ORIGINAL RESEARCH

Mesenchymal Stromal Cells Rescue Cortical Neuronsfrom Apoptotic Cell Death in an In Vitro Model of CerebralIschemia

Franziska Scheibe • Oliver Klein • Joachim Klose •

Josef Priller

Received: 4 August 2011 / Accepted: 6 January 2012 / Published online: 1 February 2012

� Springer Science+Business Media, LLC 2012

Abstract Cell therapy with mesenchymal stromal cells

(MSCs) was found to protect neurons from damage after

experimental stroke and is currently under investigation in

clinical stroke trials. In order to elucidate the mechanisms

of MSC-induced neuroprotection, we used the in vitro

oxygen–glucose deprivation (OGD) model of cerebral

ischemia. Co-culture of primary cortical neurons with

MSCs in a transwell co-culture system for 48 h prior to

OGD-reduced neuronal cell death by 30–35%. Similar

protection from apoptosis was observed with MSC-condi-

tioned media when added 48 h or 30 min prior to OGD, or

even after OGD. Western blot analysis revealed increased

phosphorylation of STAT3 and Akt in neuronal cultures

after treatment with MSC-conditioned media. Inhibition of

the PI3K/Akt pathway completely abolished the neuro-

protective potential of MSC-conditioned media, suggesting

that MSCs can improve neuronal survival by an Akt-

dependent anti-apoptotic signaling cascade. Using mass

spectrometry, we identified plasminogen activator inhibi-

tor-1 as an active compound in MSC-conditioned media.

Thus, paracrine factors secreted by MSCs protect neurons

from apoptotic cell death in the OGD model of cerebral

ischemia.

Keywords Mesenchymal stem cell � Oxygen–glucose

deprivation � Neuroprotection � Akt

Introduction

Transient focal cerebral ischemia (stroke) results in severe

irreversible loss of neuronal cells with persistent neuro-

logical deficits in affected individuals. Since current con-

cepts of stroke treatment are confined to acute systemic or

local thrombolysis therapy, cell-based therapeutic approa-

ches have received considerable attention as a delayed

treatment strategy.

Mesenchymal stromal cells (MSCs) are particularly

attractive candidates for cytotherapy due to their neuropro-

tective properties and the low immunogenicity (Gnecchi

et al. 2005; Dezawa et al. 2004). Several pre-clinical animal

studies in rats have demonstrated that administration of

MSCs after experimental stroke results in smaller infarct

volumes and improved functional recovery (Chen et al.

2001, 2003a; Kurozumi et al. 2005; Nomura et al. 2005;

Zhao et al. 2006). However, the regenerative potential of

MSCs is usually temporally restricted because MSCs tend to

disappear quickly after transplantation—usually within a

few days (Lee et al. 2009). The paradox of low cell

engraftment after intravenous MSC delivery and improved

tissue repair redirected attention to paracrine mechanisms,

by which MSCs may facilitate tissue protection (Lee et al.

2009). Today, it is widely accepted that MSCs enhance tissue

regeneration by the secretion of growth factors and cytokines

(Wagner et al. 2007), including glial-derived neurotrophic

factor (GDNF), nerve growth factor (NGF), and vascular

endothelial growth factor (VEGF) (Chen et al. 2002).

Moreover, MSCs secrete cytokines like interferon-gamma,

tumor necrosis factor-alpha, transforming growth factor-

beta (TGF-b), and interleukin (IL)-6 (Karnoub et al. 2007).

F. Scheibe � J. Priller (&)

Department of Neuropsychiatry and Laboratory of Molecular

Psychiatry, Charite—Universitatsmedizin Berlin, Chariteplatz 1,

10117 Berlin, Germany

e-mail: josef.priller@charite.de

F. Scheibe � O. Klein � J. Klose � J. Priller

Berlin-Brandenburg Center for Regenerative Therapies,

Berlin, Germany

O. Klein � J. Klose

Institute for Human Genetics, Charite—Universitatsmedizin

Berlin, Augustenburger Platz 1, 13353 Berlin, Germany

123

Cell Mol Neurobiol (2012) 32:567–576

DOI 10.1007/s10571-012-9798-2

The trophic factors secreted by MSCs prevent apoptosis and

enhance cell survival in damaged tissues (Caplan and

Dennis, 2006; Parekkadan et al. 2007). In addition, MSCs

can activate tissue self-repair by stimulating mitosis and

differentiation of endogenous stem cell populations (Munoz

et al. 2005). Importantly, intracerebral MSC administration

after an ischemic event was found to improve neovascular-

ization of damaged tissue (Chen et al. 2003b), which is

tightly linked to adult neurogenesis. MSCs also have

immunomodulatory and immunosuppressive capacities

(Maitra et al. 2004; Gerdoni et al. 2007), which may explain

some of their therapeutic effects in immune-mediated dis-

orders like multiple sclerosis (Zappia et al. 2005).

The paracrine functions of MSCs in cerebral ischemia

remain incompletely understood, although MSCs are

already administered to stroke patients in clinical trials. The

aim of this study was to identify the molecular mechanisms

mediating neuroprotection by bone marrow-derived MSCs.

Experimental Procedures

Cell Culture

Mesenchymal Stromal Cells

Murine MSCs (mMSCs) were obtained from the bone

marrow of tibias and femurs of C57BL/6 mice aged

8–12 weeks (BfR, Berlin, Germany). mMSC isolation and

culture techniques, characterization by cell surface epitope

expression and differentiation assays into fat, bone, and

cartilage are described in Scheibe et al. (2011).

Human MSCs (hMSCs) were kindly provided by the

laboratory of Dr. D. Prockop, Tulane University, Center for

Gene Therapy, New Orleans, LA, USA. hMSCs were

expanded and characterized as previously described (Colter

et al. 2000; Sekiya et al. 2002).

Neural Stem Cells (NSCs)

Subventricular region embodying the lateral ventricles was

obtained from 2-mm thick brains slices derived from male

C57BL/6 mice aged 8–10 weeks. A thin layer surrounding

the ventricles was prepared, cut into small pieces, and

incubated for 30–60 min in a papain-DNase-solution

(47.2 mg papain (Cellsystems, Troisdorf, Germany), 9 mg

cysteine (Sigma, Schnelldorf, Germany), 9 mg EDTA

(Sigma) in 50 ml EBSS (Invitrogen, Darmstadt, Germany)

at 37�C. Cells were pelleted by centrifugation at 1109g for

10 min. Tissue was dissociated in an ovomucoid solution

(0.7 mg/ml ovomucoid (Sigma) in NBM-A, 2% B27 w/o

retinoic acid, 1% L-glutamine; Invitrogen). Single cells were

centrifuged again at 1109g for 10 min and resuspended in

growth medium [NBM-A, 2% B27 w/o retinoic acid, 1%

L-glutamine, 10 ng/ml EGF, 20 ng/ml b-FGF (both from

Biochrom AG, Berlin, Germany)]. Cells were seeded in

25 cm2 flasks at a density of 4,000 cells per cm2 to obtain

neurospheres. After the first splitting of neurospheres, cells

were cultivated in low-attachment flasks (Corning, VWR

International, Darmstadt, Germany). Experiments were

performed with cells from passages 4–5.

Primary Neuronal Cells

Primary rat cortical neurons were prepared from the cerebral

cortex of Wistar rats at embryonic day 17. Animals were

purchased from FEM (Charite, Berlin, Germany), and all

media and supplements were from Biochrom AG if not

otherwise noted. Neuronal cultures were prepared according

to modified protocols from Brewer (1995) and Lautensch-

lager et al. (2000). After removal of the meninges, cerebral

cortices were dissected and incubated with 0.5%/0.2% (w/v)

trypsin/EDTA in a water bath for 15 min at 37�C, rinsed

twice with PBS, and once with dissociation medium (con-

sisting of modified Eagle’s medium (MEM) with 10% FCS,

100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM L-glu-

tamine, 10 mM HEPES, 44 mM glucose, 100 IE/l insulin).

Thereafter, they were dissociated with a Pasteur pipette

in dissociation medium, pelleted by centrifugation at

2109g for 2 min at room temperature (RT), redissociated in

starter medium (NBM supplemented with B27 (both from

Invitrogen), 100 U/ml penicillin, 100 lg/ml streptomycin,

0.5 mM L-glutamine, 25 lM glutamate) and were seeded

into 24-well plates at a density of 200,000 cells/cm2. Wells

were coated with poly-L-lysine (0.5% w/v in PBS) for 1 h at

RT, rinsed with PBS, followed by incubation with coating

medium (MEM, 5% FCS, 100 U/ml penicillin, 100 lg/ml

streptomycin, 10 mM HEPES, and 1% w/v collagen G) for

1 h at 37�C in the incubator. Wells were rinsed with PBS and

filled with 500 ll/well starter medium. Cells were added in a

volume of 100 ll/well and cultures were kept at 37�C and

5% CO2. Feeding began after 4 days in vitro (div) with

neuronal cell culture medium (starter medium without glu-

tamate) by replacing half of the medium twice a week.

Oxygen–Glucose Deprivation (OGD)

Induction of Neuroprotection by MSC Co-culture

or MSC-Conditioned Medium (CM)

Neuronal cultures were kept under the conditions described

above until a medium change was performed after 8 div.

300 ll medium were removed from each well and 250 ll

of fresh NBM ? B27 supplemented with 4% FCS (end

concentration of 2% FCS/well) were added to each well.

Finally, each well contained approximately 500 ll as total

568 Cell Mol Neurobiol (2012) 32:567–576

123

volume. In all experiments, four wells of a 24-well plate

were used for each approach and each plate contained a

control group to exclude plate to plate variability.

For investigation of MSC-mediated neuroprotective

effects, rat cortical neurons were treated either with CM from

mMSCs or hMSCs that was added in varying concentrations

(0.1, 0.25, 0.5, 1, and 5%) and at different time points

(-48 h, -30 min, immediately post-OGD) to the neurons.

Unconditioned medium consisting of NBM ? B27,

100 U/ml penicillin, 100 lg/ml streptomycin, 0.5 mM

L-glutamine, and 2% FCS served as negative control. To

generate MSC supernatants, mMSCs and hMSCs were

allowed to grow to 70–80% confluency in 145 cm2 petri

dishes in their regular growth medium. After rinsing with

PBS, 25 ml medium consisting of NBM ? B27, 100 U/ml

penicillin, 100 lg/ml streptomycin, 0.5 mM L-glutamine,

and 2% FCS were added to each dish. After 24 h, superna-

tants from MSC cultures were collected, debris was removed

by rinsing through a 0.2-lm filter system, and aliquots of

MSC supernatants were frozen at -20�C.

In another approach, cortical neurons were directly pre-

conditioned by co-culture with MSCs for 48 h. In a

transwell co-culture system, rat cortical neurons were

located in the bottom well and co-culture was initiated by

seeding of either 10,000 mMSCs or 1,000 hMSCs into

inserts. Varying MSC cell numbers resulted from different

proliferation characteristics of hMSCs and mMSCs in the

inserts, resulting in distinct factor secretion with different

dose–effect curves. In general, the cells were seeded in

150 ll medium into 0.4-lm pore size inserts (Falcon; BD

Biosciences) with a polyethylene terephthalate (PET)

membrane. The membrane was permeable to soluble fac-

tors, but prevented cell-to-cell contact between the com-

partments. 100 ll of medium were added to the bottom

well in the transwell experiments (total volume of 250 ll

was added to each well).

Blocking Experiments

To investigate molecular pathways involved in MSC-

mediated neuroprotection, the same pre-conditioning regi-

men was applied to neuronal cultures as described above.

To inhibit neuroprotective effects of 1% hMSC- or 1%

mMSC-CM, the respective CM with or without 1 lM Akt

inhibitor X (AktX; Merck, Darmstadt, Germany) were

added to neuronal cultures 48 h prior to OGD.

To block soluble factors secreted by hMSCs, 2 lg/ml

plasminogen activator inhibitor (PAI)-1 antibody (R&D

systems, Wiesbaden-Nordenstadt, Germany) were added to

neuronal cultures either with or without 1% hMSC-CM at

48 h prior to OGD. Application of vehicle or 1% hMSC-

CM served as negative or positive control in these

experiments.

OGD Experiments

Primary rat cortical neurons were used for OGD experi-

ments after a cultivation period of 10 div. Before OGD,

inserts with MSCs and medium from all wells were

removed, wells were rinsed once with 300 ll PBS, and

OGD was started by addition of 500 ll/well of a deoxy-

genated aglycemic solution (DAS: 143.8 mM Na?,

5.5 mM K?, 1.8 mM Ca2?, 1.8 mM Mg2?, 125.3 mM

Cl-, 26.2 mM HCO3-, 1.0 mM PO43-, 0.8 mM SO4

2-;

pH 7.4) in an anoxic atmosphere (0% O2). Anoxia was

generated in a humidified and gas-tight incubator (Concept

400, Ruskinn Technologies, Bridgend, UK) at 37�C that

was flushed with a gas mixture consisting of 5% CO2, 85%

N2, and 10% H2. OGD lasted for the duration of

90–120 min. In control experiments, cells were washed

with 300 ll PBS and were then exposed to 500 ll basic salt

solution (BSS: 143.8 mM Na?, 5.5 mM K?, 1.8 mM

Ca2?, 1.8 mM Mg2?, 125.3 mM Cl-, 26.2 mM HCO3-,

1.0 mM PO43-, 0.8 mM SO4

2-, 4.5 mg/l glucose, 2%

FCS; pH 7.4) in a normoxic atmosphere with 5% CO2 at

37�C. Immediately after OGD and in control experiments,

medium was removed and replaced with 400 ll neuronal

cell culture medium (50% fresh NBM ? B27 and 50%

conditioned NBM ? B27 that was collected from neuronal

cell cultures after 8 div) with or without MSC-CM.

Lactate Dehydrogenase (LDH) Assay

LDH release was measured as described previously (Bruer

et al. 1997). 24 h after OGD, the LDH activity was detected

in the supernatant of OGD-treated and control cultures to

assess neuronal cell damage. Then, 20 ll of a 10% Triton

X-100 solution were administered to each well to initiate

full kill of cells for quantification of the maximum LDH

release. Cells were incubated for 20 min at 37�C in an

incubator and LDH values were measured again. LDH

values of OGD-treated and BSS-stimulated cultures were

divided by full kill LDH values to calculate cytotoxicity.

Afterward, cytotoxicity values of control cultures were

subtracted as background from OGD-treated cultures.

Normoxic BSS stimulation was set to 0% cell damage and

OGD stimulation to 100% cell death, respectively.

Ethidium Bromide (EB) and Acridine Orange (AO)

Staining

Twenty-four hours after OGD, 2 lg/ml AO and 2 lg/ml

EB (both from Sigma) were added to neuronal cultures to

quantify apoptotic and necrotic cell death of primary

neurons. After 5 min incubation, dead cells were counted

in three wells and five visual fields (4009 magnification)

using a standard fluorescence microscope (Leica DM-RA).

Cell Mol Neurobiol (2012) 32:567–576 569

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Western Blotting

Neuronal cells with or without exposure to MSC-CM were

harvested after 5, 15, or 30 min in cell lysis buffer (New

England Biolabs, Frankfurt am Main, Germany) supple-

mented with complete mini protease inhibitor cocktail (New

England Biolabs) and 1 mM phenylmethylsulfonylfluoride

(PMSF; Sigma). After incubation on ice for 10 min and cell

scraping, lysates were centrifuged at 14,0009g at 4�C for

5 min. Protein-containing supernatants were diluted in SDS

sample buffer (end concentrations: 62.5 mM Tris–HCl, pH

6.8, 0.5% SDS, 10% glycerol, 5% b-mercaptoethanol,

0.004% bromphenol blue; all from Sigma) and boiled for

5 min before samples were stored at -80�C. Protein con-

centrations were determined by BCA assay (Pierce, Thermo

Fisher Scientific, Bonn, Germany). Approximately, 20 lg of

protein/lane were separated on SDS polyacrylamide gels by

electrophoresis. Proteins were blotted onto nitrocellulose

membranes (Roth, Karlsruhe, Germany). Blocking of

membranes was carried out with TBST (10 mM Tris, 0.05%

Tween 20, 150 mM NaCl, pH 7.6; all from Sigma) and 5%

BSA for 1 h at RT. After rinsing with TBST, membranes

were incubated with primary antibodies in TBST and 5%

BSA overnight at 4�C. The following primary antibodies

were applied: p42/44 MAPK antibody (1:1,000), pAkt

(Thr308) antibody (1:2,000), pan-Akt antibody (1:1,000),

pSTAT3 (Tyr 705) antibody (1:1,000; all from Cell Signal-

ing, Frankfurt am Main, Germany), and b-actin antibody

(1:1,000; Santa Cruz, Heidelberg, Germany). The next day,

nitrocellulose membranes were washed with TBST and a

secondary HRP-linked anti-rabbit antibody (1:1,000;

Amersham Biosciences, Freiburg, Germany) or HRP-linked

anti-goat antibody (1:10,000; Santa Cruz) was added at RT

for 2 h. Signals on membranes were visualized on X-ray

films (Kodak, Amersham Biosciences) after incubation with

a chemiluminescence kit (Santa Cruz). All experiments were

repeated at least three times.

Protein Identification and Mass Spectrometry (MS)

For preparation of hMSC supernatants, hMSCs were cul-

tured in their regular proliferation medium until 80% con-

fluency. Thereafter, cells were rinsed with PBS thrice and

serum-free proliferation medium was added for 72 h. Col-

lected hMSC-CM was filtered through a 0.2-lm filter and

frozen at -20�C. Thawed samples were concentrated with

4 ml 10 kDa MWCO Amico filters (Millipore, Billerica,

MA). For protein identification by MS, 15 ll concentrated

extract was separated by SDS-PAGE and stained using a

MS-compatible silver staining protocol (Zabel and Klose

2009). Protein bands were excised from gels and subjected

to in-gel tryptic digestion. Tryptic fragments were analyzed

by nanoflow high-performance liquid chromatography

(nanoHPLC; Proxeon Easy-nLC, Denmark, Odense)/elec-

trospray ionization (ESI)-MS and ESI-MS/MS on a LCQ

Deca XP ion trap instrument (Thermo Finnigan, Waltham,

MA, USA). Nano-HPLC was directly coupled to ESI-MS

analysis. Protein bands eluates of 18 ll were loaded onto a

SSPE traps C18 pre-column (5 lm, 120 A, 100 lm

I.D. 9 20 mm); NanoSeparations, Nieuwkoop, Nether-

lands) using 0.1% v/v trifluoroacetic acid at a flow rate of

20 ll/min. Peptides were separated onto an analytical C18

columns (5 lm, 120 A, 75 lm I.D. 9 10 cm). The elution

gradient was created by mixing 0.1% v/v formic acid in water

(solvent A) and 0.1% v/v formic acid in acetonitrile (solvent

B) and run at a flow rate of 200 nl/min. The gradient was

started at 5% v/v solvent B and increased linearly up to 50%

v/v solvent B after 40 min. ESI-MS data acquisition was

performed throughout the LC run. Three scan events: (i) full

scan, (ii) zoom scan of most intense ion in full scan, and (iii)

MS/MS scan of the most intense ion in full scan were applied

sequentially. No MS/MS scan on single charged ions was

performed. Raw data were extracted by the TurboSEQUEST

algorithm, and trypsin autolytic fragments and known

keratin peptides were subsequently filtered. All DTA files

generated by BioWorks version 3.2 (Thermo Scientific,

Waltham, MA, USA) were merged and converted to MAS-

COT generic format files (MGF). Mass spectra were ana-

lyzed using our in-house MASCOT software package

license version 2.2 automatically searching the SwissProt

database for Homo sapiens (human) (SwissProt 51.8,

513,877 sequences). MS/MS ion search was performed with

this set of parameters: (i) taxonomy: H. sapiens (human), (ii)

proteolytic enzyme: trypsin, (iii) maximum of accepted

missed cleavages: 1, (iv) mass value: monoisotopic,

(v) peptide mass tolerance 0.8 Da, (vi) fragment mass tol-

erance: 0.8 Da, and (vii) variable modifications: oxidation of

methionine and acrylamide adducts (propionamide) on

cysteine. No fixed modifications were considered. Only

proteins with scores corresponding to P \ 0.05, with at least

two independent peptides identified were considered. The

cut-off score for individual peptides using ESI identification

was equivalent to P \ 0.05 for each peptide and usually in a

MOWSE score [31. This number was calculated by the

MASCOT software.

Immunocytochemistry

Neuronal cultures grown on coverslips were fixed after

10 div with 4% paraformaldehyde (PFA) for 15 min at RT.

Blocking and permeabilization were carried out in 5%

normal donkey serum (NDS), 1% BSA, and 0.3% Triton

X-100 (Sigma) in PBS. Primary antibodies against MAP2

(1:1,000; Sigma) GFAP (1:1,000; Dako, Hamburg, Ger-

many), Iba1 (1:2,000; Wako, Neuss, Germany), and

CNPase (1:500; Covance, Munich, Germany) were

570 Cell Mol Neurobiol (2012) 32:567–576

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incubated in a PBS solution with 3% NDS at 4�C overnight.

The following secondary antibodies (all from Invitrogen)

were incubated at a 1:800 dilution in PBS with 3% NDS for

1 h at RT: Alexa 594 donkey anti-mouse, Alexa 488 donkey

anti-rabbit, and Alexa 594 donkey anti-rat. Cells were

counterstained with DAPI before mounting. Omission of

primary antibodies served as negative controls. Neuronal

cultures were examined by fluorescence microscopy using a

Leica microscope (DM-RA). For each group and experi-

ment, cells of three coverslips in 10 visual fields were

counted at 1009 magnification to quantify neuronal cell

culture composition of the respective treatment group.

Statistical Analysis

For statistical analysis of OGD data, Kruskal–Wallis one-

way ANOVA on ranks test with Dunn’s method as post-

hoc test or one-way ANOVA with Bonferroni’s post-hoc

test was used. Immunocytochemistry data were tested for

statistical significance by ANOVA with Bonferroni’s post-

hoc test. P values \0.05 were considered as statistically

significant. Results are expressed as means ? standard

deviation (SD).

Results

MSCs Rescue Cortical Neurons from OGD-Induced

Cell Death

For neuroprotection studies of MSCs and their conditioned

media, 90–120 min OGD experiments were performed to

mimic the pathophysiological conditions of cerebral ische-

mia in vitro. Figure 1a illustrates the experimental setup.

Pre-conditioning Experiments

In order to prevent cell-to-cell contacts while allowing the

diffusion of soluble factors, hMSCs, or mMSCs were co-

cultured with cortical neurons in a transwell system for

48 h prior to OGD. This resulted in a significant reduction

of OGD-induced neuronal cell death by 30–35% as deter-

mined by LDH assay (Fig. 1b). In order to investigate

whether the neuroprotective effects are specific for MSCs

or can also be conferred by other stem cell populations,

murine NSCs were co-cultured with neurons prior to OGD.

However, NSCs did not provide any protection from OGD

at up to twice the concentration of MSCs (Fig. 1c).

Fig. 1 Transwell co-culture

with MSCs increases neuronal

cell survival after OGD.

a Illustration of the

experimental setup.

b Co-culture of neuronal cells

with hMSCs or mMSCs in a

two-compartment system for

48 h resulted in neuroprotection

as shown by the significant

reduction of LDH release

(n = 5). c Co-culture of

neuronal cells with murine

NSCs failed to confer

neuroprotection (n = 3).

**P \ 0.001

Cell Mol Neurobiol (2012) 32:567–576 571

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Given the paracrine actions of MSCs, media conditioned

by hMSCs and mMSCs were added to neuronal cultures at

different time points prior to OGD (-48 h, -30 min) at

varying concentrations (0.1–5%). When CM was added to

neurons 48 h prior to OGD, dose-dependent neuroprotective

effects were observed. Low concentrations of hMSC- and

mMSC-CM (0.1%) failed to prevent neuronal cell death

after OGD, whereas higher concentrations of hMSC-CM

(0.25–5%) and mMSC-CM (1–5%) conferred significant

reductions of LDH release by 29–36% (Fig. 2a). The degree

of neuroprotection achieved with MSC-CM was comparable

to the results of direct co-culture experiments (Fig. 1b).

Addition of hMSC-CM (0.1–5%) and mMSC-CM (1–5%) to

cortical neurons only 30 min prior to OGD resulted in

reductions of LDH release by 23–25% (Fig. 2b).

Post-OGD Treatment with MSC-Conditioned Media

In order to determine whether MSC-CM also protect neu-

rons from cell death when given after OGD, varying con-

centrations of hMSC- and mMSC-CM were added to

neuronal cultures immediately after OGD. hMSC-CM and

mMSC-CM (0.25–1%) reduced neuronal cell death by

20–25% post-OGD (Fig. 2c).

MSCs and MSC-CM do not Alter the Cellular

Composition of Neuronal Cultures

The primary cultures used in this study consisted of

approximately 89% MAP2-immunoreactive neurons, 10%

GFAP-immunoreactive astrocytes, \0.3% CNPase-immu-

noreactive oligodendrocytes, and \0.05% Iba1-immuno-

reactive microglia (data not shown). The composition of

primary neuronal cell cultures was neither altered by the

co-culture with hMSCs and mMSCs nor by the addition of

hMSC-CM and mMSC-CM (Fig. 3a). Thus, the reduction

of LDH release in MSC- or MSC-CM-treated groups did

not result from altered neuronal cell culture composition

with a modified LDH release pattern.

MSC-Conditioned Media Protect Neurons

from OGD-Induced Apoptotic Cell Death

AO and EB staining revealed that apoptosis is the pre-

dominant cell death mechanism during and after OGD,

whereas necrosis only accounts for less than 5% of OGD-

induced cell death (Fig. 3b). Treatment of neurons with

hMSC-CM and mMSC-CM at 48 h prior to OGD or

immediately post-OGD resulted in significant reductions of

the rate of apoptosis, whereas the rate of necrosis was

unaffected (Fig. 3b), suggesting that MSCs protect neurons

from OGD-induced apoptotic cell death.

Molecular Mechanisms of MSC-Induced

Neuroprotection

Western blot studies were performed to investigate the

downstream molecular mechanisms involved in MSC-

mediated neuroprotection. Treatment of neuronal cultures

with hMSC-CM and mMSC-CM for 5–30 min resulted in

increased levels of pSTAT3 (Tyr705) and pAkt (Thr308) in

neurons (Fig. 4a). In contrast, p42/44 MAPK levels

remained unchanged (Fig. 4a). The data suggest that sol-

uble factors in MSC-CM activate intracellular signaling

cascades in neurons, which may be involved in anti-

apoptotic effects. To further elucidate the role of Akt in

Fig. 2 MSC-conditioned media exhibit neuroprotective effects.

a Addition of varying concentrations (0.1–5%) of hMSC-CM (leftpanel) or mMSC-CM (right panel) to neuronal cultures at 48 h prior

to OGD protected neurons from cell death (n = 5). b Significant

reduction of LDH release was also observed after addition of hMSC-

and mMSC-CM at 30 min prior to OGD (n = 5). c Post-OGD

treatment with hMSC- or mMSC-CM contributed to increased

neuronal survival after OGD (n = 5). *P \ 0.05, **P \ 0.001

572 Cell Mol Neurobiol (2012) 32:567–576

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MSC-mediated neuroprotection, neuronal cultures were

pre-treated with hMSC-CM and mMSC-CM (1%) in the

presence or absence of 1 lM AktX for 48 h prior to OGD.

AktX completely abrogated the anti-apoptotic effects of

MSC-CM (Fig. 4b), suggesting that PI3K/Akt is a major

pathway for MSC-mediated neuroprotection.

Next, we tried to analyze the secretome of hMSCs by

MS to identify putative factors, which mediate neuropro-

tection. Only factors that reached statistical significance in

the hMSC proteomic study are summarized in Table 1.

Twelve proteins were identified by MS, of which PAI-1

was considered an interesting candidate since it is known to

prevent apoptosis though the Akt pathway. We also

detected cytokines and growth factors like VEGF, TGF-b,

macrophage colony-stimulating factor (M-CSF), and

insulin-like growth factor-binding proteins (IGFBPs) in

hMSC-CM by antibody arrays (data not shown). As proof-

of-concept, 2 lg/ml anti-PAI-1 antibody was added to

neuronal cultures at 48 h prior to OGD, which partly

reverted the neuroprotective effect of hMSC-CM (Fig. 4c).

Discussion

Here, we show that human and murine MSCs rescue cor-

tical neurons from apoptotic cell death in an in vitro model

of cerebral ischemia. The neuroprotective effects are spe-

cific for MSCs and are mediated by paracrine factors, like

PAI-1.

Our data are in line with previous in vitro reports,

suggesting that astrocytes and oligodendrocytes are rescued

from cell death after OGD by transwell co-culture with

Fig. 3 Neuroprotection by MSC-conditioned media is mediated by

an anti-apoptotic mechanism. a Immunocytochemical characteriza-

tion of neuronal cell cultures revealed that transwell co-culture with

hMSCs or mMSCs for 48 h did not affect the relative percentages of

neurons and glial cells in the cultures, nor did the addition of MSC-

CM for 48 h. b AO and EB staining was used to distinguish between

apoptotic and necrotic cell death of neurons after OGD. Addition of

hMSC- and mMSC-CM at 48 h prior to OGD or post-OGD resulted

in a remarkable reduction of apoptotic neurons, whereas necrotic

neuronal cell death was not affected (n = 6)

Fig. 4 Neuroprotection by MSC-conditioned media involves the Akt

pathway and PAI-1. a Western blot studies indicated an upregulation

of pSTAT3 (Tyr 705) and pAkt (Thr 308) in neuronal cell lysates

after exposure of primary neurons to mMSC- or hMSC-CM for

5–30 min. In contrast, the p42/44 MAPK pathway was not activated

by MSC-CM. b-actin and Akt (pan) served as internal controls.

b Blocking of the Akt pathway with AktX (1 lM) abolished the

neuroprotective effects of 1% hMSC-CM and 1% mMSC-CM (pre-

conditioning time: 48 h, n = 5–6). c Blocking of PAI-1 by addition of

2 lg/ml anti-PAI-1 antibody partly reverted the neuroprotective effect

of 1% hMSC-CM at 48 h prior to OGD (n = 5). *P \ 0.05,

**P \ 0.001

Cell Mol Neurobiol (2012) 32:567–576 573

123

bone marrow stromal cells (Gao et al. 2008; Zhang et al.

2008). Moreover, intravenously administered MSCs

reduced apoptosis and improved functional recovery after

stroke in vivo (Chen et al. 2003a). MSCs were found to

protect irradiated fibroblasts from apoptosis (Block et al.

2009). However, MSC-CM did not prevent apoptosis in

this model, and MSC-mediated activation of fibroblasts by

direct cell-to-cell contact was required to induce produc-

tion of protective factors (Block et al. 2009). In our OGD

model, robust neuroprotection was achieved by addition of

diluted MSC-CM at a concentration range of 0.25–5%.

Incubation with MSC-CM at concentrations higher than

10% yielded toxic effects on neuronal cultures, as did

higher MSC numbers ([5,000 for hMSCs and [1,000 for

mMSCs, respectively) in transwell co-culture experiments

(data not shown). We performed all experiments in the

non-toxic range as demonstrated by phase-contrast

microscopy and LDH measurements of the cultures. Our

results are in line with previous observations that pre-

treatment of organotypic hippocampal slice-cultures with

undiluted or 1:2-diluted MSC supernatants induced

neurotoxicity after OGD (Horn et al. 2009). We further

observed a dose-dependent neuroprotection by 0.25–5%

MSC-CM when added to cortical neurons 48 h prior to

OGD. Neuroprotection was achieved with even lower

concentrations of MSC-CM (0.1%) when added 30 min

prior to OGD, but the degree of neuroprotection was gen-

erally lower at 30 min even with high concentrations of

MSC-CM. This may suggest a combination of direct neu-

roprotective effects of MSC-CM subject to protein degra-

dation, and delayed neuroprotection by glial cells or

secondary induction of gene expression and protein trans-

lation in neurons.

Several previous studies aimed to identify the factors

and mechanisms by which MSCs mediate not only their

cyto- and neuroprotective effects but also their immuno-

modulatory and angiogenic functions. One secretome study

using human adipose tissue-derived MSCs revealed inter-

esting molecular candidates, e.g., anti-inflammatory mol-

ecules (follistatin-like 1, pentraxin-related gene), but also

antioxidants (gluthatione-S-transferase P, peroxiredoxin 6,

thioredoxine) (Chiellini et al. 2008). Other proteomic

studies found that MSCs secrete a variety of cytokines,

chemokines, growth factors, and their receptors (Wagner

et al. 2007). One study reported that different isolation and

culture techniques of MSCs, as well as different species

and organ origins may explain the variability observed in

the secretory profiles of MSCs (Wagner et al. 2007). We

identified only 12 factors that met the criteria of statistical

significance for MS. The low number of detected mole-

cules may result from technical limitations of MS and the

serum-free culture conditions of MSCs, which were

required for precise protein identification.

The molecular complexity of MSC-CM suggests that

protection of cortical neurons after OGD may be mediated

by different mechanisms: (1) by direct effects of MSC-CM

on neurons, (2) indirectly by soluble MSC-derived factors

that stimulate contaminating astrocytes (*10%) to secrete

neuroprotective factors, and (3) by delayed conditioning

mechanisms in neurons. Besides proteins like cytokines

and growth factors, smaller molecules such as lipids,

peptides, or antioxidants are also putative candidates for

MSC-CM-mediated neuroprotection.

In our proteomic MS approach, we identified PAI-1

(*45 kDa) as a putative candidate for MSC-mediated

neuroprotection. Previous studies observed that PAI-1

Table 1 Proteomic characterization of human MSC-conditioned media

Protein ID MW (Da) Function

Collagen alpha-1 (I) chain P02452 138,827 ECM component

Collagen alpha-2 (I) chain P08123 129,209 ECM component

Chitinase-3-like protein 1 P36222 42,598 ECM component

Hyaluronan and proteoglycan link protein 1 P10915 40,140 ECM component

Basement membrane-specific heparan

sulfate proteoglycan core protein

P98160 468,501 ECM component

72 kDa type IV collagenase (MMP2) P08253 73,835 Protease, collagen metabolism

Metalloproteinase inhibitor 2 (TIMP2) P16035 24,383 Protease, metalloproteinase inhibiting activity

Plasminogen activator inhibitor 1 (PAI-1) P05121 45,031 Protease, fibrinolysis inhibitor

Vimentin P08670 53,619 Cytoskeletal component, cell growth

Profilin-1 P07737 15,045 Cytoskeletal component, cell growth

Golgin subfamily A member Q13439 260,980 Intracellular transport protein

Serum albumin P02768 69,321 Oncotic pressure in blood, transport protein

Secreted protein acidic cysteine

rich glycoprotein (SPARC)

P09486 34,610 Interaction with ECM components,

inhibition of cell cycle, cell adhesion

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123

produced by astrocytes in mixed neuronal/astrocytic cul-

tures protected neurons against N-methyl-D-aspartate

(NMDA) receptor-mediated excitotoxicity by modulating

the NMDA-evoked calcium influx (Docagne et al. 2002;

Gabriel et al. 2003). In contrast, a recent study observed

that astrocytes increase tissue plasminogen activator (tPA)

activity and downregulate PAI-1 in response to MSCs or

MSC-CM in the OGD model (Xin et al. 2010). We found

that application of blocking antibodies against PAI-1 sig-

nificantly reduced MSC-mediated neuroprotection after

OGD. The inhibition was not complete, which supports our

assumption that neuroprotection by MSCs is likely medi-

ated by a combination of different paracrine factors

secreted by MSCs. Along these lines, we also detected

SPARC/osteonectin in hMSC-CM, which has been shown

to provide protection from apoptosis via an Akt-dependent

pathway (Shi et al. 2004; Chang et al. 2010).

On the level of intracellular signal transduction, we

observed that MSC-CM increased the phosphorylation of

STAT3 and Akt in neurons. This is in line with findings

that human aortic endothelial cells are protected from

hypoxia-induced apoptotic cell death by MSC superna-

tants after activation of PI3K/Akt and STAT3 signaling

cascades (Hung et al. 2007). In this study, the authors

found relevant levels of VEGF, MCP-1, and IL-6 in

MSC-CM. However, experiments with neutralizing anti-

bodies against VEGF, MCP-1, and IL-6 failed to confirm

that these factors were involved in the prevention of

apoptotic cell death by MSC-CM (Hung et al. 2007).

Moreover, treatment of cultures with MSC-CM also

increased the levels of pERK, but an inhibitor of the

ERK1/2 pathway had no effect on hypoxia-induced

apoptosis (Hung et al. 2007). In another OGD model,

oligodendrocytes were protected from apoptosis by the

activation of Akt and p75 (Zhang et al. 2008). These data

are in line with our results, since we found that the Akt

pathway is one of the major mechanisms that underlie

the anti-apoptotic effects of MSC-CM. It remains to be

resolved whether activation of the STAT3 and PI3K/Akt

transduction pathways interrelate to provide neuropro-

tection. Thus, a recent study demonstrated that cytokines

such as IL-6 have anti-apoptotic effects in models of

NMDA-induced excitotoxicity by combined activation of

the STAT3 and PI3K/Akt signaling pathways (Liu et al.

2011). IL-10 was also found to reduce the apoptosis of

cortical neurons after OGD by up-regulation of phos-

phorylated STAT3 and Akt (Sharma et al. 2011).

In conclusion, our data suggest that MSCs provide

neuroprotection by paracrine mechanisms. MSC-derived

soluble factors either directly or indirectly activate STAT3-

and Akt-dependent anti-apoptotic pathways in neurons that

enhance survival after OGD.

Acknowledgments This study was supported by Research grant no.

01GN0508 from the German Ministry for Education and Research

(BMBF). Some of the materials employed in this study were provided

by the Tulane Center for Gene Therapy through a grant from NCRR

of the NIH, Grant # P40RR017447. The authors thank Dr. Christel

Bonnas and Dr. Dorette Freyer for their advice, and Melanie Lange,

Jasmin Jamal-el-Din and Peggy Mex for excellent technical

assistance.

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