006; Amoh

et al., 2008, Liu et al., 2011), the results in the present study may

contribute to the development of a novel SCI therapy. In addition,

MIF upregulation has been reported in stroke patients (Wang

et al., 2009). Furthermore, hypoxia-induced MIF secretion by

human endothelial cells has been reported (Simons et al., 2011).

Thus, detailed analysis of MIF function as it correlates to NSPCs

and examination of therapeutic potential of MIF in stroke may be

an important issue to address in future.

In conclusion, MIF secreted from DCs and NSPCs was

identified as a novel factor that can support the proliferation and/

or survival of NSPCs in vitro. MIF also increased self-renewal of

NSPCs. MIF activated many signaling pathways that support cell

survival, proliferation and/or maintenance of NSPCs. In the

present study, we showed that MIF stimulates DC-mediated

NSPC proliferation. Taken together, MIF may be a new potential

therapeutic factor for the treatment for degenerative brain

disorders and SCI through NSPC activation.

Materials and MethodsAnimals

Pregnant C57BL/6J and ICR mice were purchased from Sankyo Labo Service(Tokyo, Japan). ICR mice were used only in brain slice culture experiments. Allexperiments were carried out in accordance with the guidelines of theExperimental Animal Care Committee of Keio University School of Medicine.

Neurosphere culture

NSPCs were isolated from the GE and spinal cord of embryonic day 14.5 (E14.5)mice as previously described, and the cells were cultured as neurospheres (Ohtaet al., 2006). These neurospheres were cultured at a cell density of 50 cells/ml in aneurosphere culture medium (NSP medium) consisting of neurobasal medium(Invitrogen) supplemented with B27 (Invitrogen), EGF (20 ng/ml; Peprotech), andFGF2 (10 ng/ml; Peprotech). For the primary and secondary neurosphereformation assay, single, dissociated cells from neurospheres were sorted into a96-well plate at a low cell density (1 cell/ml or 4 cells/ml), using a fluorescenceactivated cell sorter (FACS) (EPICS ALTRA; Beckman Coulter). The number ofprimary and secondary neurospheres in the NSP medium was counted betweendays 10 and 14 of culture. In the MIF blockade assay, rat anti-CD74 antibody(Santa Cruz Biotechnology), rat anti-CXCR2 antibody (R&D Systems), rat-anti-CXCR2 antibody (R&D Systems), and MIF inhibitor ISO-1 (Calbiochem) wereused, and in the gene knockdown assay, retrovirally-expressed MIF shRNA was

Identification of MIF as a newly characterized growth factor of NSPCs 3217

Journ

alof

Cell

Scie

nce

used. Human neural stem/progenitor cells (NSPCs) were cultured as neurospheresin NSP medium supplemented with EGF (20 ng/ml), FGF2 (10 ng/ml), LIF(10 ng/ml; Millipore), and heparin (5 mg/ml; Sigma) as previously described(Hattori et al., 2007).

Expression cloning

cDCs were isolated from mouse splenocytes using anti-CD11c-antibody-conjugatedmagnetic beads (Miltenyi Biotec). Total RNA was prepared from the cDCs usingTRIZOL (Invitrogen), and then subjected to the oligo-cap cDNA libraryconstruction procedure following the method developed by Maruyama andSugano (Maruyama and Sugano, 1994). The full-length cDNAs generated throughthis method were cloned into a CSII-EF-BSTXI-SV40-hrGFP lentivirus expressionvector (supplementary material Fig. S1A) constructed from an original CSII-EF-MCS-IRES-hrGFP vector (kindly given by H. Miyoshi). The lentivirus wasproduced following the method described by Miyoshi (Miyoshi, 2004) andneurospheres were infected with the lentivirus (MOI51). GFP-positive singlecells dissociated from neurospheres 5 days post-infection were seeded into 96-wellplates at a low density (1 cell/ml). After 10 days of culture, highly proliferatedspheres were picked up. After expansion of the spheres, a second round of screeningwas performed based on their ability to form neurospheres compared toneurospheres infected with control lentivirus. Finally, the genomic DNA ofneurospheres was extracted using a WizardH SV Genomic DNA Purification System(Promega) and the inserted cDNA sequence was amplified using Bend-Taqpolymerase (Toyobo) using the PCR primers listed in supplementary material TableS1, The PCR conditions were as follow, 1 cycle of 5 min at 95 C, followed by 35cycles of 94 C for 30 sec, 58 C for 30 sec, and 72 C for 3 min in PCR reactionbuffer containing 2.5% DMSO. For the functional assay, obtained the cDNAs wereinserted into pMX-Ig vectors (a gift from T. Kitamura, The University Tokyo).

RNA extraction and quantitative (q) RT-PCR

Total RNA treated with RNase-free DNaseI (Takara Bio) was isolated from tissuesor cultured cells using a PureLink RNA Mini Kit (Invitrogen). Synthesis of cDNAwas performed using 1 mg of total RNA using PrimeScript RT Master Mix (TakaraBio). The intron-spanning primers were designed as shown in supplementarymaterial Table S1, and for Hes3, previously tested and optimized primer sets wereused (Kobayashi et al., 2009). Quantitative RT-PCR analysis was performed with aFastStart Universal SYBR Green Master (Roche), using the ABI prism 7900 HTSequence Detection System (Applied Biosystems). The PCR conditions were asfollows: 1 cycle of 5 min at 95 C, followed by 40 cycles of 95 C for 30 sec, 60 Cfor 60 sec, and 72 C for 60 sec. Relative gene expression levels were determinedusing the DDCt method. GAPDH mRNA levels were used as internalnormalization control. For semi-quantitative PCR analysis, cDNA samples wereamplified using Bend-Taq polymerase and the PCR products were resolved on a2% agarose gel.

Retrovirus production

To construct short hairpin RNA (shRNA)-expressing retroviral vectors,oligonucleotides targeting the coding sequence of MIF (59-GGGUCUACA-UCAACUAUUA-39) and luciferase (Clontech) were inserted into a pSIRENvector (Clontech). Recombinant retroviruses were produced through thetransfection of retrovirus vectors and a pVSVG envelope vector into packagingcell line 293GP (kindly given by T. Kitamura).

Western blot analysis

Cell lysates were prepared using RIPA buffer (25 mM Tris-HCl, 150 mM NaCl,1% NP-40, 1% sodium deoxycholate, and 0.1% SDS, pH 7.6) containing proteaseinhibitors (Cocktail Tablet; Roche). Lysates were centrifuged at 14,000 g for15 min at 4 C, and the protein concentrations of each sample were determinedusing a Bio-Rad protein assay kit (Bio-Rad) with bovine serum albumin as astandard. Identical amounts of proteins were electrophoresed in 10% SDS-PAGEgels and transferred to a nitrocellulose membrane. Blots were blocked withBlocking One (Nacalai Tesque) at RT for 1 hr, then incubated with primaryantibodies overnight at 4 C as follows: Akt (1:1000), phospho-Akt (Ser473;1:1000), AMPKa (1:1000), AMPKb (1:1000), phospho-AMPAKa (Thr172)(1:1000), phospho-AMPAKb (Ser108) (1:1000), Bcl-xL (1:1000), STAT3(1:1000), phospho-STAT3(Ser727) (1:1000), phospho-STAT3(Tyr705) (1:1000,Cell Signaling Technology), Bcl2 (1:1000), MIF (1:200; MBL), Erk (1:200),phospho-Erk (1:200, Santa Cruz Biotechnology), and actin (1:4000; Sigma). Afterthree washes in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.02% Tween-20,pH 7.4), the blots were incubated with the appropriate secondary antibodiesconjugated with horseradish peroxidase (1:4000, anti-rabbit and anti-mouse;Thermo Scientific) for 1 hr at room temperature. Signals were detected with anECL-Plus Substrate (GE Healthcare) and exposed to Hyperfilm (GE Healthcare).

Small interfering RNA and transient transfection

The sequences of the mouse and human-specific MIF siRNA, 5’-GGGUCUACAUCAACUAUUAdTdT-3’ (MIF siRNA1), mouse-specific siRNA

5’-CCGCAACUACAGUAAGCUGdTdT-3’ (MIF siRNA2) and control siRNA5’-GUACCGCACGUCAUUCGUAUC-3’ (Cntl siRNA) were used for geneknockdown experiments. Single cells dissociated from neurospheres were seededon 6-well or 96-well plates and siRNAs were transfected at a concentration of50 nM using Lipofectamine RNAiMAX (Invitrogen) according to themanufacturer’s instructions.

Cell proliferation and apoptosis assay

Cell viability assay were performed using Cell Titer-Glo Luminescent CellViability Assay kits (Promega) and a luminometer (Wallac ARVO 1420 multilabelcounter; WALLAC OY) according to the manufacturer’s protocol. For theapoptosis assay, caspase 3/7 activity was measured using Caspase-Glo 3/7 AssayKits (Promega) according to the manufacturer’s instructions. In both assays, singlecells dissociated from neurospheres were seeded onto 96-well plates at a density of56103 cells/well, and caspase activity was assayed 3 days post-infection.

Immunohistochemistry and immunocytochemistry

After transcardial perfusion with 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS), fetal brains were removed, fixed in 4% PFA for 2 hours at4 C, incubated overnight at 4 C with 30% sucrose in PBS, then frozen in OTCcompound (Sakura Finetek). In immunohistochemical studies, the coronal sections(14 mm) were incubated in blocking buffer (PBS containing 10% normal goatserum) for 60 min at room temperature. The following primary antibodies wereused: rat anti-CD74 (1:100; Santa Cruz Biotechnology), rabbit anti-MIF (1:200;Biovision), and mouse anti-Nestin (1:5; DSHB) antibodies. Brain sections wereincubated with primary antibodies overnight at room temperature, then with theappropriate Alexa Flour dye-conjugated secondary antibodies (1:1400; Invitrogen).Cell nuclei were counterstained with To-Pro-3 (Invitrogen) or 4’,6-diamino-2-phenylindole (DAPI, Invitrogen). For immunocytochemical studies, cells werefixed with PBS containing 4% PFA for 20 min at room temperature, and the cellswere subjected to a blocking procedure. Immunofluorescence staining wasperformed using the following primary antibodies: rat anti-CD74 (1:200; SantaCruz Biotechnology), mouse anti-Nestin (1:5; DSHB), mouse anti-b-III-tubulin(1:1000; Sigma), rabbit anti-MBP (1:5; DAKO) and rabbit anti-GFAP (1:200;Biomedical Technologies) antibodies. After PBS washes, antibody binding wasvisualized using the appropriate Alexa Flour dye-conjugated secondary antibodies(Invitrogen), and the nuclei were stained with To-Pro-3 or DAPI. In theimmunohistochemical and immunocytochemical studies, permeabilization wasachieved through the addition of 0.3% TritonX-100 to the primary antibodysolution (5% normal goat serum in PBS). In the differentiation assays, singledissociated cells of cultured neurospheres were plated on poly-L-lysine-coatedglass slips at a density of 26105 cells/cm2 in NSP medium without growth factorsfor 5 days, then subjected to immunocytochemical analysis. In 5-bromo-2’-deoxyuridine (BrdU) chase experiments, neurospheres were treated with 10 mMBrdU (Sigma) for 24 hrs, then mechanically dissociated and plated onto poly-Lornithine-coated coverslips at a density of 16105 cells/cm2. The cells wereprocessed for BrdU immunocytochemistry 2 hrs after plating. In order to enablethe detection of BrdU-labeled cells, these cells were fixed with 4% PFA, pretreatedwith 2 M HCl for 15 min at 37 C to denature the DNA, and stained with rat anti-BrdU antibody (1:100; Abcam). At least 10 different viewing fields were countedfor the analyses. All images were obtained using a Zeiss LSM-510 confocalmicroscope (Zeiss).

Flow cytometry

Flow cytometric analyses were performed using Flow cytometry EPICS-XL

(Beckman Coulter). Mouse neurospheres were stained with the followingantibodies: anti-CD44, anti-CXCR2 (BioLegend), anti-CXCR4, anti-GLUT1(R&D Systems), and anti-CD74 (Santa Cruz Biotechnology). Cell cycle analysisfor live cells was performed using flow cytometry. Cells were stained with Hoechst33342 for 60 min at 37 C, then subjected to flow cytometry (EPICS-ALTRA,Beckman Coulter). Retrovirally GFP-labeled cells were gated and then analyzedusing Multicycle for Windows (Beckman Coulter).

ELISA analysis

Human and mouse MIF ELISA kits (Sapporo Immuno Diagnostic Laboratory)were used to measure the amount of secreted MIF protein in culture supernatants.Mouse dendritic cells were isolated from spleens using anti-CD11c antibody-conjugated magnetic beads (Miltenyi Biotec).

Cell migration assay

Cell migration of the cells was tracked in vitro by impedance signal changes inCIM plates (Roche) measured on the back-side of the membrane using theCELLigence SP system (Roche), a real-time migration monitoring device. MIF-mediated NSPC chemotaxis was blocked by the antibodies described above. In themigration assays on brain slices (400 mm thickness), E14.5 ICR mouse brain sliceswere placed on cell inserts (Millipore). Lentivirally GFP-labeled neurospherecolonies (200 mm diameter) were placed on coronal brain slices and co-cultured in

Journal of Cell Science 125 (13)3218

Journ

alof

Cell

Scie

nce

DMEM/F12 medium containing N2 supplement and T3 (30 mg/ml). GFPfluorescence intensity of cells migrating from the neurospheres to zones I and II(2006300 mm) was measured using ZEN lite 2011 software (Zeiss). The distancefrom the center of the neurosphere to the center of zone II (outer area) was800 mm. Zone I is the contiguous inner area closer to the sphere. In theneutralization assay, mouse IgG and monoclonal anti-SDF1 neutralizing antibody(R&D) were added to the medium.

Statistical analysis

All values are expressed as means 6 standard deviation (s.d.). Student’s t-testswere used to determine the statistical significance of differences between groups.For the nonparametric multiple comparison procedure, Steel–Dwass tests wereperformed to compare levels between groups (*P,0.05, **P,0.01).

AcknowledgementsThe authors would like to acknowledge T. Kitamura (The Universityof Tokyo) for providing the pMX-Ig vector, Hiroyuki Miyoshi(RIKEN) for providing the CSII-EF-MCS-IRES-hrGFP vector, Y.Mizue (Sapporo Immuno Diagnostic Laboratory), F. Renault-Mihara,R. Kuwahara and S. Teramoto (Keio University) for technicalassistance.

FundingThis work was supported by the Ministry of Education, Culture,Sports, Science and Technology (MEXT) KAKENHI [grant number0500341 to S.O.]; and a Grant-in-Aid from the Global Center ofExcellence program (Education and Research Center for Stem CellMedicine) to Keio University.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.102210/-/DC1

ReferencesAboody, K., Capela, A., Niazi, N., Stern, J. H. and Temple, S. (2011). Translating

stem cell studies to the clinic for CNS repair: current state of the art and the need for aRosetta Stone. Neuron 70, 597-613.

Aguirre, A., Rubio, M. E. and Gallo, V. (2010). Notch and EGFR pathway interactionregulates neural stem cell number and self-renewal. Nature 467, 323-327.

Amoh, Y., Li, L., Katsuoka, K., Penman, S. and Hoffman, R. M. (2005). Multipotentnestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons. Proc.

Natl. Acad. Sci. USA 102, 5530-5534.

Amoh, Y., Li, L., Katsuoka, K. and Hoffman, R. M. (2008). Multipotent hair folliclestem cells promote repair of spinal cord injury and recovery of walking function. Cell

Cycle 7, 1865-1869.

Andreu-Agullo, C., Morante-Redolat, J. M., Delgado, A. C. and Farinas, I. (2009).Vascular niche factor PEDF modulates Notch-dependent stemness in the adultsubependymal zone. Nat. Neurosci. 12, 1514-1523.

Androutsellis-Theotokis, A., Leker, R. R., Soldner, F., Hoeppner, D. J., Ravin, R.,Poser, S. W., Rueger, M. A., Bae, S. K., Kittappa, R. and McKay, R. D. (2006).Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 442, 823-826.

Bacher, M., Deuster, O., Aljabari, B., Egensperger, R., Neff, F., Jessen, F., Popp, J.,

Noelker, C., Reese, J. P., Al-Abed, Y. et al. (2010). The role of macrophagemigration inhibitory factor in Alzheimer’s disease. Mol. Med. 16, 116-121.

Bernhagen, J., Calandra, T., Mitchell, R. A., Martin, S. B., Tracey, K. J., Voelter,

W., Manogue, K. R., Cerami, A. and Bucala, R. (1993). MIF is a pituitary-derivedcytokine that potentiates lethal endotoxaemia. Nature 365, 756-759.

Bryan, K. J., Zhu, X., Harris, P. L., Perry, G., Castellani, R. J., Smith, M. A. and

Casadesus, G. (2008). Expression of CD74 is increased in neurofibrillary tangles inAlzheimer’s disease. Mol. Neurodegener. 3, 13.

Bucala, R. and Donnelly, S. C. (2007). Macrophage migration inhibitory factor: aprobable link between inflammation and cancer. Immunity 26, 281-285.

Carpentier, P. A. and Palmer, T. D. (2009). Immune influence on adult neural stemcell regulation and function. Neuron 64, 79-92.

Chojnacki, A., Shimazaki, T., Gregg, C., Weinmaster, G. and Weiss, S. (2003).Glycoprotein 130 signaling regulates Notch1 expression and activation in the self-renewal of mammalian forebrain neural stem cells. J. Neurosci. 23, 1730-1741.

Culmsee, C., Monnig, J., Kemp, B. E. and Mattson, M. P. (2001). AMP-activatedprotein kinase is highly expressed in neurons in the developing rat brain and promotesneuronal survival following glucose deprivation. J. Mol. Neurosci. 17, 45-58.

Dasgupta, B. and Milbrandt, J. (2009). AMP-activated protein kinase phosphorylatesretinoblastoma protein to control mammalian brain development. Dev. Cell 16, 256-270.

Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M. and Alvarez-Buylla, A.(2002). EGF converts transit-amplifying neurogenic precursors in the adult brain intomultipotent stem cells. Neuron 36, 1021-1034.

Einstein, O., Karussis, D., Grigoriadis, N., Mizrachi-Kol, R., Reinhartz, E.,

Abramsky, O. and Ben-Hur, T. (2003). Intraventricular transplantation of neuralprecursor cell spheres attenuates acute experimental allergic encephalomyelitis. Mol.

Cell. Neurosci. 24, 1074-1082.

Gage, F. H. (2000). Mammalian neural stem cells. Science 287, 1433-1438.

Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C. and Gage, F. H. (2010).Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918-934.

Hattori, Y., Ohta, S., Hamada, K., Yamada-Okabe, H., Kanemura, Y., Matsuzaki,

Y., Okano, H., Kawakami, Y. and Toda, M. (2007). Identification of a neuron-specific human gene, KIAA1110, that is a guanine nucleotide exchange factor for

ARF1. Biochem. Biophys. Res. Commun. 364, 737-742.

Imitola, J., Raddassi, K., Park, K. I., Mueller, F. J., Nieto, M., Teng, Y. D., Frenkel,

D., Li, J., Sidman, R. L., Walsh, C. A. et al. (2004). Directed migration of neuralstem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC

chemokine receptor 4 pathway. Proc. Natl. Acad. Sci. USA 101, 18117-18122.

Iwanami, A., Yamane, J., Katoh, H., Nakamura, M., Momoshima, S., Ishii, H.,

Tanioka, Y., Tamaoki, N., Nomura, T., Toyama, Y. et al. (2005). Establishment ofgraded spinal cord injury model in a nonhuman primate: the common marmoset.J. Neurosci. Res. 80, 172-181.

Kalluri, H. S., Vemuganti, R. and Dempsey, R. J. (2007). Mechanism of insulin-like

growth factor I-mediated proliferation of adult neural progenitor cells: role of Akt.Eur. J. Neurosci. 25, 1041-1048.

Kim, H. R., Park, M. K., Cho, M. L., Yoon, C. H., Lee, S. H., Park, S. H., Leng, L.,

Bucala, R., Kang, I., Choe, J. et al. (2007). Macrophage migration inhibitory factorupregulates angiogenic factors and correlates with clinical measures in rheumatoid

arthritis. J. Rheumatol. 34, 927-936.

Kobayashi, T., Mizuno, H., Imayoshi, I., Furusawa, C., Shirahige, K. and

Kageyama, R. (2009). The cyclic gene Hes1 contributes to diverse differentiationresponses of embryonic stem cells. Genes Dev. 23, 1870-1875.

Kokovay, E., Goderie, S., Wang, Y., Lotz, S., Lin, G., Sun, Y., Roysam, B., Shen, Q.

and Temple, S. (2010). Adult SVZ lineage cells home to and leave the vascular niche

via differential responses to SDF1/CXCR4 signaling. Cell Stem Cell 7, 163-173.

Kuhn, H. G., Winkler, J., Kempermann, G., Thal, L. J. and Gage, F. H. (1997).

Epidermal growth factor and fibroblast growth factor-2 have different effects onneural progenitors in the adult rat brain. J. Neurosci. 17, 5820-5829.

Leng, L., Metz, C. N., Fang, Y., Xu, J., Donnelly, S., Baugh, J., Delohery, T., Chen,

Y., Mitchell, R. A. and Bucala, R. (2003). MIF signal transduction initiated bybinding to CD74. J. Exp. Med. 197, 1467-1476.

Li, J., Hu, X., Selvakumar, P., Russell, R. R., 3rd, Cushman, S. W., Holman, G. D.

and Young, L. H. (2004). Role of the nitric oxide pathway in AMPK-mediated

glucose uptake and GLUT4 translocation in heart muscle. Am. J. Physiol. Endocrinol.

Metab. 287, E834-E841.

Liu, F., Uchugonova, A., Kimura, H., Zhang, C., Zhao, M., Zhang, L., Koenig, K.,

Duong, J., Aki, R., Saito, N. et al. (2011). The bulge area is the major hair follicle

source of nestin-expressing pluripotent stem cells which can repair the spinal cordcompared to the dermal papilla. Cell Cycle 10, 830-839.

Lue, H., Thiele, M., Franz, J., Dahl, E., Speckgens, S., Leng, L., Fingerle-Rowson,

G., Bucala, R., Luscher, B., Bernhagen, J. (2007). Macrophage migration inhibitoryfactor (MIF) promotes cell survival by activation of the Akt pathway and role for

CSN5/JAB1 in the control of autocrine MIF activity. Oncogene. 26, 5046-5059.

Maruyama, K. and Sugano, S. (1994). Oligo-capping: a simple method to replace the

cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174.

Meyer-Siegler, K. L., Iczkowski, K. A., Leng, L., Bucala, R. and Vera, P. L. (2006).

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuatesgrowth and invasion of DU-145 prostate cancer cells. J. Immunol. 177, 8730-8739.

Mikami, Y., Okano, H., Sakaguchi, M., Nakamura, M., Shimazaki, T., Okano, H. J.,

Kawakami, Y., Toyama, Y. and Toda, M. (2004). Implantation of dendritic cells in

injured adult spinal cord results in activation of endogenous neural stem/progenitorcells leading to de novo neurogenesis and functional recovery. J. Neurosci. Res. 76,453-465.

Miller, E. J., Li, J., Leng, L., McDonald, C., Atsumi, T., Bucala, R. and Young, L. H.

(2008). Macrophage migration inhibitory factor stimulates AMP-activated protein

kinase in the ischaemic heart. Nature 451, 578-582.

Miyoshi, H. (2004). Gene delivery to hematopoietic stem cells using lentiviral vectors.

Methods Mol. Biol. 246, 429-438.

Mizutani, K., Yoon, K., Dang, L., Tokunaga, A. and Gaiano, N. (2007). DifferentialNotch signalling distinguishes neural stem cells from intermediate progenitors.Nature 449, 351-355.

Morand, E. F., Leech, M. and Bernhagen, J. (2006). MIF: a new cytokine linkbetween rheumatoid arthritis and atherosclerosis. Nat. Rev. Drug Discov. 5, 399-411.

Nagao, M., Sugimori, M. and Nakafuku, M. (2007). Cross talk between notch andgrowth factor/cytokine signaling pathways in neural stem cells. Mol. Cell. Biol. 27,

3982-3994.

Oda, S., Oda, T., Nishi, K., Takabuchi, S., Wakamatsu, T., Tanaka, T., Adachi, T.,

Fukuda, K., Semenza, G. L. and Hirota, K. (2008). Macrophage migrationinhibitory factor activates hypoxia-inducible factor in a p53-dependent manner. PLoS

ONE 3, e2215.

Ohta, S., Gregg, C. and Weiss, S. (2006). Pituitary adenylate cyclase-activating

polypeptide regulates forebrain neural stem cells and neurogenesis in vitro and invivo. J. Neurosci. Res. 84, 1177-1186.

Ohta, S., Ueda, Y., Yaguchi, M., Matsuzaki, Y., Nakamura, M., Toyama, Y.,

Tanioka, Y., Tamaoki, N., Nomura, T., Okano, H. et al. (2008). Isolation and

Identification of MIF as a newly characterized growth factor of NSPCs 3219

Journ

alof

Cell

Scie

nce

characterization of dendritic cells from common marmosets for preclinical celltherapy studies. Immunology 123, 566-574.

Okano, H. (2002). Stem cell biology of the central nervous system. J. Neurosci. Res. 69,698-707.

Okano, H. and Sawamoto, K. (2008). Neural stem cells: involvement in adultneurogenesis and CNS repair. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363, 2111-

2122.

Okano, H., Sakaguchi, M., Ohki, K., Suzuki, N. and Sawamoto, K. (2007).

Regeneration of the central nervous system using endogenous repair mechanisms.J. Neurochem. 102, 1459-1465.

Otaegi, G., Yusta-Boyo, M. J., Vergano-Vera, E., Mendez-Gomez, H. R., Carrera,

A. C., Abad, J. L., Gonzalez, M., de la Rosa, E. J., Vicario-Abejon, C. and de

Pablo, F. (2006). Modulation of the PI 3-kinase-Akt signalling pathway by IGF-I andPTEN regulates the differentiation of neural stem/precursor cells. J. Cell Sci. 119,

2739-2748.

Park, M., Song, K. S., Kim, H. K., Park, Y. J., Kim, H. S., Bae, M. I. and Lee, J.

(2009). 2-Deoxy-d-glucose protects neural progenitor cells against oxidative stressthrough the activation of AMP-activated protein kinase. Neurosci. Lett. 449, 201-206.

Rafalski, V. A. and Brunet, A. (2011). Energy metabolism in adult neural stem cell

fate. Prog. Neurobiol. 93, 182-203.

Ren, Y., Tsui, H. T., Poon, R. T., Ng, I. O., Li, Z., Chen, Y., Jiang, G., Lau, C., Yu,

W. C., Bacher, M. et al. (2003). Macrophage migration inhibitory factor: roles inregulating tumor cell migration and expression of angiogenic factors in hepatocellular

carcinoma. Int. J. Cancer 107, 22-29.

Reynolds, B. A. and Weiss, S. (1992). Generation of neurons and astrocytes from

isolated cells of the adult mammalian central nervous system. Science 255, 1707-1710.

Russo, I., Barlati, S. and Bosetti, F. (2011). Effects of neuroinflammation on theregenerative capacity of brain stem cells. J. Neurochem. 116, 947-956.

Schwartz, V., Lue, H., Kraemer, S., Korbiel, J., Krohn, R., Ohl, K., Bucala, R.,

Weber, C. and Bernhagen, J. (2009). A functional heteromeric MIF receptor formed

by CD74 and CXCR4. FEBS Lett. 583, 2749-2757.

Shi, X., Leng, L., Wang, T., Wang, W., Du, X., Li, J., McDonald, C., Chen, Z.,

Murphy, J. W., Lolis, E. et al. (2006). CD44 is the signaling component of themacrophage migration inhibitory factor-CD74 receptor complex. Immunity 25, 595-

606.

Shimazaki, T. (2003). Biology and clinical application of neural stem cells. Horm. Res.

60 Suppl. 3, 1-9.

Shimizu, T., Abe, R., Nakamura, H., Ohkawara, A., Suzuki, M. and Nishihira, J.

(1999). High expression of macrophage migration inhibitory factor in human

melanoma cells and its role in tumor cell growth and angiogenesis. Biochem. Biophys.

Res. Commun. 264, 751-758.Simons, D., Grieb, G., Hristov, M., Pallua, N., Weber, C., Bernhagen, J. and

Steffens, G. (2011). Hypoxia-induced endothelial secretion of macrophage migrationinhibitory factor and role in endothelial progenitor cell recruitment. J. Cell. Mol. Med.

15, 668-678.Starlets, D., Gore, Y., Binsky, I., Haran, M., Harpaz, N., Shvidel, L., Becker-

Herman, S., Berrebi, A. and Shachar, I. (2006). Cell-surface CD74 initiates asignaling cascade leading to cell proliferation and survival. Blood 107, 4807-4816.

Stumptner-Cuvelette, P. and Benaroch, P. (2002). Multiple roles of the invariant chainin MHC class II function. Biochim. Biophys. Acta 1542, 1-13.

Suzuki, T., Ogata, A., Tashiro, K., Nagashima, K., Tamura, M. and Nishihira, J.

(1999). Augmented expression of macrophage migration inhibitory factor (MIF) inthe telencephalon of the developing rat brain. Brain Res. 816, 457-462.

Temple, S. (2001). The development of neural stem cells. Nature 414, 112-117.Thuret, S., Moon, L. D. and Gage, F. H. (2006). Therapeutic interventions after spinal

cord injury. Nat. Rev. Neurosci. 7, 628-643.van der Kooy, D. and Weiss, S. (2000). Why stem cells? Science 287, 1439-1441.Wada, T., Haigh, J. J., Ema, M., Hitoshi, S., Chaddah, R., Rossant, J., Nagy, A. and

van der Kooy, D. (2006). Vascular endothelial growth factor directly inhibitsprimitive neural stem cell survival but promotes definitive neural stem cell survival.J. Neurosci. 26, 6803-6812.

Wang, L., Zis, O., Ma, G., Shan, Z., Zhang, X., Wang, S., Dai, C., Zhao, J., Lin, Q.,Lin, S. et al. (2009). Upregulation of macrophage migration inhibitory factor geneexpression in stroke. Stroke 40, 973-976.

Whitney, N. P., Eidem, T. M., Peng, H., Huang, Y. and Zheng, J. C. (2009).Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesisof brain injury and neurodegenerative disorders. J. Neurochem. 108, 1343-1359.

Wu, Y., Peng, H., Cui, M., Whitney, N. P., Huang, Y. and Zheng, J. C. (2009).CXCL12 increases human neural progenitor cell proliferation through Akt-1/FOXO3asignaling pathway. J. Neurochem. 109, 1157-1167.

Wyss-Coray, T. and Mucke, L. (2002). Inflammation in neurodegenerative disease - adouble-edged sword. Neuron 35, 419-432.

Yaguchi, M., Tabuse, M., Ohta, S., Ohkusu-Tsukada, K., Takeuchi, T., Yamane, J.,

Katoh, H., Nakamura, M., Matsuzaki, Y., Yamada, M. et al. (2009).Transplantation of dendritic cells promotes functional recovery from spinal cordinjury in common marmoset. Neurosci. Res. 65, 384-392.

Zhao, C., Deng, W. and Gage, F. H. (2008). Mechanisms and functional implicationsof adult neurogenesis. Cell 132, 645-660.

Ziv, Y. and Schwartz, M. (2008). Orchestrating brain-cell renewal: the role of immunecells in adult neurogenesis in health and disease. Trends Mol. Med. 14, 471-478.

Journal of Cell Science 125 (13)3220