TRANSLATIONAL AND CLINICAL RESEARCH

Transduction of Neural Precursor Cells with TAT-Heat Shock Protein

70 Chaperone: Therapeutic Potential Against Ischemic Stroke After
Intrastriatal and Systemic Transplantation
THORSTEN R. DOEPPNER,a,b TOBIAS A. S. EWERT,c LARS T€oNGES,b JOSEPHINE HERZ,a ANIL ZECHARIAH,a
AYMAN ELALI,a ANNA-KRISTIN LUDWIG,d BERND GIEBEL,d FLORIAN NAGEL,e GUNNAR P. H. DIETZ,b,f,g
JENS WEISE,h DIRK M. HERMANN,a MATHIAS B€aHRb,g
a
Department of Neurology, University of Duisburg-Essen Medical School, Essen, Germany; bDepartment of
Neurology, University of Goettingen Medical School, Goettingen, Germany; cDepartment of Neurology,
University Medical Center Hamburg-Eppendorf, Hamburg, Germany; dInstitute for Transfusion Medicine,
University of Duisburg-Essen Medical School, Essen, Germany; eCenter for Molecular Neurobiology, Hamburg,
Germany; fDepartment 851, Neurodegeneration II, H. Lundbeck A/S, Valby, Denmark; gDFG Research Center for
the Molecular Physiology of the Brain (CMPB), Germany; hDepartment of Neurology, University of Jena Medical
School, Jena, Germany

Key Words. Adult stem cells • Cell transplantation • Cellular therapy • Nervous system • Neural stem cell • Progenitor cells

ABSTRACT
Novel therapeutic concepts against cerebral ischemia focus
on cell-based therapies in order to overcome some of the
side effects of thrombolytic therapy. However, cell-based
therapies are hampered because of restricted understanding
regarding optimal cell transplantation routes and due to
low survival rates of grafted cells. We therefore trans-
planted adult green fluorescence protein positive neural
precursor cells (NPCs) either intravenously (systemic) or
intrastriatally (intracerebrally) 6 hours after stroke in
mice. To enhance survival of NPCs, cells were in vitro pro-
tein-transduced with TAT-heat shock protein 70 (Hsp70)
before transplantation followed by a systematic analysis of
brain injury and underlying mechanisms depending on cell
delivery routes. Transduction of NPCs with TAT-Hsp70
resulted in increased intracerebral numbers of grafted
NPCs after intracerebral but not after systemic transplan-
tation. Whereas systemic delivery of either native or trans-
duced NPCs yielded sustained neuroprotection and induced
neurological recovery, only TAT-Hsp70-transduced NPCs
Disclosure of potential conflicts of interest is found at the end of this article.
prevented secondary neuronal degeneration after intracere-
bral delivery that was associated with enhanced functional
outcome. Furthermore, intracerebral transplantation of
TAT-Hsp70-transduced NPCs enhanced postischemic neu-
rogenesis and induced sustained high levels of brain-derived
neurotrophic factor, glial cell line-derived neurotrophic fac-
tor, and vascular endothelial growth factor in vivo. Neuro-
protection after intracerebral cell delivery correlated with
the amount of surviving NPCs. On the contrary, systemic
delivery of NPCs mediated acute neuroprotection via stabi-
lization of the blood-brain-barrier, concomitant with
reduced activation of matrix metalloprotease 9 and
decreased formation of reactive oxygen species. Our
findings imply two different mechanisms of action of intra-
cerebrally and systemically transplanted NPCs, indicating
that systemic NPC delivery might be more feasible for
translational stroke concepts, lacking a need of in vitro
manipulation of NPCs to induce long-term neuroprotection.
STEM CELLS 2012;30:1297–1310

cellular mediators into the extracellular space that promote

INTRODUCTION
Neurogenesis persists in the adult rodent brain within the
subventricular zone (SVZ) [1–3]. Cerebral ischemia stimu-
lates neurogenesis [4–6], and newly formed neurons release
neurological recovery [7, 8]. Unfortunately, survival and dif-
ferentiation rates of new-born cells are low [4–6, 9, 10].
Therefore, exogenous NPC transplantation strategies have
been used to overcome limitations of endogenous neurogene-
sis [7, 11, 12].

Author contributions: T.R.D.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript
writing, and final approval of manuscript; T.A.E.: collection and/or assembly of data, provision of study material or patients, and final
approval of manuscript; L.T. and F.N.: provision of study material or patients and final approval of manuscript; J.H., A.Z., A.E., and A.-
K.L.: collection and/or assembly of data and final approval of manuscript; B.G. and G.P.D.: manuscript writing and final approval of
manuscript; J.W.: conception and design, financial support, and final approval of manuscript; D.M.H.: administrative support, manuscript
writing, and final approval of manuscript; M.B.: financial support, administrative support, and final approval of manuscript.
Correspondence: Thorsten R. Doeppner, M.Sc., M.D., Department of Neurology, University of Duisburg-Essen Medical School,
Hufelandstr. 55, 45147 Essen, Germany. Telephone: þ49-201-723-83133; Fax: þ49-201-723-1660; e-mail: thorsten.doeppner@uk-essen.
de Received October 6, 2011; accepted for publication March 16, 2012; first published online in STEM CELLS EXPRESS March 30, 2012.
V
C AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1098

STEM CELLS 2012;30:1297–1310 www.StemCells.com

1298
Table 1. groups and numbers of animals used for statistical analysis
Systemic delivery
PBS NPCs NPCs TAT-HA NPCs TAT-Hsp70
Day 1 14 14 14 14
Day 4 15 14 14 14
Day 14 7 7 7 7
Day 28 8 9 9 9
Day 56 11 11 11 11
Animals were assigned to four treatment groups for both systemic and intrastriatal delivery of adult NPCs or PBS, respectively. All
injections were performed 6 hours after stroke onset. Five survival periods were defined for each group. Time points given refer to induction
of stroke, which was assigned as day 0. The number of animals given reflects the amount of surviving mice used for final analysis of
experimental results.
Abbreviations: Hsp70, heat shock protein 70; NPCs, neural precursor cells; PBS, phosphate buffered saline; TAT-HA, TAT-hemagglutinin.
Transplantation of NPCs ameliorates ischemic brain injury
in rodents [7, 8, 13–17], but its therapeutic benefit is hampered
by low survival rates of grafted cells [18, 19]. Since cell trans-
plantation is usually done after stroke [20], grafted cells are not
exposed to ischemic insults directly. Rather, cells are trans-
planted into a nonfavorable tissue environment, in which they
are exposed to oxidative stress and inflammation [21, 22],
which may cause protein misfolding and cell death [23, 24].
Regarding clinical translation, the most appropriate route of
NPC transplantation is an important but unresolved question.
Among different transplantation routes, intravenous cell delivery
has been used due to its simplicity and clinical practicability [8,
25, 26], although achieving low intracerebral cell numbers [15,
27]. Therefore, local intracerebral injections are widely used,
which can enhance the number of transplanted intracerebral
cells but lack clinical utility due to invasive delivery procedures
[28–32]. Until recently, only two studies systematically com-
pared the effect of different NPC transplantation routes on post-
ischemic outcome. However, these studies are limited to a maxi-
mal observation period of 1 week and lack an analysis of
underlying cellular mechanisms [33, 34].
To elucidate consequences of NPC delivery strategies, we
herein submitted mice to cerebral ischemia and transplanted
NPCs 6 hours later either intravenously (systemically) or
intrastriatally (intracerebrally), followed by an analysis of
brain injury and remodeling for up to 8 weeks. In order to
promote survival of grafted cells, NPCs were protein-trans-
duced with the chaperone Hsp70, which reduces apoptosis
and inflammation after hypoxic-ischemic injury [23, 24].
Since cellular entry of Hsp70 is poor, transduction was
achieved in vitro using the membrane-permeable and neuro-
protective TAT-Hsp70 fusion protein before transplantation
[10, 35]. This study has explicit meaning for future cell-based
stroke therapies, since it highlights the importance of cell
transplantation routes, implying different mechanisms by
which transplanted NPCs induce poststroke neuroprotection.
MATERIALS AND METHODS
Animals and Experimental Groups
Experimental procedures were in accordance with the National
European Institutes of Health guidelines for the care and use of
laboratory animals and approved by local authorities. For all
experiments, male C57BL/6 mice (11–13 weeks, 23–27 g;
Charles River, Sulzfeld, Germany (www.criver.com) were used.
The total number of animals was 437, which were assigned to
eight treatment paradigms (Table 1). Treatment was performed in
a blinded manner. A schematic display for intracerebral transplan-
Transplantation of TAT-Hsp70-Transduced NPCs
Intrastriatal delivery
PBS NPCs NPCs TAT-HA NPCs TAT-Hsp70
14 14 14 14
14 14 14 14
7 7 7 7
8 8 9 8
11 11 11 11
tations and the in vivo experimental paradigm is given in Sup-
porting Information (Supporting Information Fig. S1). Survival
rates were 100% for all animals surviving 1, 4, 14, or 28 days for
each experimental condition, and the amount of animals used per
experimental group as described in Materials and Methods. The
survival rate of animals receiving systemic or intracerebral injec-
tions of phosphate buffered saline (PBS) was 78.6% (11/14) on
day 56 for both groups. Survival rate after intracerebral transplan-
tation of native NPCs, TAT-HA-transduced, or TAT-Hsp70-trans-
duced NPCs was 84.6% (11/13) for each group. For animals that
had received systemic delivery of TAT-Hsp70-transduced NPCs
survival was 91.6% (11/12), whereas for the remaining experi-
mental groups with a 56 days survival, the survival rate was
100%. Assessment of cell proliferation was performed on animals
surviving either 28 days or 56 days. These mice received 11 con-
secutive intraperitoneal (i.p.) injections of 5-bromo-2-desoxyuri-
dine (BrdU; 50 mg/kg b.wt.) on days 8–18.
Preparation of Recombinant Proteins
TAT-Hsp70 and TAT-hemagglutinin (TAT-HA), which is also
included in the TAT-Hsp70 construct serving as a negative con-
trol, were prepared under native conditions as described [36].
Briefly, the recombinant genes were expressed in Escherichia coli
strain BL21 (DE3) pLysS (Novagen, Madison, WI, www.
novagen.com) and proteins were isolated in 10 mM Tris, pH 10,
20% glycerol, 274 mM NaCl, 0.1% Pluronic, and 0.02% Tween
80. Bacterial debris was removed by centrifugation and the cell
extracts were purified by affinity chromatography using Ni-tris-
carboxymethyl-ethylene-diamine [37]. Protein was eluted by step-
wise addition of binding buffer containing increasing concentra-
tions of imidazole. The column eluate was purified from imidaz-
ole by gel filtration (Sephadex G-25 M, GE Healthcare Bio-
Sciences AB, Munich, Germany, www.gehealthcare.com).
According to this procedure, TAT-Hsp70 is highly stable after
cell transduction as no protein degradation could be detected 24
hours after protein application, suggesting that the intracellular
half-life time is at least a few days [36]. For transplantation of
NPCs, in vitro incubation period with either TAT-Hsp70 (250
nM) or TAT-HA (250 nM) was 4 hours.
Preparation and Cultivation of Adult SVZ-Derived
NPCs
NPCs were isolated from the SVZ of 19 6–8 weeks old male
nontransgenic C57BL/6 mice for in vitro experiments [19]. For in
vivo experiments, 346 transgenic green fluorescence protein posi-
tive (GFPþ) animals (C57BL/6-Tg ACTB-enhanced green fluo-
rescence protein (EGFP), 1Osb/J; JAX Laboratory, Bar Harbor,
Maine, www.jax.org; male, 6–8 weeks old) were used. GFP
expression was under control of the actin promoter, which allows
reliable and stable tracking of transplanted NPCs. The SVZ was
microdissected under stereomicroscopic control (Zeiss, Jena, Ger-
many, www.zeiss.de), minced into small pieces, and then
Doeppner, Ewert, T€onges et al.
mechanically triturated and dissociated into a single-cell suspen-
sion. Cells were cultured in serum-free basic Dulbecco’s modified
Eagle’s medium (DMEM)/F12 (PAA, Linz, Austria, www.paa.
com) supplemented with epidermal growth factor (EGF, 2 lg/
ml), basic fibroblast growth factor (bFGF, 2 lg/ml), and penicil-
lin-streptomycin (Invitrogen, Frankfurt, Germany, www.
invitrogen.com). Cells were incubated with 5% CO2 at 37
C. The
growth factors were supplemented every 2–3 days and cells were
passaged via accutase (Invitrogen) digestion for 30 minutes at
37
C with a resuspension every 10 minutes. Thereafter, cells
were centrifuged and resuspended in conditioned media. Neuro-
sphere passages were done every 7–10 days and cells used for
transplantation were derived from passage 4.
Quantitative Analyses of NPCs and Neurosphere
Differentiation Assay
For estimation of neurosphere numbers, 10,000 primary NPCs
(P0) and cells from passage 4 (P4) were plated in six-well plates
and cultured for 7 days with/without TAT-HA (250 nM) or TAT-
Hsp70 (250 nM). Thereafter, neurosphere numbers and neuro-
sphere diameters were quantified. Cells were then treated with
accutase in order to achieve single-cell suspensions followed by
determination of total cell numbers. For differentiation analysis,
NPCs from P0 and P4 that had been cultured for 7 days (native
or transduced with 250 nM TAT-HA or 250 nM TAT-Hsp70)
were gently triturated and plated onto 24-well plates (40 cells per
microliter) containing coverslips coated with 1 mg/ml poly(D-ly-
sine) (Sigma-Aldrich, Taufkirchen, Germany, www.sigmaaldrich.
com) according to modified protocols [38, 39]. Differentiation
medium consisted of DMEM/F12 supplemented with 2% B27
(Invitrogen), 1% fetal bovine serum, and penicillin-streptomycin.
For Western blot analysis, cells were lysed (50 mM Tris at pH
8.0, 150 mM NaCl, and Triton 1%) on day 7 postplating. Lysates
were centrifuged and supernatants were used for SDS-PAGE.
Equal amounts of protein (75 lg) were diluted in 6 sample
buffer, boiled and loaded onto 10% polyacrylamide gels. Proteins
were transferred onto polyvinylidene difluoride membranes,
which were immersed in blocking solution (5% dry milk powder
in TBS-T [0.1% Tween 20 þ Tris-buffered saline]; 1 hour at
room temperature [RT]). The following primary antibodies were
used: polyclonal rabbit antinestin (1:2,000), polyclonal rabbit
antiglial fibrillary acidic protein (GFAP; 1:10,000), polyclonal
rabbit anti-20 ,30 -cyclic nucleotide 30 -phosphodiesterase (CNPase;
1 lg/ml), and antibeta tubulin III (1 lg/ml; all obtained from
Abcam, Cambridge, UK, www.abcam.com). Membranes were
incubated with a peroxidase-coupled, goat anti-rabbit secondary
antibody (1:2,000; Abcam), washed several times, immersed in
enhanced chemiluminescence solution, and exposed to enhanced
chemiluminescence-Hyperfilm (Amersham Biosciences, Freiburg,
Germany, www.amershambiosciences.com).
Oxygen-Glucose-Deprivation of Cultured NPCs
Experimental procedures were performed as previously described
[19]. NPCs were passaged and 100,000 cells were first preincu-
bated for 4 hours with conditioned cell culture medium contain-
ing TAT-Hsp70 (250 nM). Then, the medium was substituted by
a glucose-free crystalloid solution (‘‘Thomajodin’’ plus 1 mM
mannitol; Deltapharm, Dortmund, Germany) containing TAT-
Hsp70 (250 nM). Cells were incubated in a hypoxic chamber
(1% O2; remainder 5% CO2 and 94% N2) for 45 minutes and
reincubated in normal glucose and TAT-Hsp70 (250 nM) contain-
ing cell culture medium for 24 hours. Cell viability was assessed
using a LIVE/DEAD-Viability/Cytotoxicity-Assay kit (Lonza,
Basel, Switzerland, www.lonza.com). TAT-HA (250 nM; nega-
tive control) and PBS served as controls for TAT-Hsp70. Con-
trols were kept under normal cell conditions without oxygen-glu-
cose-deprivation (OGD) for the duration of the experiment.
www.StemCells.com
1299
Induction of Transient Focal Cerebral Ischemia
Cerebral ischemia was induced using middle cerebral artery
(MCA) occlusion [19]. Animals were anesthetized (0.8%–1.5%
isofluran, 30% O2, and remainder N2O), and rectal temperature
was maintained at 36.5
C–37.0
C using a feedback-controlled
heating system under continuous control of blood flow changes
by means of a laser Doppler flow (LDF) system (Perimed, Jar-
falla, Sweden, www.perimed-instruments.com). Occlusion of the
left MCA was achieved using a 7-0 silicon-coated nylon monofil-
ament (180 lm tip diameter; Doccol, Redlands, CA, www.
doccol.com), which was withdrawn after 45 minutes to induce
transient ischemia. LDF recordings continued for additional 15
minutes to monitor appropriate reperfusion.
Transplantation of NPCs
Mice received either intravenous (systemic) or intrastriatal (intra-
cerebral) injections of NPCs or PBS 6 hours after induction of
stroke. Systemic injection of NPCs (106 cells in 100 ll PBS) or
PBS (100 ll) was performed via cannulation of the right femoral
vein. For intracerebral stereotactic injections of PBS (5 ll) or
NPCs (5  105 cells in 5 ll PBS), surgeries were performed in
mice that were anesthetized with ketamine (10 mg/kg) and
xylazine (25 mg/kg). Thereafter, animals were placed in a
stereotactic apparatus (Kopf Instruments, Tujunga, CA, www.
kopfinstruments.com) and fixed accordingly. The skull was
exposed, and a hole was drilled at the appropriate position on the
ischemic left hemisphere. Injections (0.4 mm anterior, 1.8 mm
lateral and 3.5 mm ventral from bregma) were performed using
10 ll Hamilton syringes (Bonaduz, Switzerland, www.hamilton-
company.com) with a rate of 1 ll/minute. The syringe was kept
in place for additional 5 minutes after injection before removal.
Analysis of Poststroke Brain Injury and
Immunohistochemistry
Infarct volumes were analyzed on day 4 (n ¼ 5 per group), for
which brains were removed and cut into slices of 2 mm each. Sli-
ces were stained with 2,3,5-triphenyltetrazolium chloride (2%),
and a computer-based analysis of infarct volumes was done using
the freely available software ImageJ (NIH; http://rsbweb.nih.gov/
ij) by subtracting the area of the nonlesioned ipsilateral hemi-
sphere from that of the contralateral side. Infarct volume sizes
were calculated by integration of the lesioned areas. Postischemic
brain edema was measured as the increase of ipsilateral hemi-
spheric volume in comparison to the contralateral hemisphere.
For immunohistochemical analysis, animals were i.p. injected
with chloralhydrate (420 mg/kg b.wt.) and transcardially perfused
with 4% paraformaldehyde at days 4 (n ¼ 5–6), 14 (n ¼ 7), 28
(n ¼ 8–9), and 56 (n ¼ 7). The brains were removed, shock-fro-
zen in liquid nitrogen, and 16-lm thick coronal cryostat sections
were prepared. Quantitative analyses for immunohistochemical
stainings were performed defining regions of interest (ROIs)
within the ischemic basal ganglia. Stereotactic coordinates were
0.14 mm anterior, 2.5–3.25 mm ventral, and 1.5–2.25 mm lateral
from bregma. Three sections per animal and ROI were used. For
quantitative analysis of proliferating BrdUþ cells, sections were
exposed to blocking solution and subsequently stained with a
monoclonal mouse anti-BrdU antibody (1:400; Roche, Mannheim,
Germany, www.roche.de) or a monoclonal rat anti-BrdU antibody
(1:400; Abcam). Since quenching of fluorescence signal of GFPþ
NPCs occurs during section processing, a polyclonal rabbit anti-
GFP antibody (1:2,500; Abcam) was used to enhance GFP signal
intensity. For differentiation analysis of GFPþ or BrdUþ cells, dou-
ble staining was performed against BrdU/GFP and a polyclonal
goat antidoublecortin antibody (1:50; Santa Cruz Biotechnology,
Heidelberg, Germany, www.scbt.com), a polyclonal rat anti-GFAP
antibody (1:500; Zymed, Germany), a monoclonal mouse anti-
CNPase antibody (1:400; Millipore, Abingdon, UK, www.
millipore.com), a monoclonal mouse anti-NeuN antibody (1:1,000;
Millipore), or a monoclonal mouse antinestin antibody (GFP
1300
counterstaining only; 1:500; Millipore). In order to exclude engulf-
ment of GFPþ-transplanted NPCs by microglia, double staining
against GFP and IB4 was done on day 4 using a rat biotin-conju-
gated anti-IB4 antibody (1:100; Vector, Peterborough, UK,
www.vectorlabs.com). All antibodies were incubated for 18 hours
at 4
C. Thereafter, the sections were incubated for 1 hour at RT.
For double staining with BrdU, the following secondary antibodies
were used: goat anti-mouse Cy-3 (1:400; Dianova, Hamburg, Ger-
many, www.dianova.com) or goat anti-rat Alexa 594 (1:400; Dia-
nova) for BrdU staining, goat anti-rat Alexa 488 (1:250; Invitro-
gen, Germany) or donkey anti-goat Alexa 488 (1:250; Invitrogen)
for GFAP or doublecortin (Dcx) staining, goat anti-mouse Alexa
488 (1:100; Jackson ImmunoResearch, Newmarket, UK, www.
jireurope.com) for CNPase staining, and goat anti-mouse Alexa
488 (1:400; Invitrogen) for NeuN staining. For double staining
with GFP, the secondary antibodies were as follows: goat anti-
mouse Cy-3 (1:100; Jackson ImmunoResearch) for NeuN, CNPase
and nestin staining as well as goat anti-rat Cy-3 antibody (1:200,
Abcam) for GFAP staining. Double staining against Dcx was done
using a donkey anti-goat Cy-3 secondary antibody (1:500; Dia-
nova). Photos for differentiation analysis with subsequent three-
dimensional reconstruction were made using a Zeiss microscope
equipped with an Apotome and the corresponding AxioVision
software.
For assessment of brain injury on day 4, terminal deoxynucleo-
tidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)
was done. The staining was performed incubating the sections with
proteinase K (7 minutes at 37
C) followed by exposure to the TdT
enzyme reaction according to the manufacturer’s manual (Roche).
Sections were stained with a streptavidin-Alexa-488-conjugated
secondary antibody (2 hours at RT; Invitrogen) and analyzed. In
order to assess viability of BrdUþ cells, double staining using the
rat anti-BrdU antibody and the TUNEL-protocol was performed.
Further analysis of brain injury on days 4, 28, and 56 was per-
formed by determination of neuronal density, that is, after quantita-
tive analysis of NeuNþ cells within defined ROIs as stated above.
Cell numbers for each specific staining were recalculated and are
given as total amount of cells per square millimeter.
Assessment of glial scar surrounding the ischemic territory
was measured in four defined ROIs after GFAP-staining using a
modified protocol as previously described [8]. Images were
obtained from the indicated ROIs, and the gliotic area was meas-
ured for each ROI using Image J software. Data are given as
mean area (mm2) out of four ROIs.
Assessment of Poststroke Functional Recovery
Motor coordination deficits were analyzed using the rota rod,
tight rope, and the corner turn test. All behavioral tests were per-
formed on the same animals, which were also used for immuno-
histochemical analysis on day 56. Behavioral tests were per-
formed in a blinded manner. One day before induction of stroke,
animals were trained before the beginning of the actual tests on
day 7, 14, 28, and 56. Both rota rod and tight rope test were per-
formed as previously described [40]. Using the rota rod test, ani-
mals were put on an accelerating treadmill (TSE Systems, Bad
Homburg, Germany, www.tse-systems.com; 3-cm diameter) with
an accelerating speed of 4–40 rpm. The maximum speed was
achieved after 260 seconds, and maximum testing time was 300
seconds. The time until animals dropped was registered and stat-
istically analyzed. For the tight rope test, animals were placed on
a 60-cm long rope grasping the string with their forepaws. Maxi-
mum test time was 60 seconds, and results were scored from 0
(minimum) to 20 (maximum) according to a validated score [40],
depending on the time animals spent on the rope and whether or
not they reached the platform. Rota rod and tight rope tests were
performed twice at each time point and means were calculated.
For the corner turn test, two vertical boards were attached at one
side with an angle of 30
, and each mouse was tested for the side
chosen over 10 trials per test day. Whereas healthy animals leave
the corner without side preference, mice suffering from stroke
Transplantation of TAT-Hsp70-Transduced NPCs
preferentially turn to the left, nonimpaired body side [41, 42].
The laterality index was calculated according to the following
formula: (number of left turns  number of right turns)/10.
Zymography of Matrix Metalloproteases
Left ischemic hemispheres (n ¼ 4 per condition; 24 hours post-
stroke) were homogenized in cold lysis buffer (basic buffer) con-
taining 50 mmol/l Tris-HCl (pH 7.6), 150 mmol/l NaCl, 5 mmol/l
CaCl2, 0.05% BRIJ-35, 0.02% NaN3, and 1% Triton X-100. Ho-
mogenates were centrifuged at 4
C at 12,000g for 5 minutes and
supernatants were incubated with a 1:10 volume of gelatine-
Sepharose 4B for 1 hour at 4
C. After centrifugation, pellets
were resuspended in elution buffer (basic buffer containing 10%
dimethyl sulfoxide and 20% volume of lysis buffer); purified
samples were analyzed by zymography. Protein concentrations
were determined by the bicinchoninic acid method (BCA kit,
Thermo Scientific, Karlsruhe, Germany, www.thermoscientific.
com). Separation of matrix metalloprotease (MMP)-2 and MMP-9
as pro-form and active form was performed using Novex Zymo-
gram Gels (Invitrogen) according to the manufacturer’s instruc-
tions. Samples were incubated in nonreducing sample buffer (0.4
mol/l Tris, pH 6.8, 5% SDS, 20% glycerol, and 0.05% bromphe-
nol blue) for 10 minutes at RT and then loaded onto 10% SDS
polyacrylamide electrophoresis gels containing 0.1% gelatin. Af-
ter electrophoresis, samples were incubated with 2.5% Triton X-
100 twice for 20 minutes, equilibrated with developing buffer
(Invitrogen), and incubated for 18 hours at 37
C. Gels were
stained with Coomassie Blue for 30 minutes and destained in
washing solution (30% methanol and 10% acetic acid). White
bands on a dark background indicated zones of digestion corre-
sponding to the presence of pro-MMPs and activated MMPs on
the basis of their molecular weight. As standards, 0.1 ng of
human pro-MMP-9 and human pro-MMP-2 (Merck Biosciences,
Darmstadt, Germany, www.merckgroup.com) and 0.01 ng of acti-
vated MMP-9 and activated MMP-2 (Merck Biosciences) were
used. Gels were scanned and densitometrically analyzed.
Analysis of Blood-Brain-Barrier Permeability
Mice (n ¼ 6 per condition) received intravenous bolus injections
of 2% Evans blue dye (2 ml/kg b.wt.) via tail vein cannulation
22 hours poststroke [43]. Two hours later, animals were sacrificed
by transcardiac perfusion with PBS. Left hemispheres from nonis-
chemic animals that had received no treatment served as refer-
ence for extravasal Evans blue dye contents. Brains were
removed and separated into hemispheres. Left (ischemic) hemi-
spheres were weighed, homogenized in 2 ml of 50% trichloroace-
tic acid, and centrifuged at 10,000 rpm for 20 minutes. The
extracted Evans blue dye was further diluted with ethanol, and
the fluorescence signal was measured with a luminescence spec-
trophotometer (exc. ¼ 620 nm, em. ¼ 680 nm). An external
standard (62.5–500 ng/ml) was used for calculation of Evans blue
dye contents, which is given as (lg) Evans blue dye per (g)
tissue.
Determination of Thiobarbituric Acid-Reactive
Substances
Oxidative stress was assessed 24 hours poststroke in brain ho-
mogenates from left ischemic hemispheres (n ¼ 4 per condition),
which were obtained using lysis buffer as described above. For-
mation of reactive oxygen species (ROS) leads to peroxidation of
fatty acids of phospholipids contained within the cell membrane.
During peroxidation, thiobarbituric acid (TBA) reactive substan-
ces (TBARS) are generated such as malondialdehyde (MDA).
MDA reacts with TBA resulting in a chromogenic compound
whose absorption is photometrically measured at k ¼ 532 nm
[44]. Samples were incubated with cold 80% trichloroacetate so-
lution (volume ratio of 5:1) and centrifuged at 7,500g for 5
minutes. Supernatants were incubated with 1% TBA solution (pH
7.0; volume ratio of 2:1) at 95
C for 10 minutes followed by
additional centrifugation at 5,000g and subsequent photometric
Doeppner, Ewert, T€onges et al.
analysis. The extent of TBARS formation is expressed as MDA
equivalents using 1,1,3,3-tetramethoxypropan as standard.
ELISA for Measurement of Growth Factors
For ELISA experiments (n ¼ 4), samples were obtained on days
4 and 56 as described for measurement of TBARS. Levels of
growth factors such as vascular endothelial growth factor (VEGF;
R&D Systems, Minneapolis, MN, www.rndsystems.com), nerve
growth factor (Promega, Mannheim, Germany, www.promega.-
com), brain-derived neurotrophic factor (BDNF; Promega), glial
cell line-derived neurotrophic factor (GDNF; Promega), bFGF
(R&D Systems), and EGF (R&D Systems, Minneapolis, MN,
www.rndsystems.com) were measured using commercial mouse
ELISA kits according to the manufacturer’s instructions.
Statistics
All data are given as mean 6 SD. For comparison between two
groups, the Student’s t test was used, whereas for comparison
between multiple groups, a one-way analysis of variance followed
by the Tukey’s post hoc test was performed. A p value of <.05
was considered to be statistically significant.
RESULTS
TAT-Hsp70 Does Not Affect NPC Proliferation and
Differentiation but Protects from Hypoxic-Hypogly-
cemic Cell Injury
Protein delivery using TAT technology is highly efficient and
has been successfully tested on various cell types, with often
100% transduction rates in cultivated cells [45]. Previously,
treatment of neuroblastoma cells with TAT-Hsp70 (250 nM)
achieved at least fourfold higher intracellular levels of TAT-
Hsp70 compared to endogenous Hsp70. In this study, we
again detected TAT-Hsp70 in cellular lysates after treatment
with recombinant protein (Supporting Information Materials
and Methods; Supporting Information Fig. S2). Thereafter,
effects of both the recombinant protein and cell passaging on
NPC proliferation, differentiation, and susceptibility to OGD
were analyzed in vitro before cell transplantation.
Neurosphere formation rates were independent of both
TAT-Hsp70 and cell passaging (Fig. 1A–1C). Likewise, neu-
rosphere diameters did not significantly differ between treat-
ment groups. As for P0, mean neurosphere diameters were
89.9 6 27.2 (native NPCs), 107.3 6 34.1 (NPCs þ TAT-
HA), and 98.5 6 24.7 nm (NPCs þ TAT-Hsp70). In P4,
mean diameters were 93.6 6 19.4 (native NPCs), 96.9 6
27.8 (NPCs þ TAT-HA), and 101.3 6 29.1 nm (NPCs þ
TAT-Hsp70). Furthermore, total cell numbers did not signifi-
cantly differ between nontreated and transduced NPCs from
both passages (Fig. 1D).
Assessment of NPC differentiation (Fig. 1E, 1F) revealed
high protein abundance of the neural stem/progenitor cell
marker nestin and the astroglial marker GFAP, whereas pro-
tein abundance of the neuronal marker b-tubulin III and the
oligodendroglial marker CNPase was low. However, no dif-
ference between the experimental groups was observed, that
is, neither cell passaging nor TAT transduction affected cell
differentiation.
TAT-Hsp70-mediated neuroprotection was analyzed in
NPCs from P0 and P4 that were exposed to a 45-minute
OGD with subsequent recultivation under standard cell culture
conditions (Fig. 1G). Whereas TAT-Hsp70-transduced NPCs
from passage P0/P4 showed little cell injury, less than 30% of
native and TAT-HA-transduced NPCs survived OGD injury.
www.StemCells.com
1301
TAT-Hsp70 Enhances Numbers of Transplanted
NPCs After Intracerebral Injection Without Affect-
ing Cell Differentiation
Native or transduced GFPþ NPCs were transplanted systemi-
cally or intracerebrally 6 hours after stroke (Fig. 2). Cells
were treated ex vivo before transplantation with either TAT-
HA or TAT-Hsp70. Intracerebral transplantation within the is-
chemic striatum resulted in high numbers of GFPþ NPCs
under each condition (Fig. 2A). Although the number of
grafted NPCs gradually declined over time in all experimental
groups, animals that had received TAT-Hsp70-transduced
NPCs always showed significantly more transplanted cells
(Fig. 2A). The latter typically formed agglomerations at the
site of transplantation without significant migration (Fig. 2C–
2I). On the contrary, systemic transplantation of NPCs
(native/transduced) yielded lower numbers of NPCs under
each experimental condition as compared to intracerebral cell
delivery (Fig. 2B). Likewise, the number of transplanted cells
gradually decreased over time. Transduction of NPCs with
TAT-Hsp70, however, did not significantly increase the num-
ber of transplanted cells after systemic injection within the is-
chemic hemisphere.
Differentiation states of transplanted NPCs (Supporting In-
formation Fig. S3) were not affected by TAT-Hsp70 on days 4
and 56 (Fig. 2J, 2K). No colocalization between GFP and the
mature neuronal marker NeuN was observed. Noteworthy, coun-
terstaining between GFP and the microglial (and also monocyte)
marker IB4 [46] revealed very low rates of colocalizations on
day 4 after stroke (Fig. 2J), suggesting that transplanted NPCs
are not significantly engulfed by activated microglia.
NPC-Mediated Poststroke Neuroprotection
Infarct volume analysis revealed that both intracerebral and
systemic transplantation of NPCs yielded significant neuropro-
tection on day 4 poststroke, which was independent of NPC
transduction states (Fig. 3A–3C). Likewise, edema formation
was significantly decreased in animals that had received either
intracerebral or systemic transplantation of native or trans-
duced NPCs (Fig. 3D). In line with this, analysis of neuronal
density and TUNELþ cells on day 4 yielded increased neuro-
nal density and reduced numbers of TUNELþ cells after ei-
ther intracerebral or systemic injection of native/transduced
NPCs (Fig. 3E, 3F). Assessment of long-term neuroprotection
after intracerebral transplantation, however, revealed that only
mice receiving TAT-Hsp70-transduced NPCs showed a signif-
icantly increased neuronal density (Fig. 3G, 3H). On the other
hand, sustained neuroprotection after systemic transplantation
of NPCs was observed in animals that had received either
native or transduced NPCs. These data suggest that sustained
neuroprotection by intracerebral transplantation of NPCs is
only achieved via in vitro transduction with TAT-Hsp70,
whereas systemic transplantation of NPCs induces long-term
neuroprotection independent of cell transduction.
Transplantation of NPCs Reduces Poststroke Neuro-
logical Impairment
Neurological deficits in mice were assessed using the rota rod
(Fig. 4A, 4B), the tight rope (Fig. 4C, 4D), and the corner
turn test (Fig. 4E, 4F). Although intracerebral transplantation
of either native or transduced NPCs reduced functional
impairment in all behavioral tests as early as day 7, only ani-
mals that were treated with TAT-Hsp70-transduced NPCs
showed sustained reduced functional deficits. However, sys-
temic injection of either native or transduced NPCs resulted
in sustained improved functional outcome in these animals as
compared to PBS controls.
1302 Transplantation of TAT-Hsp70-Transduced NPCs

Figure 1. In vitro characterization of cultivated adult NPCs. (A–C): Neurosphere formation rates were analyzed in both primary passage (P0)
cells (i.e., 7 days after cell culturing) and in NPCs from passage 4 (P4). Typical examples of neurosphere formation in P0 and in P4 are depicted
in (A) and (B), respectively. (D): Neurospheres used for experiments described in (A–C) were dissociated by means of accutase followed by
determination of total NPC numbers within each six wells. (E, F): Cell differentiation was analyzed using Western blotting against nestin,
CNPase, GFAP, and b-tubulin III in P0 and P4 neurospheres. (F): Data from (E) are presented as arbitrary units after densitometric analysis. (G):
NPCs (P0 and P4) were exposed to 45 minutes of oxygen-glucose-deprivation (OGD) and recultivated for 24 hours at standard cell culture condi-
tions followed by subsequent analysis of cell injury. Controls were incubated under standard cell culture conditions without OGD. Scale bars ¼
100 lm. *Significantly different from controls, p < .05. Abbreviations: CNPase, 20 ,30 -cyclic nucleotide 30 -phosphodiesterase; GFAP, glial fibril-
lary acidic protein; NPC, neural precursor cell; TAT-HA, TAT-hemagglutinin.

Neurogenesis Is Enhanced in Mice Stereotactically
Grafted with TAT-Hsp70-Transduced NPCs
Cerebral ischemia was associated with detection of enhanced
numbers of proliferating BrdUþ cells for up to 8 weeks within
the ischemic hemisphere of each experimental group (Fig.
5A, 5B). Noteworthy, colocalizations of proliferating BrdUþ
cells with TUNELþ cells on day 4 were less than 3% for
each experimental condition (representative image in Fig.
5C), suggesting that BrdUþ cells do survive initially. BrdUþ
cells were typically located in the SVZ and scattered in the is-
chemic striatum (Fig. 5D–5N). Systemic transplantation of
native or transduced NPCs did not alter the amount of BrdUþ
cells within the ischemic hemisphere (Fig. 5A, 5B). However,
intracerebral transplantation of TAT-Hsp70-transduced NPCs
resulted in a sustained postischemic increase of BrdUþ cells
Doeppner, Ewert, T€onges et al. 1303

Figure 2. TAT-Hsp70 increases numbers of grafted NPCs after intracerebral transplantation without affecting cell differentiation. NPCs were
transplanted 6 hours after stroke via stereotactic injection of 500,000 GFPþ cells into the ischemic striatum (intracerebral injection; A) or via in-
travenous injection of 106 cells into the femoral vein (systemic injection; B). Photographs depicted represent typical orientations of TAT-Hsp70-
transduced NPCs after intracerebral injection on day 56 in low magnification (C–E; green ¼ GFP and blue ¼ DAPI; scale bar ¼ 50 lm) and
high magnification (F–H; scale bar ¼ 20 lm). (I): Three-dimensional reconstruction from merged photo (H). (J, K): Differentiation profile of
transplanted GFPþ NPCs on day 4 (J) and day 56 (K) after stroke. Note that GFP/IB4 staining was only performed on day 4. Colocalization of
GFPþ cells with NeuN was not observed at any time point analyzed. *Significantly different from animals treated with native or TAT-HA-trans-
duced NPCs, p < .05. Abbreviations: CNPase, 20 ,30 -cyclic nucleotide 30 -phosphodiesterase; GFAP, glial fibrillary acidic protein; GFP, green fluo-
rescence protein; NPC, neural precursor cell; TAT-HA, TAT-hemagglutinin; DAPI, 4’,6-diamidino-2-phenylindole.

(Fig. 5A, 5B). The latter might be a consequence of either
enhanced stimulation of postischemic cell proliferation or
enhanced survival of proliferating cells due to intracerebrally
grafted TAT-Hsp70-transduced NPCs. In line with this, a dif-
ferentiation analysis of BrdUþ cells (Fig. 5O–5Z) revealed
that only intracerebral transplantation of TAT-Hsp70-trans-
duced NPCs yielded enhanced colocalizations with the imma-
ture neuronal marker Dcx and the mature neuronal marker
NeuN. On the other hand, systemic transplantation of trans-
www.StemCells.com
duced NPCs did not significantly affect neuronal differentia-
tion of proliferating cells.
NPC-Induced Poststroke Mechanisms Vary with
Transplantation Routes
We next analyzed putative mechanisms underlying NPC-
mediated neuroprotection after intracerebral and systemic
transplantation. Among different events, which contribute to
early ischemic injury, MMP-induced breakdown of the blood-
1304 Transplantation of TAT-Hsp70-Transduced NPCs

Figure 3. Analysis of brain injury after cerebral ischemia. Infarct volume analysis after occlusion of the left middle cerebral artery was performed
on day 4 after intracerebral (A, C) or systemic (B, C) injection of NPCs or PBS, respectively. Likewise, edema formation (D) and number of
TUNELþ cells within the lesion site (E) were analyzed on day 4 after intracerebral and systemic injection protocols. Neuronal density, that is, the
number of NeuNþ cells within the ischemic lesion site, was determined on day 4 (F), day 28 (G), and on day 56 (H). *Significantly different from
PBS control mice, p < .05. #Significantly different from mice treated with intracerebral injections of native NPCs or TAT-HA-transduced NPCs,
p < .05. Abbreviations: NPC, neural precursor cell; PBS, phosphate buffered saline; TAT-HA, TAT-hemagglutinin; TUNEL, dUTP nick end labeling.

brain-barrier (BBB) is one decisive key factor [47]. As such, nor TAT-Hsp70-transduced NPCs affected MMP-9 activity,
analysis of MMP gel zymography at 24 hours poststroke (Fig. whereas systemic injection of NPCs (native/transduced) yielded
6A, 6B) revealed a detection of MMP-9 activity in control ani- decreased MMP-9 activity. MMP-2 activity was below detec-
mals. However, intracerebral transplantation of neither native tion threshold under all experimental paradigms (Fig. 6A).
Doeppner, Ewert, T€onges et al. 1305

Figure 4. NPC-mediated functional recovery after cerebral ischemia. Postischemic functional deficits were analyzed for up to 8 weeks after trans-
plantation of NPCs or injection of PBS, respectively. Data are given for animals that received either intracerebral (A, C, E) or systemic (B, D, F)
injections. Motor coordination was analyzed using the rota rod (A, B), the tight rope (C, D), and the corner turn test (E, F). Using the rota rod test,
maximal test time was 300 seconds, whereas for the tight rope test, a validated score between 0 (min) and 20 (max) was defined. For the corner turn
test, the laterality index (LI) was defined as (number of left turns  number of right turns)/10. Whereas healthy animals (not depicted here) show
random side preference (i.e., a LI of approximately 0.5), mice suffering from stroke leave the corner toward the nonimpaired left side of the body.
*Significantly different from PBS animals, p < .05. #Significantly different from mice treated with intracerebral injections of native NPCs or
TAT-HA-transduced NPCs, p < .05. Abbreviations: NPC, neural precursor cell; PBS, phosphate buffered saline; TAT-HA, TAT-hemagglutinin.

Further analysis revealed that BBB integrity was significantly
enhanced after systemic application of native/transduced NPCs
at 24 hours poststroke (Fig. 6C). Moreover, measurement of
TBARS formation at 24 hours showed fewer TBARS in mice
systemically treated with native/transduced NPCs (Fig. 6D). In-
tracerebral injection of NPCs had, however, no effect on TBARS
formation. Analysis of long-term effects like astroglial scar for-
mation revealed that scar formation was independent from trans-
duction states of systemically transplanted cells; both native and
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transduced NPCs reduced glial scar formation (Fig. 6E). On the
contrary, only TAT-Hsp70-transduced NPCs mediated reduced
glial scar formation after intracerebral transplantation.
Since transplanted NPCs might affect sustained changes
within the ischemic lesion site via by-stander effects, we
analyzed intracerebral contents of growth factors on day 4
(Fig. 6F) and on day 56 (Fig. 6G). Whereas systemic transplan-
tation of native/transduced NPCs did not significantly affect
growth factor contents, intracerebral transplantation of NPCs
1306 Transplantation of TAT-Hsp70-Transduced NPCs

Figure 5. Analysis of postischemic cell proliferation and differentiation. Poststroke cell proliferation was analyzed using BrdU staining on day
28 (A) and on day 56 (B). (C): Double staining of BrdUþ cells (red) with TUNELþ cells (green) within the ischemic striatum on day 4 as shown
exemplarily from animals treated with intracerebral transplantation of TAT-Hsp70-transduced NPCs (scale bar ¼ 20 lm). (D–G): Typical local-
ization of BrdUþ cells (red) with DAPI signal (blue) on day 56 within the SVZ (arrows; scale bar ¼ 10 lm) including three-dimensional (3D)
reconstruction (G). Representative image after BrdU staining (red) plus DAPI signal (blue) taken from the aforementioned animal within the is-
chemic striatum both in low (H–J; scale bar ¼ 50 lm) and high magnification (K–M; scale bar ¼ 20 lm). (N): 3D reconstruction from merged
photo (M). Differentiation analysis of BrdUþ cells (O–Z) was performed using staining against Dcx (Q–U) and NeuN (V–Z) with subsequent
quantitative analysis within the SVZ and the ischemic striatum followed by a calculation of total cell numbers. Closed columns from (O–P) represent
intracerebral injections, whereas open columns reflect systemic injections. Stainings include BrdU signal (red), DAPI (blue), and Dcx or NeuN (green)
signal with subsequent 3D-reconstruction of merged photos (U and Z). Data are given as percentages of colocalizations of BrdU and the specific marker
referred to total numbers of BrdUþ cells. Scale bars ¼ 20 lm. *Significantly different from mice treated with PBS, native NPCs, or TAT-HA-trans-
duced NPCs, p < .05. Abbreviations: BrdU, 5-bromo-2-desoxyuridine; NPC, neural precursor cell; PBS, phosphate buffered saline; SVZ, subventricular
zone; TAT-HA, TAT-hemagglutinin.

(native/transduced) increased contents of BDNF, VEGF, and

GDNF on day 4. No significant difference between the various
intracerebral treatment groups was observed at that time point.
On the contrary, growth factor levels on day 56 were signifi-
cantly increased only in mice treated with intracerebral trans-
plantation of TAT-Hsp70-transduced NPCs. These results sug-
gest that NPC-mediated beneficial effects against stroke depend
on the transplantation route chosen.
DISCUSSION
The efficacy of stem and progenitor cell-based therapies is
well established and has already lead to clinical trials [13, 28,
29, 31, 48–50]. In this context, NPCs that can differentiate
into both glia and neurons might be advantageous, albeit neu-
ral cell replacement seems to be not a prerequisite for cell-
Doeppner, Ewert, T€onges et al. 1307

Figure 6. NPC-induced neuroprotective mechanisms vary with transplantation routes. (A): Gelatin zymography of brain homogenates at 24 hours after
induction of cerebral ischemia. Human pro-MMPs (0.1 ng) and activated MMPs (0.01 ng) served as standards. (B): Densitometric analysis of activated
MMP-9 from gelatin zymography at 24 hours poststroke. (C): Assessment of blood-brain-barrier permeability was assessed 24 hours poststroke by means of
Evans blue dye extravasation. Nonischemic animals (‘‘No ischemia’’) that had received no treatment served as reference. (D): Oxidative stress was indirectly
measured determining the amount of TBARS in brain homogenates of left ischemic hemispheres at 24 hours after induction of stroke. (E): Glial scar forma-
tion was analyzed using GFAP-staining on day 56 after stroke. (F, G): Determination of selected growth factors on day 4 and day 56 after stroke. Brain
homogenates from left ischemic hemispheres were prepared for measurement of BNDF, EGF, FGF, GDNF, NGF, and VEGF by means of ELISA. Closed
columns represent data after intracerebral injections, whereas open columns show data for systemic injections. Data are presented as means 6 SD. #Signifi-
cantly different from mice treated with systemic injections of any kind as well as from mice treated with intracerebral injection of PBS, p < .05. *Significantly
different from mice treated with intracerebral injections of PBS, native NPCs, or TAT-HA-transduced NPCs as well as from mice receiving any kind of
systemic injection, p < .05. Abbreviations: BDNF, brain-derived neurotrophic factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; GDNF,
glial cell line-derived neurotrophic factor; MMP, matrix metalloprotease; NGF, nerve growth factor; NPC, neural precursor cell; PBS, phosphate buffered
saline; SVZ, subventricular zone; TAT-HA, TAT-hemagglutinin; TBARS, thiobarbituric acid-reactive substances; VEGF, vascular endothelial growth factor.

www.StemCells.com
1308
based stroke therapy [20]. Nevertheless, therapeutic
approaches are limited due to low survival of transplanted
cells. We therefore transduced adult NPCs in vitro with the
chaperone TAT-Hsp70 in order to enhance resistance of
grafted cells within the postischemic milieu. Bearing in mind
that the best cell delivery route still remains unknown, NPCs
were injected either systemically or intracerebrally. Our data
show that sustained neuroprotection after intracerebral trans-
plantation of NPCs correlates with increased survival of
grafted cells due to TAT-Hsp70 transduction. On the other
hand, systemic transplantation of NPCs mediates acute neuro-
protection via different mechanisms, which may include stabi-
lization of the BBB and reduction of ROS despite low intra-
cerebral numbers of grafted cells.
Cell-penetrating peptides like TAT-Hsp70 are an elegant
tool to deliver cargo across intact biological membranes [51].
In this context, we have previously shown that in vitro trans-
duction of NPCs with TAT-Bcl-xL is efficient and results in
sustained neuroprotection against stroke after intracerebral
transplantation of TAT-Bcl-xL-transduced NPCs [19].
Although Hsp70-induced neuroprotection has been shown
before [10, 52–54], this study shows for the first time that the
fusion protein TAT-Hsp70 enhances resistance of NPCs
against hypoxic-hypoglycemic injury. Since neuroprotection
by the TAT domain itself has been described in vitro recently
[55, 56], we used TAT-HA as a negative control in order to
exclude effects of the TAT domain itself on cell viability,
neurosphere formation rates, NPC numbers, and differentia-
tion patterns of NPCs.
Although the number of transplanted NPCs gradually
declined under each experimental condition, intracerebral cell
delivery always yielded higher intracerebral NPC numbers
than systemic cell delivery. However, intracerebral transplan-
tation of only TAT-Hsp70-transduced NPCs resulted in signif-
icantly enhanced cell numbers within the ischemic striatum
for up to 8 weeks when compared with controls. Despite the
fact that transplanted NPCs were not exposed to cerebral is-
chemia itself, secondary cell death of transplanted NPCs in
the process of a proinflammatory and proapoptotic ischemic
milieu has already been described before [18, 22]. Therefore,
enhanced numbers of grafted NPCs after TAT-Hsp70 trans-
duction are most likely due to the antiapoptotic properties of
the fusion protein itself.
Analysis of brain injury and functional impairment after
intracerebral transplantation of NPCs correlated with the
amount of grafted cells within the peri-infarct area. As such,
only animals that had been transplanted with TAT-Hsp70-
transduced NPCs showed sustained neuroprotection that was
associated with improved motor coordination. On the other
hand, systemic delivery of native and transduced NPCs signif-
icantly reduced brain injury and motor coordination deficits at
any time point analyzed, albeit only a small number of scat-
tered NPCs were found within the ischemic hemisphere.
Although stem cell transplantation extends the therapeutic
time window as compared to thrombolysis, the optimal time
point for transplantation is still elusive and depends on the
focus of the therapeutic aim. If reduction of brain injury and
infarct volume is to be achieved, acute cell delivery of cells
might be most critical [20]. Consequently, we have chosen a
6-hour time window for this study. On the other hand, manip-
ulation of endogenous repair mechanisms such as neuroplas-
ticity might allow cell transplantation at subacute or even
later time points [8, 29, 57, 58]. In this context, different cell
delivery routes also affect the time window for transplanta-
tion. Whereas systemic transplantation of NPCs could benefit
from cell homing via inflammation, the latter might induce
cell death after local cell transplantation pointing toward dif-
Transplantation of TAT-Hsp70-Transduced NPCs
ferent mechanisms by which systemic and intracerebral trans-
plantation of NPCs reduce postischemic brain injury.
Systematic studies on NPC-induced neuroprotection
against cerebral ischemia after both systemic and local trans-
plantation do not exist apart from the aforementioned works
[33, 34]. Since our study indicates that systemic transplanta-
tion of native and transduced NPCs resulted in sustained neu-
roprotection despite low intracerebral numbers of grafted
cells, NPC-induced modulation of extracerebral injurious
mechanisms might be intriguing. Among mechanisms leading
to acute poststroke brain injury, breakdown of the BBB is one
key factor. This study shows that systemic transplantation of
NPCs induces a reduced formation of ROS during reperfusion
and a reduction of BBB leakage. Along with this, activation
of MMP-9, which is critically involved in BBB breakdown
[47], was significantly reduced in animals that had received
either native or TAT-transduced NPCs. Nevertheless, one has
to keep in mind that a 45-minute stroke induces relatively
small infarct sizes that affect both the striatum and parts of
the dorsolateral cortex resulting in rather mild breakdown of
the BBB [59].
Intracerebral transplantation of NPCs did not affect BBB
leakage or formation of ROS as compared to systemic trans-
plantation of NPCs. Rather, transplantation of TAT-Hsp70-
transduced NPCs resulted in sustained enhancement of post-
stroke neurogenesis as suggested by expression of the neuro-
nal markers Dcx and NeuN in BrdUþ-proliferating cells.
However, TAT-Hsp70 itself does neither induce neuronal
differentiation of SVZ-derived NPCs in vitro nor affect dif-
ferentiation of grafted cells in vivo. Thus, enhanced post-
stroke neurogenesis as observed by differentiation analysis
of BrdUþ cells is most likely due to the antiapoptotic prop-
erties of the fusion protein itself. In other words, TAT-
Hsp70 protects intracerebrally transplanted NPCs from sec-
ondary cell death resulting in sustained neuroprotection.
Taken into account that general mature neuronal differentia-
tion rates of BrdUþ cells after intracerebral transplantation
of TAT-Hsp70-transduced NPCs were low with no mature
neuronal phenotype of exogenous NPCs observable, neuronal
cell replacement or integration of grafted cells within the
neural network is not likely. Rather, indirect by-stander
effects orchestrating neurorestorative responses of the ische-
mic milieu might be responsible for NPC-induced neuropro-
tection after intracerebral transplantation. As such, intracere-
bral transplantation of TAT-Hsp70-transduced NPCs was
associated with increased levels of growth factors after 8
weeks poststroke, possibly being critically involved in NPC-
mediated long-term neuroprotection and stimulation of en-
dogenous neurogenesis as observed by enhanced numbers of
new-born mature neuronal cells. Enhanced secretion of
growth factors is also likely to be involved in mediation of
subacute neuroprotection after intracerebral transplantation
of NPCs, where growth factor levels were always signifi-
cantly increased independent of transduction states.
Increased growth factor levels on day 4 after intracerebral
transplantation correlated with intracerebral cell numbers,
which were at that time point still high under each experi-
mental condition. On the contrary, reduction of growth fac-
tor contents after intracerebral transplantation of native or
TAT-HA-transduced NPCs 8 weeks poststroke is likely due
to secondary cell loss of these nontransduced NPCs. There-
fore, it is well conceivable that intracerebrally grafted NPCs
act via indirect by-stander mechanisms as discussed by us
and others [7, 8, 17, 19, 20], for which sufficiently high
numbers of residing grafted cells as induced by TAT-Hsp70
are a prerequisite. Nevertheless, a causal relation between
growth factor secretion and NPC-mediated neuroprotection
Doeppner, Ewert, T€onges et al.
still has to be established. On the other hand, systemic trans-
plantation of NPCs mediates acute neuroprotection from the
luminal side of the vessels for which high intracerebral cell
numbers seem to be not necessary. This observation is in
line with previous reports where systemic transplantation of
stem and precursor cells initiated beneficial effects despite
low cell numbers or even no detectable cells within the is-
chemic brain [60–63].
CONCLUSIONS
This work shows that TAT-Hsp70 successfully enhances sur-
vival of NPCs after intracerebral transplantation culminating in
long-term neuroprotection against cerebral ischemia. Although
cell dosages for intracerebral transplantation did not vary
within this study, it is intriguing that the effects seen after in-
tracerebral transplantation depend on high intracerebral num-
bers of grafted cells. On the other hand, systemic NPC delivery
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www.StemCells.com
1309
initiates sustained neuroprotection despite low intracerebral
numbers of grafted cells via different mechanisms, like stabili-
zation of the BBB and reduction of ROS during early reperfu-
sion. Therefore, systemic NPC delivery might be more feasible
for clinical stroke concepts because of its simplicity and due to
lower intracerebral cell numbers needed, lacking a need of in
vitro manipulation of NPCs such as TAT-Hsp70 transduction.
Our study points out the need of a more adequate recognition
of NPC actions related to cell delivery routes.
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
G.P.H.D. is currently affiliated with the Department 851,
Neurodegeneration II, H. Lundbeck A/S, Valby, Denmark. No
conflict of interest results from this employment. The remaining
authors have nothing to disclose and also have no conflict of
interest.
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