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Therapeutic Potential of Human Induced Pluripotent Stem Cells in Experimental Stroke

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Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) is the 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 differentiated iPSCs derived from human fibroblasts into NPCs and found their robust therapeutic potential in a rodent MCAo stroke model. We observed the significant graftinduced behavioral recovery, as well as extensive neural tissue formation. Animal MRI results indicated that the majority of contra-laterally 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.
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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
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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
... Immunocytochemistry data of in vivo MCAO models of ischemic stroke have also found similar anti-inflammatory and neuroregenerative potential of iPSC-NSCs. Studies in rodent models have found significant reductions in IBA1 + (Chang et al., 2013a;Eckert et al., 2015), ED1 + (Chang et al., 2013a;Heo et al., 2013;Lee et al., 2017), and GFAP + cells (Chang et al., 2013a;Lee et al., 2017) in the ischemic core of iPSC-NSC-treated animals compared to non-treated animals. Additionally, the number of apoptotic cells post-MCAO measured through TUNEL assay and the number of activated inflammatory cells has been shown to be significantly reduced in iPSC-NSC treated animals (Chang et al., 2013a;Lee et al., 2017) as far as 8 weeks post-transplantation. ...
... Immunocytochemistry data of in vivo MCAO models of ischemic stroke have also found similar anti-inflammatory and neuroregenerative potential of iPSC-NSCs. Studies in rodent models have found significant reductions in IBA1 + (Chang et al., 2013a;Eckert et al., 2015), ED1 + (Chang et al., 2013a;Heo et al., 2013;Lee et al., 2017), and GFAP + cells (Chang et al., 2013a;Lee et al., 2017) in the ischemic core of iPSC-NSC-treated animals compared to non-treated animals. Additionally, the number of apoptotic cells post-MCAO measured through TUNEL assay and the number of activated inflammatory cells has been shown to be significantly reduced in iPSC-NSC treated animals (Chang et al., 2013a;Lee et al., 2017) as far as 8 weeks post-transplantation. ...
... Immunocytochemistry data of in vivo MCAO models of ischemic stroke have also found similar anti-inflammatory and neuroregenerative potential of iPSC-NSCs. Studies in rodent models have found significant reductions in IBA1 + (Chang et al., 2013a;Eckert et al., 2015), ED1 + (Chang et al., 2013a;Heo et al., 2013;Lee et al., 2017), and GFAP + cells (Chang et al., 2013a;Lee et al., 2017) in the ischemic core of iPSC-NSC-treated animals compared to non-treated animals. Additionally, the number of apoptotic cells post-MCAO measured through TUNEL assay and the number of activated inflammatory cells has been shown to be significantly reduced in iPSC-NSC treated animals (Chang et al., 2013a;Lee et al., 2017) as far as 8 weeks post-transplantation. ...
Article
Full-text available
Inflammation has proven to be a key contributing factor to the pathogenesis of ischemic and hemorrhagic stroke. This sequential and progressive response, marked by proliferation of resident immune cells and recruitment of peripheral immune populations, results in increased oxidative stress, and neuronal cell death. Therapeutics aimed at quelling various stages of this post-stroke inflammatory response have shown promise recently, one of which being differentiated induced pluripotent stem cells (iPSCs). While direct repopulation of damaged tissues and enhanced neurogenesis are hypothesized to encompass some of the therapeutic potential of iPSCs, recent evidence has demonstrated a substantial paracrine effect on neuroinflammation. Specifically, investigation of iPSCs, iPSC-neural progenitor cells (iPSC-NPCs), and iPSC-neuroepithelial like stem cells (iPSC-lt-NESC) has demonstrated significant immunomodulation of proinflammatory signaling and endogenous inflammatory cell populations, such as microglia. This review aims to examine the mechanisms by which iPSCs mediate neuroinflammation in the post-stroke environment, as well as delineate avenues for further investigation.
... NSCs ameliorate ischemic damage by lowering proinflammatory mediators including TNF-α, IL-1β, IL-6, MCP-1, and Iba-1 [223,253]. For instance, hiPSC-NSC transplantation is associated with significantly decreased Iba-1 positive cells in the stroke mouse model [254]. Transplanted mice also had decreased numbers of GFAP-positive astrocytes [254]. ...
... For instance, hiPSC-NSC transplantation is associated with significantly decreased Iba-1 positive cells in the stroke mouse model [254]. Transplanted mice also had decreased numbers of GFAP-positive astrocytes [254]. Similarly, NSC-engrafted mice displayed lower CD45 + and Iba-1 + /major histocompatibility II immune cells after ischemia [253]. ...
Article
Full-text available
Clinical treatments for ischemic stroke are limited. Neural stem cell (NSC) transplantation can be a promising therapy. Clinically, ischemia and subsequent reperfusion lead to extensive neurovascular injury that involves inflammation, disruption of the blood-brain barrier, and brain cell death. NSCs exhibit multiple potentially therapeutic actions against neurovascular injury. Currently, tissue plasminogen activator (tPA) is the only FDA-approved clot-dissolving agent. While tPA’s thrombolytic role within the vasculature is beneficial, tPA’s non-thrombolytic deleterious effects aggravates neurovascular injury, restricting the treatment time window (time-sensitive) and tPA eligibility. Thus, new strategies are needed to mitigate tPA’s detrimental effects and quickly mediate vascular repair after stroke. Up to date, clinical trials focus on the impact of stem cell therapy on neuro-restoration by delivering cells during the chronic stroke stage. Also, NSCs secrete factors that stimulate endogenous repair mechanisms for early-stage ischemic stroke. This review will present an integrated view of the preclinical perspectives of NSC transplantation as a promising treatment for neurovascular injury, with an emphasis on early-stage ischemic stroke. Further, this will highlight the impact of early sub-acute NSC delivery on improving short-term and long-term stroke outcomes.
... The bystander effects [29] of NSCPs may serve essential roles in reducing infarct volume in the early stroke stage, revealed by TTC staining and MRI (Fig. 4). The primary functions of NSCPs include protecting the blood-brain barrier [30], promoting the secretion of neurotrophic factors such as BDNF, NTFs, and VEGF [31], inhibiting local neuroinflammation [32], and enhancing angiogenesis [33]. Therefore, activation of NSPCs by ART treatment could reduce the infarct volume at the early stage of stroke. ...
Article
Full-text available
Promoting neurogenesis and proliferation of endogenous neural stem/progenitor cells (NSPCs) is considered a promising strategy for neurorehabilitation after stroke. Our previous study revealed that a moderate dose of artesunate (ART, 150 mg/kg) could enhance functional recovery in middle cerebral artery occlusion (MCAO) mice. This study aimed to investigate the effects of ART treatment on neurogenesis and proliferation of NSPCs using a rodent MCAO model. MRI results indicated that the ischemic brain volume of MCAO mice was reduced by ART treatment. The results of diffusion tensor imaging, electron microscopic, and immunofluorescence of Tuj-1 also revealed that ischemia-induced white matter lesion was alleviated by ART treatment. After ischemia/reperfusion, the proportion of Brdu + endogenous NSPCs in the ipsilateral subventricular zone and peri-infarct cortex was increased by ART treatment. Furthermore, the neuro-restorative effects of ART were abolished by the overexpression of FOXO3a. These findings suggested that ART could rescue ischemia/reperfusion damage and alleviate white matter injury, subsequently contributing to post-stroke functional recovery by promoting neurogenesis and proliferation of endogenous NSPCs via the FOXO3a/p27Kip1 pathway.
... Retinoic acid-pretreated ESCs have been shown to be effective for the treatment of rodent models of ischemia [26], in which neurological and behavioral tests indicated functional restoration. Motor neurons derived from ESCs have demonstrated the ability to alter motor functions in a rodent model of hereditary amyotrophic lateral sclerosis (ALS) [27], and multipotent neural progenitor cells (NPCs) have demonstrated the ability to reduce the clinical indications of MS in a mouse model of encephalomyelitis by reducing immune-mediated inflammation [28]. The utilization of undifferentiated ESCs has been associated with concerns about the potential to develop tumors and teratomas due to their capacity for continual proliferation. ...
Article
Full-text available
Neurodegenerative diseases are a global health issue with inadequate therapeutic options and an inability to restore the damaged nervous system. With advances in technology, health scientists continue to identify new approaches to the treatment of neurodegenerative diseases. Lost or injured neurons and glial cells can lead to the development of several neurological diseases, including Parkinson’s disease, stroke, and multiple sclerosis. In recent years, neurons and glial cells have successfully been generated from stem cells in the laboratory utilizing cell culture technologies, fueling efforts to develop stem cell-based transplantation therapies for human patients. When a stem cell divides, each new cell has the potential to either remain a stem cell or differentiate into a germ cell with specialized characteristics, such as muscle cells, red blood cells, or brain cells. Although several obstacles remain before stem cells can be used for clinical applications, including some potential disadvantages that must be overcome, this cellular development represents a potential pathway through which patients may eventually achieve the ability to live more normal lives. In this review, we summarize the stem cell-based therapies that have been explored for various neurological disorders, discuss the potential advantages and drawbacks of these therapies, and examine future directions for this field.
... Along with PD, several lines of research have aimed to replace dead or damaged cells in the respective rodent model of disease in order to bring a stem cell-based disease-modifying therapy to the clinic. For example, transplantation of human iPSCs differentiated to neural stem cells in rodent models of spinal cord injury [164][165][166] and ischemic stroke [167][168][169] have been successful. These cells survive, differentiate, migrate, and result in functional recovery in some cases. ...
Article
Full-text available
Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism's somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been twofold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer's disease and Parkinson's disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.
... Many studies using animal models mimicking different neurological conditions with brain damage have shown that stem cell-based treatment might improve recovery through two types of action mode. First mode, the so-called bystander effect, is proposed to be caused by release of different factors leading to immunomodulation, reduction of brain-blood-barrier damage (Eckert et al., 2015), stimulation of angiogenesis, endogenous neurogenesis, and neuronal plasticity (Chang et al., 2013;Mine et al., 2013). The second mode is based on cell replacement and justified by recent publications demonstrating the ability of grafted pluripotent stem cells (PSCs) to morphologically differentiate into different types of neurons, establish synaptic connections with the host circuitry and get integrated in damaged neuronal network (Grade and Gotz, 2017). ...
Article
Full-text available
Stem cell therapy using human skin-derived neural precursors holds much promise for the treatment of stroke patients. Two main mechanisms have been proposed to give rise to the improved recovery in animal models of stroke after transplantation of these cells. First, the so called by-stander effect, which could modulate the environment during early phases after brain tissue damage, resulting in moderate improvements in the outcome of the insult. Second, the neuronal replacement and functional integration of grafted cells into the impaired brain circuitry, which will result in optimum long-term structural and functional repair. Recently developed sophisticated research tools like optogenetic control of neuronal activity and rabies virus monosynaptic tracing, among others, have made it possible to provide solid evidence about the functional integration of grafted cells and its contribution to improved recovery in animal models of brain damage. Moreover, previous clinical trials in patients with Parkinson’s Disease represent a proof of principle that stem cell-based neuronal replacement could work in humans. Our studies with in vivo and ex vivo transplantation of human skin-derived cells neurons in animal model of stroke and organotypic cultures of adult human cortex, respectively, also support the hypothesis that human somatic cells reprogrammed into neurons can get integrated in the human lesioned neuronal circuitry. In the present short review, we summarized our data and recent studies from other groups supporting the above hypothesis and opening new avenues for development of the future clinical applications.
... However, this approach has not yet been tested in humans. Most of the current studies were conducted in animal models ( Table 1; Brenneman et al., 2010;Chang et al., 2013;Kawabori et al., 2013;Cheng et al., 2015;Webb et al., 2018;Tian et al., 2019;Tobin et al., 2020;Asgari Taei et al., 2021). Further studies are needed to determine the best route for stem cell transplantation in treating stroke patients. ...
Article
Full-text available
Strokes are the most common types of cerebrovascular disease and remain a major cause of death and disability worldwide. Cerebral ischemic stroke is caused by a reduction in blood flow to the brain. In this disease, two major zones of injury are identified: the lesion core, in which cells rapidly progress toward death, and the ischemic penumbra (surrounding the lesion core), which is defined as hypoperfusion tissue where cells may remain viable and can be repaired. Two methods that are approved by the Food and Drug Administration (FDA) include intravenous thrombolytic therapy and endovascular thrombectomy, however, the narrow therapeutic window poses a limitation, and therefore a low percentage of stroke patients actually receive these treatments. Developments in stem cell therapy have introduced renewed hope to patients with ischemic stroke due to its potential effect for reversing the neurological sequelae. Over the last few decades, animal tests and clinical trials have been used to treat ischemic stroke experimentally with various types of stem cells. However, several technical and ethical challenges must be overcome before stem cells can become a choice for the treatment of stroke. In this review, we summarize the mechanisms, processes, and challenges of using stem cells in stroke treatment. We also discuss new developing trends in this field.
Preprint
Full-text available
Promoting neurogenesis and proliferation of endogenous neural stem/progenitor cells (NSPCs) is considered a promising strategy for neurorehabilitation after stroke. Our previous study revealed that a moderate dose of artesunate (ART, 150mg/kg) could enhance functional recovery in middle cerebral artery occlusion (MCAO) mice. This study aimed to investigate the effects of ART treatment on neurogenesis and proliferation of NSPCs using a rodent MCAO model. MRI results indicated that the ischemic brain volume of MCAO mice was reduced by ART treatment. The results of diffusion tensor imaging, electron microscopic, and immunofluorescence of Tuj-1 also revealed that ischemia-induced white matter lesion was alleviated by ART treatment. After ischemia/reperfusion, endogenous NSPCs were activated by ART, which was displayed by comparing the proportion of Brdu+ neuronal precursor cells in the ipsilateral subventricular zone and peri-infarct cortex. Furthermore, the neuro-restorative effects of ART were abolished by the overexpression of FOXO3a. These findings suggested that ART could rescue penumbra damage and alleviate white matter injury, subsequently contributing to post-stroke functional recovery by promoting neurogenesis and proliferation of endogenous NSPCs via the FOXO3a/p27 Kip1 pathway.
Article
Full-text available
Neurological disorders are big public health challenges that are afflicting hundreds of millions of people around the world. Although many conventional pharmacological therapies have been tested in patients, their therapeutic efficacies to alleviate their symptoms and slow down the course of the diseases are usually limited. Cell therapy has attracted the interest of many researchers in the last several decades and has brought new hope for treating neurological disorders. Moreover, numerous studies have shown promising results. However, none of the studies has led to a promising therapy for patients with neurological disorders, despite the ongoing and completed clinical trials. There are many factors that may affect the outcome of cell therapy for neurological disorders due to the complexity of the nervous system, especially cell types for transplantation and the specific disease for treatment. This paper provides a review of the various cell types from humans that may be clinically used for neurological disorders, based on their characteristics and current progress in related studies.
Chapter
Neural repair is a therapeutic strategy distinct from acute stroke strategies, as the goal is to boost function in surviving brain elements rather than salvage threatened tissue. Many classes of therapy are under study in animals and in human trials to improve stroke recovery, including drugs, biologic agents, brain stimulation, activity-based therapies, cognitive-based therapies, and lesion bypass. This chapter reviews progress with these therapies. Principles of neural repair after stroke are also reviewed. (1) Repair-based therapies are not a one-size-fits-all program—a single treatment is unlikely to improve outcomes across all infarct sizes or behavioral deficits. Instead, repair-based therapies often benefit from being individualized: based on measures of brain structure and function or genetics, akin to what is currently done in many other fields of medicine. (2) Time is an important factor for repair-based therapies, especially during the initial days and weeks post-stroke, given the evolution of brain biology during this time period. Neural repair is experience dependent. Many forms of therapy galvanize brain circuits and foster clinically useful plasticity, and concomitant training is often useful to maximize this therapeutically generated enhanced potential for plasticity. Implications of these principles are considered in the context of clinical trials of repair-based therapies.
Article
Full-text available
Ischemic stroke remains a major health problem associated with high mortality and severe morbidity. In spite of the extensive research in the field of stroke biology, there is little effective treatment for a completed disease onset. Numerous neuroprotective strategies have failed in clinical trials because of lack of efficacy or unacceptable side-effects. The challenge of clinical trial design is now to understand the process leading to ischemic brain injury and thus identify the targets for intervention in stroke. Therefore, uncovering cellular and molecular processes involved in ischemic brain injury is of critical importance. The review discusses the current understanding of these processes engaged in pathogenesis of stroke including excitotoxicity and inflammation. In addition recruitment of endogenous progenitors engaged in neurogenesis and vascular regeneration has been implicated. All of the aforementioned changes opted for therapeutic intervention to protect neurons in the region adjacent to the ischemic cerebral tissue and enhance cell recovery. Stem cell transplantation seems to offer a major promise of this therapy in stroke disorders.
Article
Full-text available
Transplantation of human mesenchymal stem cells has been shown to reduce infarct size and improve functional outcome in animal models of stroke. Here, we report a study designed to assess feasibility and safety of transplantation of autologous human mesenchymal stem cells expanded in autologous human serum in stroke patients. We report an unblinded study on 12 patients with ischaemic grey matter, white matter and mixed lesions, in contrast to a prior study on autologous mesenchymal stem cells expanded in foetal calf serum that focused on grey matter lesions. Cells cultured in human serum expanded more rapidly than in foetal calf serum, reducing cell preparation time and risk of transmissible disorders such as bovine spongiform encephalomyelitis. Autologous mesenchymal stem cells were delivered intravenously 36-133 days post-stroke. All patients had magnetic resonance angiography to identify vascular lesions, and magnetic resonance imaging prior to cell infusion and at intervals up to 1 year after. Magnetic resonance perfusion-imaging and 3D-tractography were carried out in some patients. Neurological status was scored using the National Institutes of Health Stroke Scale and modified Rankin scores. We did not observe any central nervous system tumours, abnormal cell growths or neurological deterioration, and there was no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following stem cell infusion. The median daily rate of National Institutes of Health Stroke Scale change was 0.36 during the first week post-infusion, compared with a median daily rate of change of 0.04 from the first day of testing to immediately before infusion. Daily rates of change in National Institutes of Health Stroke Scale scores during longer post-infusion intervals that more closely matched the interval between initial scoring and cell infusion also showed an increase following cell infusion. Mean lesion volume as assessed by magnetic resonance imaging was reduced by >20% at 1 week post-cell infusion. While we would emphasize that the current study was unblinded, did not assess overall function or relative functional importance of different types of deficits, and does not exclude placebo effects or a contribution of recovery as a result of the natural history of stroke, our observations provide evidence supporting the feasibility and safety of delivery of a relatively large dose of autologous mesenchymal human stem cells, cultured in autologous human serum, into human subjects with stroke and support the need for additional blinded, placebo-controlled studies on autologous mesenchymal human stem cell infusion in stroke.
Article
Full-text available
Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.
Article
Full-text available
Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5 x 10(5)) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.
Article
Full-text available
For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.
Article
We have examined the incidence and size of infarction after occlusion of different portions of the rat middle cerebral artery (MCA) in order to define the reliability and predictability of this model of brain ischemia. We developed a neurologic examination and have correlated changes in neurologic status with the size and location of areas of infarction. The MCA was surgically occluded at different sites in six groups of normal rats. After 24 hr, rats were evaluated for the extent of neurologic deficits and graded as having severe, moderate, or no deficit using a new examination developed for this model. After rats were sacrificed the incidence of infarction was determined at histologic examination. In a subset of rats, the size of the area of infarction was measured as a percent of the area of a standard coronal section. Focal (1-2 mm) occlusion of the MCA at its origin, at the olfactory tract, or lateral to the inferior cerebral vein produced infarction in 13%, 67%, and 0% of rats, respectively (N = 38) and produced variable neurologic deficits. However, more extensive (3 or 6 mm) occlusion of the MCA beginning proximal to the olfactory tract--thus isolating lenticulostriate end-arteries from the proximal and distal supply--produced infarctions of uniform size, location, and with severe neurologic deficit (Grade 2) in 100% of rats (N = 17). Neurologic deficit correlated significantly with the size of the infarcted area (Grade 2, N = 17, 28 +/- 5% infarction; Grade 1, N = 5, 19 +/- 5%; Grade 0, N = 3, 10 +/- 2%; p less than 0.05). We have characterized precise anatomical sites of the MCA that when surgically occluded reliably produce uniform cerebral infarction in rats, and have developed a neurologic grading system that can be used to evaluate the effects of cerebral ischemia rapidly and accurately. The model will be useful for experimental assessment of new therapies for irreversible cerebral ischemia.
Article
Although grafted cells may be promising therapy for stroke, survival of implanted neural cells in the brains of stroke patients has never been documented. Human NT2N (hNT) neurons derived from the NTera2 (NT2) teratocarcinoma cell line were shown to remain postmitotic, retain a neuronal phenotype, survive >1 year in host rodent brains and ameliorate motor and cognitive impairments in animal models of ischemic stroke. Here we report the first postmortem brain findings of a phase I clinical stroke trial patient implanted with human hNT neurons adjacent to a lacunar infarct 27 months after surgery. Neurofilament immunoreactive neurons were identified in the graft site, fluorescent in situ hybridization revealed polyploidy in groups of cells at this site just like polyploid hNT neurons in vitro, and there was no evidence of a neoplasm. These findings indicate that implanted hNT neurons survive for >2 years in the human brain without deleterious effects.
Article
Stem cell-based approaches hold much promise as potential novel treatments to restore function after stroke. Studies in animal models have shown that stem cell transplantation can improve function by replacing neurons or by trophic actions, modulation of inflammation, promotion of angiogenesis, remyelination and axonal plasticity, and neuroprotection. Endogenous neural stem cells are also potential therapeutic targets because they produce new neurons after stroke. Clinical trials are ongoing but there is currently no proven stem cell-based therapy for stroke. Preclinical studies and clinical research will be needed to optimize the therapeutic benefit and minimize the risks of stem cells in stroke.