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1
Received March 3, 2012; final acceptance June 20, 2012. Online prepub date: October 3, 2012.
1These authors provided equal contribution to this work.
Address correspondence to Jihwan Song, D.Phil., CHA Stem Cell Institute, Department of Biomedical Science, CHA University,
606-16 Yeoksam 1-dong, Kangnam-gu, Seoul 135-081, Korea. Tel: +82 2 3468 3393; Fax: +82 2 538 4102; E-mail: jsong@cha.ac.kr
Cell Transplantation, Vol. 22, pp. 000–000, 2013 0963-6897/13 $90.00 + .00
Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368912X657314
Copyright 2013 Cognizant Comm. Corp. E-ISSN 1555-3892
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Therapeutic Potential of Human Induced Pluripotent Stem Cells
in Experimental Stroke
Da-Jeong Chang,*1 Nayeon Lee,*1 In-Hyun Park,†‡1 Chunggab Choi,* Iksoo Jeon,* Jihye Kwon,*
Seung-Hun Oh,* Dong Ah Shin,§ Jeong Tae Do,* Dong Ryul Lee,* Hyunseung Lee,¶
Hyeyoung Moon,¶ Kwan Soo Hong,¶ George Q. Daley,† and Jihwan Song*
*CHA Stem Cell Institute, Department of Biomedical Science, CHA University, Seoul, Korea
†Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA
‡Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
§Department of Neurosurgery, Yonsei University College of Medicine, Seoul, Korea
¶Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Korea
Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) is a major type of stroke, but there
are currently very limited therapeutic options for its cure. Neural stem cells (NSCs) or neural precursor cells
(NPCs) derived from various sources are known to survive and improve neurological functions when they are
engrafted in animal models of stroke. Induced pluripotent stem cells (iPSCs) generated from somatic cells of
patients are novel cells that promise the autologous cell therapy for stroke. In this study, we successfully dif-
ferentiated iPSCs derived from human fibroblasts into NPCs and found their robust therapeutic potential in a
rodent MCAo stroke model. We observed the significant graft-induced behavioral recovery, as well as exten-
sive neural tissue formation. Animal MRI results indicated that the majority of contralaterally transplanted
iPSC-derived NPCs migrated to the peri-infarct area, showing a patho-tropism critical for tissue recovery. The
transplanted animals exhibited the significant reduction of stroke-induced inflammatory response, gliosis and
apoptosis, and the contribution to the endogenous neurogenesis. Our results demonstrate that iPSC-derived
NPCs are effective cells for the treatment of stroke.
Key words: Stroke; Induced pluripotent stem cells (iPSCs); Behavioral recovery; Endogenous neurogenesis
and circuitry. Secondly, various kinds of stem cells can
be injected systemically to achieve neuroprotection and
modulation of inflammation. Thirdly, drugs or chemical
compounds can be infused to promote neurogenesis from
endogenous stem/progenitor cells in the sub-ventricular
zone (SVZ).
Clinical trials have been performed using various cell
sources, from immortalized human teratocarcinoma cell
line to autologous bone marrow-derived mesenchymal
stem cells (BM-MSCs) (2,8,17,18,23). Outcomes have
been variable with some reports describing no benefit
while others have indicated transient clinical improve-
ment (19). More recently, another clinical trial in stroke
patients is ongoing by ReNeuron using clonal, condi-
tionally immortalized neural stem cells (NSCs) isolated
from human fetal cortex, which were previously shown
to ameliorate motor impairments in the rat stroke model
INTRODUCTION
Ischemic stroke mainly caused by middle cerebral
artery occlusion (MCAo) represents the major type of
stroke, which leads to the death of multiple neuronal
cell types, as well as astrocytes, oligodenderocytes, and
endothelial cells in the brain (20,33). After stroke, spon-
taneous recovery may occur to varying degrees, but most
patients suffer from persistent motor, sensory, or cogni-
tive impairments. Despite extensive research efforts,
there are still very limited therapeutic options for stroke-
damaged patients. Based on numerous animal transplan-
tation experiments using various cell sources, as well as
new observations on the stroke-induced neurogenesis,
the action modes of stem cell-based approaches in stroke
can be proposed largely in the following ways (20).
Firstly, stem cell-derived neural precursor cells (NPCs)
can be transplanted to restore damaged neural tissues
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PROOF
2 CHANG ET AL.
(29). In any case, while autologous MSCs provide mini-
mal clinical outcomes, human fetal tissue transplants
raise ethical issues and inevitably are genetically dissimi-
lar to the recipient with the associated risk of immune
rejection. Therefore, other suitable nonfetal cell source
of syngeneic donor tissue with high neurogenic potential
would be advantageous.
In this study, we show that an iPSC derived from nor-
mal human fibroblasts can form NPCs efficiently. After
intrastriatal implantation of these cells into a rodent model
of MCAo, the grafted animals exhibit clear functional
recovery with neural tissue formation in the damaged
brain by the transplanted cells. They also show significant
reduction in inflammation, gliosis, and apoptosis, as well
as significant contribution to the endogenous neurogen-
esis, implying that iPSC-derived NPCs have the unique
properties of treating stroke using all the several modes
of action as described above (20).
MATERALS AND METHODS
Culture and Neuronal Differentiation of 551-8 hiPSCs
We cultured and maintained human iPSCs (Detroit
551-iPS8, called 551-8 hiPSC thereafter) according to
the method described previously (26–28). We previously
established the 551-8 hiPSC line (26), but its neuronal dif-
ferentiation has not been studied extensively. As a control,
H9 human ES cells (obtained from WiCell, Madison, WI,
USA) were used. Neuronal differentiation of iPSCs was
induced by coculturing the cells with mouse PA6 stromal
cells (obtained from RIKEN Cell Bank, Ibaraki, Japan) as
described previously (15). Undifferentiated iPSC colonies
were mechanically dissected and transferred onto freshly
prepared PA6 cells in differentiation medium (DM-PA6)
that consists of Glasgow minimum essential medium
(GMEM) containing 10% KO-SR (knockout serum
replacement, both Invitrogen, Carlsbad, CA, USA), and
4 days later, the KO-SR in DM-PA6 was replaced by N2
supplements (Invitrogen). In the following 11–13 days,
definitive neural rosette-like structures containing neu-
roepithelial (NE) cells were formed, which were mechani-
cally detached and transferred onto a nonsticky Petri dish
(BD Biosciences, San Jose, CA, USA ) for suspension cul-
ture for 7 days to induce neurosphere formation.
Stable Generation of Neural Precursor Cells (NPCs)
To prepare NPCs for transplantation, we dissociated
neurospheres into single cells following treatment with
AccutaseTM (Chemicon, Temecula, CA, USA) and plated
them onto poly l-ornithine (PLO; 15 μg/ml, Sigma, St.
Louis, MO, USA)/fibronectin (FN; 1 μg/ml, Sigma)-coated
60 mm2 tissue culture dishes (BD Biosciences). NPCs
were maintained in GMEM supplemented with 1% peni-
cillin, 1% streptomycin (both Welgene, Daegu, Korea),
1% nonessential amino acids, 0.1% b-mercaptoethanol,
N2 supplements, and 20 ng/ml basic fibroblast growth
factor (bFGF; all Invitrogen).
Differentiation Into Mature Neurons (MNs)
To differentiate iPSCs into mature neurons, we plated
neurospheres directly onto PLO/FN-coated dishes in DM
supplemented with 20 ng/ml brain-derived neurotrophic
factor (BDNF, R&D Systems, Minneapolis, MN, USA)
in the absence of bFGF.
Immunocytochemical Analysis
To analyze the marker expression of NPCs and differ-
entiated neuronal cells, we carried out immunocytochemi-
cal analyses using the following primary antibodies:
octamer-binding transcription factor 4 (OCT4; 1:250, Santa
Cruz Biotechnology, Dallas, TX, USA), stage-specific
embryonic antigen 4 (SSEA-4; 1:100, Developmental
Studies Hybridoma Bank, Iowa City, IA, USA), TRA-1-81
(1:250, Chemicon), human-specific nuclei (hNu; 1:200,
Chemicon), human-specific mitochondria (hMito; 1:200,
Chemicon), human-specific NESTIN (1:200, Chemicon),
sex-determining region Y box 2 (SOX2; 1:200, Chemicon),
type III b-tubulin (Tuj1) (1:500, Chemicon), orthodenticle
homeobox 2 (OTX2; 1:500, Chemicon), brain factor-1
(BF-1; 1:100, Santa Cruz), microtubule-associated pro-
tein 2 (MAP2; 1:200, Chemicon), neuronal nuclei (NeuN;
1:500, Chemicon), g-aminobutyric acid (GABA; 1:5000,
Sigma), glutamic acid decarboxylase 65/67 (GAD65/67;
1:200, Chemicon), dopamine- and cAMP-regulated phos-
phoprotein, Mr 32 kDa (DARPP-32; 1:100, Cell Signaling
Technology, Danvers, MA, USA), tyrosine hydroxylase (TH;
1:1000, Pel-Freez, Rodgers, AR, USA), glial fibrillary
acidic protein (GFAP; 1:500, BD Biosciences), oligoden-
drocyte marker 4 (O4; 1:250, Chemicon), ionized calcium-
binding adapter molecule 1 (Iba-1; 1:250, Wako, Osaka,
Japan), ED1 [cluster of differentiation 68 (CD68); 1:250,
Serotec; Raleigh, NC, USA], doublecortin (DCX; 1:200,
Cell Signaling), polysialylated-neural cell adhesion mol-
ecule (PSA-NCAM; 1:250, Chemicon), and chemokine
(C-X-C motif) receptor 4 (CXCR4; 1:40, R&D Systems).
Secondary antibodies used were goat anti-mouse IgG-
conjugated Alexa-555 (1:200, Molecular Probes, Eugene,
OR, USA), goat anti-rabbit IgG-conjugated Alexa-488
(1:200, Molecular Probes), and goat anti-mouse IgM-
conjugated Alexa-555 (1:200, Molecular Probes). Staining
patterns were examined and photographed using a confo-
cal laser scanning microscope imaging system (LSM510,
Carl Zeiss, Inc., Thornwood, NY, USA).
RNA Isolation and Reverse Transcription-Polymerase
Chain Reaction (RT-PCR) Analysis
We isolated total RNA from cells using Trizol RNA
extraction method (Gibco, Gaithersburg, MD, USA).
cDNA was synthesized using Moloney Murine Leukemia
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PROOF
FUNCTIONS OF iPSC-DERIVED NEURONS IN STROKE 3
Virus M-MLV reverse transcriptase (Promega, Madison,
WI, USA) at 42°C for 1 h. PCR amplification was per-
formed using Taq polymerase according to the manufac-
turer’s instructions (Intron Biotechnology, Kyungki-Do,
Korea). Primer sequences, number of cycles, annealing
temperatures are described in Table 1. The housekeep-
ing gene glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as an internal loading control.
MCAo Animal Model and Cell Transplantation
We carried out animal experiments in accordance with
the CHA University IACUC (Institutional Animal Care
and Use Committee; IACUC090012). MCAo was induced
according to the method of Longa (21) using adult male
Sprague–Dawley rats (Orient, Seoul, Korea), weighing
270–300 g. Blunt-ended monofilament (4–0, Ethicon,
Livingston, Scotland, UK) was inserted approximately
19 mm from the carotid bifurcation into the internal carotid
artery to occlude middle cerebral artery (MCA) for 90 min,
after which monofilament was carefully removed. In order
to determine and select the appropriate stroke animal mod-
els, we employed the acute neurological assessment during
the first 24 h after 90 min of MCAo, which include fore-
limb and hindlimb placement (5) and circling behavior (3).
Animals scored 3 points or lower (normal: 5 points) were
used for transplantation experiments. At 7 days post-
MCAo induction, we injected a total of 10 rats with 2 ml
of 551-8 hiPSC-derived NPCs (100,000 cells/ml) into the
contralateral side of the infarct region using a Hamilton
syringe (Reno, NV, USA), targeting the following coor-
dinates: AP +1.0 mm, ML +3.0 mm, and DV –5.0 mm
from the Bregma. In the control group (n = 10), 2 ml of sus-
pension medium (GMEM) were injected in parallel. For
animal magnetic resonance imaging (MRI) experiments
ferumoxide (Feridex®) (Taejoon Pharm, Seoul, Korea)
and protamine sulfate (Sigma) were initially prepared at a
Table 1. RT-PCR Primers Used in This Study
Gene Name Forward (F) and Reverse (R) Primer Sequences
Product
Size (bp)
Annealing
Temp. (°C)
No.
Cycles
Gene Bank
Accession No.
Oct4 F: 5¢-CTGAAGCAGAAGAGGATCAC-3¢
R: 5¢-GACCACATCCTTCTCGAGCC-3¢
366 60 30 NM_002701.4
Nanog F: 5¢-TTCTTGACCGGGACCTTGTC-3¢
R: 5¢-GCTTGCCTTGCTTTGAAGCA-3¢
256 60 30 NM_024865
Sox2 F: 5¢-GCTGCAAAAGAGAACACCAA-3¢
R: 5¢-CTTCCTGCAAAGCTCCTACC-3¢
232 59 30 NM_003106
Nestin F: 5¢-TCCAGAAACTCAAGCACCA-3¢
R: 5¢-AAATTCTCCAGGTTCCATGC-3¢
183 59 30 NM_006617
Musashi F: 5¢-ACAGCCCAAGATGGTGACTC-3¢
R: 5¢-CCACGATGTCCTCACTCTCA-3¢
191 59 30 NM_002442
bIII tubulin F: 5¢-ATGAGGGAGATCGTGCACAT-3¢
R: 5¢-GCCCCTGAGCGGACACTGT-3¢
239 59 30 BC000748.2
Map2 F: 5¢-GCATATGCGCTGATTCTTCA-3¢
R: 5¢-CTTTCCGTTCATCTGCCATT-3¢
202 59 30 U01828
Bf-1 F: 5¢-CTCAGAACTCGCTGGGCAAC-3¢
R: 5¢-CGTGGGGGAAAAAGTAACTGG-3¢
225 59 30 NM_005249
Otx2 F: 5¢-CGCCTTACGCAGTCAATGGG-3¢
R: 5¢-CGGGAAGCTGGTGATGCATAG-3¢
618 60 30 AF093138
En-1 F: 5¢-CTAGCCAAACCGCTTACGAC-3¢
R: 5¢-GCAGAACAGACAGACCGACA-3¢
358 59 30 NM_001426.3
Darpp-32 F: 5¢-CCTGAAGGTCATCAGGCAGT-3¢
R: 5¢-GGTCTTCCACTTGGTCCTCA-3¢
131 50 30 AF464196.1
GAD67 F: 5¢-AGCAAACCGTGCAATTCCTC-3¢
R: 5¢-AAATCGAGGATGACCTGTGC-3¢
230 55 30 L16888.1
GFAP F: 5¢-GCAGAGATGATGGAGCTCAATGACC-3¢
R: 5¢-GTTTCATCCTGGAGCTTCTGCCTCA-3¢
266 59 30 BC041765.1
MBP F: 5¢-ATTAGCTGAATTCGCGTGTG-3¢
R: 5¢-CTCTGAGTGCGCAAGTCTTC-3¢
235 59 30 M13577.1
GAPDH F: 5¢-GTCATACCAGGAAATGAGCT-3¢
R: 5¢-TGACCACAGTCCATGCCATC-3¢
422 60 30 BC083511.1
Oct4, octamer-binding transcription factor 4; Sox2, sex-determining region Y box 2; Map2, microtubule-associated protein 2; Bf-1, brain factor-1;
Otx2, orthodenticle homeobox 2; En-1, engrailed-1; Darpp-32, dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa; GAD67, glutamate decar-
boxylase 67; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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PROOF
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concentration of 10 mg/ml in DMEM (Dulbecco’s modi-
fied Eagle’s medium, Invitrogen) without serum, which
were mixed for 30 min at room temperature and subse-
quently added at an equal volume to the culture medium
containing the cells for 12–16 h at 37°C. Transplanted
animals were immunosuppressed with cyclosporine A
(10 mg/kg, Sigma) intraperitoneally 24 h before trans-
plantation and then everyday for up to 8 weeks.
Behavioral Tests
To determine that MCAo models were properly made
and to evaluate whether rats recovered functionally fol-
lowing cell transplantation, we performed rotarod test (13),
stepping test (24), and modified neurological severity
score (mNSS) test (30) every week, and apomorphine-
induced rotation test (32) every 2 weeks following trans-
plantation. To determine the baseline scores, each test
was performed 1–3 days prior to transplantation.
Statistical Analysis
We performed statistical analyses on the behavior data
using the Statistical Analysis System (Enterprise 4.1; SAS,
Seoul, Korea) on a CHA University mainframe computer.
Performance measures were analyzed using the PROC
MIXED program, a linear mixed models procedure for con-
ducting repeated measures analyses of both normal data.
The results are presented as the mean ± SEM (standard error
of mean). A value of p <0.05 was regarded as significant.
MRI Detection of Transplanted Cells
We employed a 4.7T Bio Spec (Bruker, Ettlingen,
Germany) for animal MRI analysis using T2- and T2*-
weighted imaging techniques. The MRI setting and
detection methods were the same as previously described
(16). To detect the Feridex®-labeled 551-8 hiPSC-derived
NPCs, Prussian blue staining was carried out by incubat-
ing the cells with a mixture of 4% potassium ferrocyanide
(Sigma) and 20% hydrochloric acid (Sigma) for 30 min
at room temperature, followed by immunohistochemical
staining using antibodies against human-specific nuclei
or mitochondria, in combination with markers of interest.
Prior to transplantation, we confirmed that the cells were
completely labeled with Feridex®, as demonstrated by the
Prussian blue staining pattern.
Immunohistochemical Analysis
For immunohistochemical analysis, we sacrificed the
rats at 8 weeks posttransplantation, perfused, and fixed
their brains with 4% paraformaldehyde (Sigma). Frozen
coronal sections (40 mm) were prepared using a cryostat
(Microm, Walldorf, Germany). Antibodies used for immu-
nohistochemistry were the same as described in immunocy-
tochemical analysis (see above). To detect the endogenous
stem cells, we injected 5¢-bromo-2¢-deoxyuridine (BrdU)
(50 mg/kg, Sigma) intraperitoneally three times at 12-h
intervals prior to sacrifice. BrdU-positive cells were
detected by immunohistochemistry using an antibody
against BrdU (1:500, BD Biosciences) following denatur-
ation of DNA using 1M HCl for 30 min at 45°C.
Terminal Deoxynucleotidyl Transferase dUTP Nick End
Labeling (TUNEL) Assay
To detect apoptotic cells, frozen sections were stained
using In Situ Cell Detection Kit according to the manu-
facturer’s instructions (Roche, Indianapolis, IN, USA).
All sections were counterstained with 4¢,6-diamidino-2-
phenylindole (DAPI; Roche). The number of TUNEL-
positive cells were counted using Image J (NIH, Bethesda,
MD, USA) and processed for statistical analysis.
RESULTS AND DISCUSSION
Neuronal Differentiation of 551-8 Cells
We have previously isolated an iPSC line from Detroit
551 human fibroblasts (551-iPS8, called 551-8 hiPSCs
thereafter) by retroviral infection of four reprogramming
factors [OCT4, SOX2, Krüppel-like factor 4 (KLF4),
Figure 1. Comparison of undifferentiated 551-8 hiPSCs and H9 ESCs. Comparison of undifferentiated 551-8 human induced pluri-
potent stem cells (hiPSCs) (A) with H9 human embryonic stem cells (ESCs) (B), in terms of morphology, semiquantitative RT-PCR
[octamer binding transcription factor 4 (Oct4) and Nanog], and immunocytochemical [OCT4, stage-specific embryonic antigen
(SSEA-4) and TRA-1-81] analyses. Scale bar: 100 mm. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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PROOF
FUNCTIONS OF iPSC-DERIVED NEURONS IN STROKE 5
and V-myc myelocytomatosis viral oncogene homolog
(MYC)] (25). 551-8 cells showed very similar proper-
ties with H9 hESCs, in terms of hESC-like morphology,
and pluripotency marker expression by semiquantitative
RT-PCR and immunocytochemical analyses (Fig. 1A, B),
confirming their pluripotency. In order to test the thera-
peutic effect of hiPSCs for stroke, we initiated the neu-
ronal differentiation of 551-8 iPSCs and H9 hESCs (Fig.
2A). Our differentiation protocol involves the mainte-
nance of the undifferentiated H9 or 551-8 cells on mouse
embryonic fibroblasts (MEFs) (Stage 1), coculture with
PA6 stromal cells (Stage 2), the isolation of neural rosette-
like structure (Stage 3, Fig. 2B), and the neurosphere for-
mation (Stage 4, Fig. 2C). Afterwards, neurospheres were
either dissociated into single cells to make neural precur-
sor cells (NPCs) (Stage 5-NP, Fig. 2D) for transplanta-
tion or further differentiated into mature neurons (Stage
5-MN, Fig. 2E). Following the Stage 3 of differentiation,
we found that the 551-8 iPSCs and H9 hESCs showed the
difference in their rosette-forming capacity. While more
Figure 2. Neuronal differentiation of 551-8 hiPSCs. (A) Experimental scheme showing a step-wise neuronal differentiation proce-
dure. (B) Neural rosette-like structures formed through coculturing 551-8 hiPSCs with mouse PA6 stromal cells (Stage 3, see text
for details). (C) Neurospheres (NS) formed though suspension culture of isolated neural rosette-like structures (Stage 4). (D) Neural
precursor (NP) cells undergoing expansion as single cells (Stage 5-NP), which were prepared by dissociation of neurospheres using
accutase. (E) Differentiated neuronal cells formed from the attached neurospheres (Stage 5-MN). MEF, mouse embryonic fibroblasts;
DMEM, Dulbecco’s modified Eagle’s medium; GMEM, Glasgow minimum essential medium; KO-SR, knockout serum replacement;
AA, ascorbic acid; bFGF, basic fibroblast growth factor; BDNF, brain-derived growth factor.
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PROOF
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than 80% colonies of H9 cells showed the rosette-like
structure, around 20% colonies of 551-8 iPSCs showed
the rosette-like structure. The difference may be due to
the intrinsic clonal variation among pluripotent stem cells
or specific to iPSCs (10,25). A larger number of hESCs
and iPSCs will be needed to further investigate the issue
involved in the difference in neuronal differentiation (4).
Although the initial rosette-forming efficiency of 551-8
cells was relatively lower than H9 cells, we isolated the
rosette-like structures at Stage 3 and further differentiated
them into later stages. Interestingly, we found that high
levels of NPC markers, such as NESTIN, SOX2, Musashi,
were robustly expressed at Stage 5-NPCs in 551-8 iPSCs,
comparably as those in H9 cells (Fig. 3A, B). At mature
neuronal differentiation stage (Stage 5-MN), we observed
that the cultures express markers for various neuronal cell
types that include general neurons (Tuj1), mature neurons
(MAP2 and NeuN), forebrain/midbrain (Otx2), forebrain
(BF-1), medium spiny projection neurons (DARPP-32),
GABAergic (GABA and GAD67), and dopaminergic (TH)
neurons, as well as markers for astrocytes (GFAP) and
oligodendrocytes [O4 and myelin basic protein (MBP)]
(Fig. 4A, B). By contrast, markers for pluripotency (Oct4
and Nanog) or midbrain/hindbrain [homeobox protein
engrailed-1 (En-1)] were not detectable (Fig. 4B). Taken
together, these results indicate that 551-8 iPSCs are highly
efficient in completing the neuronal differentiation despite
their lower rosette-forming capacity.
Behavioral Improvement Following Transplantation of
551-8 hiPSC-Derived NPCs Into MCAo Animal Models
We next investigated whether the iPSC-derived NPCs
(iPSC-NPCs) could exert functional effects after trans-
plantation into a rodent model of ischemic stroke (Fig.
5A). For in vivo studies, we first induced 90-min MCAo
and selected appropriate animals by behavioral standards.
One week later we grafted 200,000 551-8 hiPSC-NPCs
(Stage 5), in a volume of 2 ml, into the striatum of con-
tralateral side of infarct area (n = 10). In sham control rats
(n = 10), we only injected vehicle (GMEM). We chose the
contralateral side, as opposed to ipsilateral side, because
our previous MRI study using human bone marrow-
derived mesenchymal stem cells (BM-MSCs) demon-
strated that most stem cells have the capacity to migrate
to the injury site (16). In addition, the ipsilateral injec-
tion risks introducing secondary injury to the infarct area,
when the Hamilton syringe is inserted into the infarct area,
which will inevitably cause tissue damage and inflam-
matory response. Moreover, the contralateral injection
avoids the misinterpretation of MRI results, because the
injection site itself can be interpreted as false positive.
To evaluate the functional effects of the grafts, we
employed four different behavioral tests (i.e., rotarod, step-
ping, mNSS, and apomorphine-induced rotation tests).
When we followed the transplantation up to 8 weeks, sig-
nificant behavioral recovery (p < 0.05) was observed as early
as 1 week (in the rotarod test) or 2 weeks (in the staircase,
mNSS, and apomorphine-induced rotation tests) (Fig. 5B).
Among the tests we employed, we observed more steady
and gradual improvements in the rotarod and mNSS tests,
compared with stepping and apomorphine-induced rotation
tests. In addition to improvement of motor functions, we
also observed the improvement of sensory functions in the
mNSS test. Taken together, these results strongly indicate
that the transplanted 551-8 hiPSC-NPCs contribute to the
behavioral improvement in the MCAo stroke animal model.
Formation of Neural Tissues From the
Transplanted iPSC-NPCs
After completion of the behavioral tests at 8 weeks, we
sacrificed the rats and performed the hisological analysis
of the brains. To identify the donor human cells, we used
antibodies against human-specific nuclear antigen (hNu)
and human mitochondria (hMito) (Fig. 5C). Using con-
focal microscopy and double immunostaining, we found
that some transplanted 551-8 hiPSC-derived cells were still
NESTIN-positive neural precursors. Importantly, a large
number of the surviving human cells formed MAP2- and
NeuN-positive mature neurons. They also formed medium
spiny projection neurons (MSN), essential for striatal func-
tion. Moreover, we found that some neurons expressed
markers for GABAergic (GAD65/67) and dopaminergic
(TH) neurons. We also observed small populations of
Figure 3. Characterization of 551-8 hiPSC-derived neural pre-
cursor cells (Stage 5-NP). (A) Immunocytochemical staining
showing high levels of NESTIN and sex-determining region Y
box 2 (SOX2) expression and low levels of bIII tubulin (Tuj1)
expression. 4¢,6-diamidino-2-phenylindole (DAPI) was used
for counterstaining the individual cells. (B) Semiquantitative
RT-PCR analysis showing the expression of pluripotency mark-
ers (i.e., Oct4, Nanog), neural precursor markers (i.e., Sox2,
Nestin, Musashi), early and late neuronal markers [bIII tubulin
and microtubule-associated protein 2 (Map2), respectively],
and an astrocyte marker [glial fibrillary acidic protein (GFAP)].
Scale bar: 50 mm.
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FUNCTIONS OF iPSC-DERIVED NEURONS IN STROKE 7
transplanted cells forming astrocytes (GFAP) and oligo-
dendrocytes (O4).
Recently it was reported that NPCs differentiated from
mouse iPSCs can give rise to tumors when transplanted
into a mouse model of MCAo (14), raising the safety
concerns in using iPSC-NPCs for cell therapy in stroke.
However, among the total of 20 cell transplanted and
sham control groups in our study, we did not detect any
signs of graft overgrowth or tumor formation. Contrary
to the previous report of the mouse iPSCs, our NPC dif-
ferentiation protocol may be more robust through a series
of selection procedures and contain no undifferentiated
cells. Alternatively, the intrinsic difference in mouse and
human iPSCs in terms of neuronal differentiation may
be attributed to the less tumorigenecity of human iPSC-
derived NPCs. However, since 8 weeks of observation
period may not be long enough to exclude the possibility
of tumor formation, it may be necessary to monitor the
tumor formation over more extended time periods such as
six months or longer to ensure the safety of transplanted
cells. Taken together, these results strongly suggest that
551-8 hiPSC-NPCs survive and contribute to the neural
tissue formation and possibly neuronal regeneration in
the infarct area without tumor formation.
In Vivo Tracking of 551-8 hiPSC-Derived NPCs
Using Animal MRI
To monitor the fates of 551-8 cells following trans-
plantation into the contralateral side of infarct area, we
employed a 4.7-T animal MRI using T2 and T2*-weighted
imaging techniques (Fig. 6A). Animal MRI was taken at
1 day after transplantation and right before sacrificing
the animals after completion of behavior tests at 8 weeks
posttransplantation. Figure 6A shows T2 and T2* images
at 8 weeks following transplantation. Interestingly, we
found that the great majority of transplanted cells migrate
to the peri-infacrt area, exhibiting the patho-tropism of
stem cells in stroke animal models. We have previously
observed this phenomenon as well when BM-MSCs were
used (16). More recently, similar phenomenon was also
observed in our group when other cell types, such as
human ESC-derived NPCs, neural stem cells (NSCs), and
cord blood-derived MSCs, were transplanted and exam-
ined by MRI (data not shown). Importantly, we found that
Figure 4. Characterization of 551-8 hiPSC-derived mature neurons (Stage 5-MN). (A) Immunocytochemical staining showing the
expression of markers for early (Tuj1), mature (MAP2 and NeuN), forebrain/midbrain (OTX2), forebrain (BF-1), medium spiny
projection (DARPP-32), GABAeric (GABA), dopaminergic (TH) neurons, and astrocytes (GFAP), and oligodendrocytes (O4).
(B) Semiquantitative RT-PCR analysis showing the expression of markers for pluripotency (i.e., Oct4, Nanog), early and late neu-
rons (Tuj1 and Map2, respectively), forebrain (Bf-1), forebrain/midbrain (Otx2), midbrain/hindbrain (En2), GABAergic (GAD67),
and medium spiny projection (DARPP-32) neurons, and astrocytes (GFAP) and oligodendrocytes (MBP). Scale bar: 50 mm. Otx2,
orthodenticle homeobox 2; Bf-1, brain factor-1; Darpp-32, dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa; GABA,
g-aminobutyric acid; TH, tyrosine hydroxylase; GFAP, glial fibrillary acidic protein; O4; oligodendrocyte marker 4; En-1, engrailed-1;
GAD67, glutamate decarboxylase 67; MBP, myelin basic protein.
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8 CHANG ET AL.
the transplanted cells not only migrate tangentially across
the brain hemisphere, mainly via corpus callosum, but
also encompass the infarct area, judged by the widespread
distribution of Feridex®-labeled cells in anterior to poste-
rior position, regardless of injection site (Fig. 6A–C).
We also found that most of the migrated cells (80.10 ±
8.50%) express CXCR4 (Fig. 6C), the cognate receptor for
the inflammatory chemoattractant SDF-1a (stromal cell-
derived factor 1-a) that is known to be upregulated by
local astrocytes and endothelium in response to ischemic
Figure 5. In vivo functional analyses of 551-8 hiPSC-derived NP cells following transplantation into a rodent model of MCAo.
(A) Experimental scheme showing time schedules for making middle cerebral artery occlusion (MCAo) animal models and determination
of prescores for behavioral tests (MCAo), cell transplantation (Cell Tx), posttransplantation follow-up studies including various behav-
ioral tests (behavioral tests) and animal magnetic resonance imaging (MRI), followed by sacrifice of animals and histological analysis
(perfusion and IHC). (B) Behavioral improvements following transplantation of 551-8 hiPSC-derived NP cells. Rotarod test, stepping
test, and modified neurological severity score (mNSS) test were carried out every week, and apomorphine test was performed biweekly
up to 8 weeks. *p < 0.05; **p < 0.001. w, week. (C) Immunohistochemical (IHC) staining to visualize the transplanted cells and their
differentiated cell types. To detect the fate of transplanted cells, antibodies against either human-specific nuclei (hNu) or human-
specific mitochondria (hMito) were used. Transplanted cells were shown to form human-specific NESTIN (hNESTIN), MAP2, neu-
ronal nuclei (NeuN), DARPP-32, GAD65/67, TH, GFAP, and O4. DAPI was used to counterstain the cells. Arrows, colocalized
marker expression. Scale bar: 20 mm.
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PROOF
FUNCTIONS OF iPSC-DERIVED NEURONS IN STROKE 9
injury (11,22). Therefore, results from the animal MRI
study and CXCR4 expression in transplanted cells from
551-8 NPCs strongly suggest that hiPSC-derived NPCs
have the capacity to migrate to the injured brain, which
fulfills the first critical step in stem cell homing and
engraftment for tissue repair and regeneration.
Due to the limited accessibility to animal MRI facility,
animal MRI was taken only twice in this experiment. But
it will be more useful if MRI observation can be carried
out routinely, so that selection of animals based on MRI-
guided infarct volume measurement prior to transplan-
tation, as well as more detailed follow-ups on the graft
mobility or changes of infarct volume after transplanta-
tion, can be carried out.
Reduced Inflammation, Gliosis, and Apoptosis
Inflammation is a critical mediator of brain damage
in response to ischemic injury. As demonstrated both in
animal models (31) and human stroke patients (7), the
post-ischemic inflammatory cascade involves a rapid
activation of microglia and astrocytes. In this study, we
first carried out quantitative analysis of two representa-
tive microglial markers, Iba-1 and ED1 expression in
both iPSC-transplanted and sham control groups. While
Iba-1 is expressed both in the ischemic core and the pen-
umbra, ED1 is expressed in the ischemic core only (31).
Penumbra is a region of constrained blood supply in which
energy metabolism is preserved (9). Compared with the
sham control, animals transplanted with 551-8 hiPSC-
derived NPCs showed the significant reduction in the num-
ber of Iba-1+ cells in the ischemic core (53.40 ± 4.66% vs.
17.31 ± 1.40%, p < 0.05) (Fig. 7A, first panel), although
their difference in the penumbra was shown to be insig-
nificant (14.86 ± 1.10% vs.12.51 ± 1.27%, p = 0.31, figures
not shown). Compared with the sham control, the num-
ber of round-shaped ED1-positive cells was also shown
to be significantly reduced in the transplanted group
(23.84 ± 2.71% vs. 12.50 ± 0.10%, p < 0.05) (Fig. 7A,
second panel). Since this analysis was made 8 weeks fol-
lowing transplantation, it is currently unknown exactly
when the reduction in inflammatory cells first occurred.
However, it will be likely that this histological finding is
the result of an earlier event (data not shown).
We also examined the number of astrocytes in both
hiPSC-derived NPC-transplanted and sham control
groups and found that GFAP-positive astrocytes in the
Figure 6. In vivo tracking of 551-8 hiPSC-derived neural precursor cells following transplantation into the contralateral side of infarct
in a rodent model of MCAo. (A) 4.7T animal MRI analyses showing the coronal view of T2- and T2*-weighted images in antero-
posterior sequences (from 4.2 to -1.20 mm, measured from the bregma). Note the migration of Feridex®-labeled transplanted cells to
the infarct area (arrowheads). (B) Prussian blue staining showing the detection of Feridex®-labeled transplanted cells in the infarct area.
(C) Immunocytochemical staining showing the presence of transplanted cells, judged by expression of human-specific mitochondria
(hMito), which are also colocalized with chemokine (C-X-C motif) receptor 4 (CXCR4). Scale bar: 50 mm.
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PROOF
10 CHANG ET AL.
Figure 7. Transplantation effects of 551-8 hiPSC-derived NP cells. (A) Reduction of inflammation
(Iba-1, ED1), gliosis (GFAP), and apoptosis (TUNEL) in transplanted animals, judged by immuno-
histochemical staining and cell counting, followed by statistical analysis. DAPI was used for counter-
staining the cells. (B) Immunohistochemical staining showing the enhancement of endogenous
neurogenesis (BrdU) and formation of migrating neuroblasts (DCX and PSA-NCAM) in the trans-
planted animals. Cell counting and statistical analysis was also performed. *p < 0.05; **p < 0.001.
Scale bar: 50 mm. Iba-1, Ionized calcium binding adaptor molecule 1; ED1, antibody for cluster of
differentiation 68 (CD68); TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling;
BrdU, 5¢-bromo-2¢-deoxyuridine; DCX, doublecortin; PSA-NCAM, polysialylated neural cell adhe-
sion molecule.
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PROOF
FUNCTIONS OF iPSC-DERIVED NEURONS IN STROKE 11
penumbra were greatly reduced in the transplanted group
(87.70 ± 5.7% vs. 32.14 ± 3.53%, p < 0.001) (Fig. 7A,
third panel). Proliferation of GFAP-positive astrocytes
(gliosis) after ischemic injury usually leads to the for-
mation of a glial scar. Therefore, it is likely that the
transplanted iPSCs can also reduce the risk of glial scar
formation. We also observed that the number of TUNEL-
positive apoptotic cells was significantly reduced in
the transplanted group (22.15 ± 0.60% vs. 4.59 ± 0.90%,
p < 0.001) (Fig. 7A, last panel). At this stage, only few
neurons undergo apoptosis as the major apoptotic events
take place much earlier, during first 2 weeks after stroke.
Therefore, it is likely that the TUNEL-positive cells are
mostly represented by apoptotic microglia that might be
involved in neural repair during later stages.
It is still controversial whether the activation of micro-
glia and astrocytes are detrimental or beneficial to neu-
ronal survival, neurogenesis, and functional recovery
after stroke (6,12). Nevertheless, our results strongly sug-
gest that the transplanted hiPSC-derived NPCs contribute
to the reduction of inflammation, gliosis, and apoptosis in
animal stroke model. Further study to understand how the
transplanted cells play a role in each process at molecular
levels will be important to optimize the use of hiPSC-
derived NPCs for the treatment of stroke.
Promotion of Endogenous Neurogenesis Process
Stroke-induced neurogensis has been reported in adult
brains, in which a pool of BrdU-positive endogenous
NSCs or NPCs in the subventricular zone (SVZ) starts to
proliferate and generate neuroblasts upon stroke-mediated
injury (1). These newly generated neuroblasts have the
capacity to migrate to the damaged area in the striatum
and to differentiate into functional neurons (20,33). For
this reason, it is now widely accepted that enhancement of
endogenous neurogenesis would be one of most important
targets for stroke therapeutics. Having this in mind, we
addressed whether the transplanted hiPSC-derived NPCs
have an influence on the endogenous neurogenesis. To do
this, we injected the transplanted and sham control animals
with BrdU three times at 12-h intervals prior to sacrifice.
BrdU-positive cells were detected by immunohistochem-
istry using an antibody against BrdU, which were also
double-stained with either DCX or PSA-NCAM, both of
which are associated with neuronal migration.
While there was no difference of BrdU-positive cells
in the contralateral side of SVZ between the iPSC-NPC
transplanted and sham control groups (12.12 ± 2.39 vs.
19.56 ± 2.39, figures not shown), there was a significant
difference in the ipsilateral side of SVZ (49.89 ± 7.03 vs.
83.83 ± 12.14, p < 0.05) (Fig. 7B) between them. Among
the BrdU-positive cells, we found that significantly high
numbers of cells positive for either DCX (12.17 ± 2.39 vs.
19.56 ± 2.39, p < 0.05) or PSA-NCAM (42.20 ± 16.38 vs.
70.67 ± 16.27, p < 0.05) were detected in the hiPSC-
transplanted group, compared with the sham control. No
BrdU-positive human cells were detected, meaning that
the transplanted hiPSC-derived NPCs were unrelated to
BrdU-positive cells. Taken together, these results strongly
suggest that the transplanted hiPSC-derived NPCs
greatly contribute to the proliferation of endogenous
neural stem cells in the SVZ, as well as to their migration
Figure 8. Schematic diagram showing the action mode of 551-8 hiPSC-derived NPCs following
transplantation into MCAo animal models. See text for details.
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12 CHANG ET AL.
to the infarct area in the striatum. However, although we
observed a significant increase in SVZ cell proliferation
and DCX-positive neuroblast formation, there was no
evidence that DARPP-32-positive functional neurons
were directly formed from endogenous neural stem cells.
Therefore, it can be only said at this stage that the trans-
planted hiPSC-derived NPCs can promote some aspects
of endogenous neurogenesis (i.e., proliferation and migra-
tion) but not the entire process (including mature neuron
formation). How exactly the transplanted iPSC-NPCs
can induce or mediate the enhancement of endogenous
neurogenesis is currently unknown, but it will be likely
that secretion of neurotrophic factors, modulation of
inflammation, or promotion of angiogenesis by hiPSC-
derived NPCs would play major roles in this process. For
this, we have observed that several kinds of neurotrophic
factors such as BDNF are highly expressed in the trans-
planted animals, as compared with sham controls (data
now shown).
In summary, we have carried out the detailed analy-
sis on the neurogenic and therapeutic potentials of NPCs
derived from human iPSCs to treat stroke. Our results
show that iPSCs can form NPCs efficiently and signifi-
cant behavioral recovery can be achieved when these
NPCs are transplanted into a rodent stroke model. As
summarized in Figure 8, the transplanted hiPSC-derived
NPCs led to not only behavioral recovery and neural tis-
sue formation but also significant reduction in stroke-
induced inflammatory response, gliosis, and apoptosis.
More importantly, they contributed to the major events in
endogenous neurogenesis. No tumor was found from the
transplanted animals. So far, various types of stem cells
have exhibited neurogenic and therapeutic potentials in
animal models of stroke, but our work provides encour-
aging evidence showing that human hiPSC-derived NPCs
have the capacity to cure the damaged brain by exerting
three important modes of action simultaneously. Firstly,
they migrate to the infarct area and form neural tissues in
the stroke-damaged brain. Secondly, they reduce inflam-
mation, gliosis, and apoptosis in the stroke-damaged
brain. Thirdly, they contribute to the endogenous neu-
rogenesis in the SVZ of stroke-induced brain. Although
more evidence will be required to prove the effects of
hiPSCs in neuronal restoration, neuroprotection, neural
circuit connection, mature neuron formation, etc., given
the importance of iPSCs as autologous sources for cell
therapy, the current study provides the important possibil-
ity on the therapeutic potential of iPSCs in treating stroke.
Obviously, it will be necessary to test multiple iPSC lines
derived from viral or nonviral methods for the generaliza-
tion of our results, and more extended follow-up study
will be required to ensure the persistence and safety of
transplantation effects.
ACKNOWLEDGMENTS: This work was supported by grants
from the Korea Health Technology R&D Project, Ministry
of Health and Welfare (A111016), the National Research
Foundation of Korea (2010-0008719), and the Korea Food and
Drug Administration (S-11-04-2-SJV-993-0-H), Republic of
Korea to J.S. We are grateful to the MRI facility in the Division
of Magnetic Resonance, Korea Basic Science Institute, Ochang,
Korea. We also thank WiCell and RICKEN Cell Bank for pro-
viding us with H9 human ES cells and PA6 cells, respectively.
J.S. is highly indebted to Professor Olle Lindvall who provided
the initial support and the great inspiration on stroke research.
The authors declare no conflicts of interest.
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PROOF
PROOF
