Human Neural Stem Cell Grafts Modify Microglial

Response and Enhance Axonal Sprouting in Neonatal

Hypoxic–Ischemic Brain Injury
Marcel M. Daadi, PhD; Alexis S. Davis, MD; Ahmet Arac, MD; Zongjin Li, MD, PhD;
Anne-Lise Maag, BS; Rishi Bhatnagar, BS; Kewen Jiang, MD, PhD; Guohua Sun, MD;
Joseph C. Wu, MD, PhD; Gary K. Steinberg, MD, PhD

Background and Purpose—Hypoxic–ischemic (HI) brain injury in newborn infants represents a major cause of cerebral
palsy, development delay, and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic
tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional
recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells
derived from human embryonic stem cells in a rat model of neonatal HI and the mechanisms enhancing brain repair.
Methods—The human neural stem cells were genetically engineered for in vivo molecular imaging and for postmortem
histological tracking. Twenty-four hours after the induction of HI, animals were grafted with human neural stem cells into the
forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and
neuroanatomical tracing to analyze neural differentiation, axonal sprouting, and microglia response. Treatment-induced
changes in gene expression were investigated by microarray and quantitative polymerase chain reaction.
Results—Bioluminescence imaging permitted real time longitudinal tracking of grafted human neural stem cells. HI
transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the Rotorod test. The
grafts showed good survival, dispersion, and differentiation. We observed an increase of uniformly distributed microglia cells
in the grafted side. Anterograde neuroanatomical tracing demonstrated significant contralesional sprouting. Microarray
analysis revealed upregulation of genes involved in neurogenesis, gliogenesis, and neurotrophic support.
Conclusions—These results suggest that human neural stem cell transplants enhance endogenous brain repair through
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multiple modalities in response to HI. (Stroke. 2010;41:516-523.)

Key Words: axonal sprouting 䡲 cell therapy 䡲 hypoxia ischemia 䡲 microglia 䡲 neural stem cells

H ypoxic–ischemic (HI) brain injury causes brain damage

in the fetus and newborn infants and represents a major
cause of cerebral palsy, cognitive impairment, learning dis-
ability, and epilepsy.1 Although mild body hypothermia has
been shown to improve the outcome after neonatal HI
encephalopathy when initiated within 6 hours of birth,2,3 there
are no other effective interventions to improve the chronic
sequelae of perinatal asphyxia. Neural stem cell-based therapy
offers the prospect to rescue damaged tissue, to replace lost cells,
and to restore neurological function after cerebral HI.
Early imaging studies in patients with stroke4 –7 and micro-
stimulation in experimental models of stroke8 –10 reported that
in response to ischemic injury, the brain undergoes limited
compensatory changes in an effort to recover from structural
and functional loss.11 These changes or neuroplasticity are
most prominent in early weeks and include axonal, dendritic,
and synaptic changes; inflammatory and immune adaptation;
neurogenesis; gliogenesis; and angiogenesis.12 Axonal
sprouting and reorganization manifest by changes in dendritic
arborization, spine remodeling, branching into the denervated
areas, and de novo formation of novel projections.13–17
Although limited, this spontaneous plasticity-mediated recov-
ery is a promising target for drug and cell therapeutic
interventions.17–21
In parallel to endogenous central nervous system plasticity,
inflammation and immune components are markedly acti-
vated de novo in the neonatal brain and peripheral organs
after HI.22,23 Noteworthy, this inflammatory response to
ischemic injury may exert neuroprotective and regenerative
effects on the central nervous system.24,25
In the present study, we evaluated functional recovery after
transplantation of multipotent human embryonic stem cell-

Received November 20, 2009; accepted December 1, 2009.

From the Department of Neurosurgery and Stanford Stroke Center (M.M.D., A.S.D., A.A., A.-L.M., R.B., K.J., G.S., G.K.S.), Stanford University
School of Medicine, Stanford, Calif; and Department of Radiology and Molecular Imaging Program (Z.L., J.C.W.), Stanford University School of
Medicine, Stanford, Calif.
Present address for K.J.: Department of Neurology, Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Correspondence to Marcel M. Daadi, PhD, Stanford University, Department of Neurosurgery, MSLS P304, 1201 Welch Road, Stanford, CA
94305-5487. E-mail mdaadi@stanford.edu
© 2010 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.109.573691

516
 Daadi et al Neural Stem Cells in Neonatal Hypoxia Ischemia 517

derived human neural stem cells (hNSCs) into the rat model of
neonatal HI. We also investigated modalities by which grafted
hNSCs provide therapeutic benefits to the HI-damaged brain.

Materials and Methods

Derivation of Multipotent hNSCs
Human neural stem cells were derived from human embryonic stem
cells and perpetuated as previously described.26,27

Induction of HI and Cell Transplantation

All animal experimentations were conducted according to the Na-
tional Institutes of Health guidelines and approved by the Institu-
tional Animal Care and Use Committee. Seven-day-old Sprague-
Dawley rats (Charles River Laboratories; Wilmington, Mass) were
subjected to permanent ligation of the left carotid artery followed by
90 minutes in a hypoxic chamber (8% O2 and 92% N2, at 37°C).28
The newborns were divided into HI vehicle (total n⫽12) and HI
transplant (total n⫽12) groups. Twenty-four hours after the induc-
tion of HI, animals were placed in a stereotaxic apparatus with
neonatal rat adaptor (Kopf Instruments). The skull bregma was
determined and a single cell suspension (50 000 cell/␮L) of hNSCs
(passages P9 to P15) was transplanted, using a Hamilton syringe,
into 3 sites (2 ␮L/site) in the left ischemic hemisphere at the
following stereotaxic coordinates in millimeters: anteroposterior: ⫹0.0,
mediolateral: ⫹3.0, dorsoventral: ⫺4.0; anteroposterior: ⫹0.5, me-
diolateral: ⫹2.5, dorsoventral: ⫺3.5; anteroposterior: ⫺1.0, medio-
lateral: ⫹2.0, dorsoventral: ⫺4.0. The injection rate was 1 ␮L/min,
and the cannula was left in place for 5 minutes before retraction.

Bioluminescence Imaging of Grafted hNSCs

In Vivo
Bioluminescence imaging was performed using the Xenogen in vivo
imaging system. Due to space limitation, we refer the reader to our
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recent publication27 where we described in detail the biolumines-

cence imaging technique.

Behavioral Tests
The animals were evaluated 4 weeks after transplantation for their
sensorimotor skills in the cylinder and in the Rotorod tests, as we
previously reported.26,29

Transcriptome Analysis
Total RNA was isolated from the ipsilateral hemisphere of normal, HI,
and HI transplant groups (n⫽3) using the RNAeasy kit (Qiagen)
according to the manufacturer’s instructions. The Oligo GEArray
neurogenesis and neural stem cell microarrays were purchased from
SuperArray (SA Biosciences Corporation). The protocol for the microarray
analyses followed the manufacturer’s recommendations. Array spot density
and differential probe expression were calculated using SuperArray’s
GEArray Expression Analysis Suite software. Spot density was normalized
to select positive and negative controls spotted onto each array.

Quantitative Reverse Transcriptase–Polymerase

Chain Reaction
Total RNA was extracted from tissue dissected from the ipsilateral
hemisphere of vehicle and hNSC-treated animals (n⫽3). Aliquots (1
␮g) of total RNA from the cells were reverse transcribed as Figure 1. Imaging of human embryonic stem cell-derived
previously described.30 Real-time polymerase chain reaction was hNSCs in HI neonates. A, Neural stem cells were isolated
performed using Maxima SYBR Green qPCR Master Mix (Fermen- through a multistep process previously described.26,27 Flow
tas) in the Stratagene thermocycler MX3000P (Stratagene) according cytometry analysis demonstrated the purity of the eGFP-
to the supplier’s instructions. Data were expressed as mean⫾SEM. expressing hNSCs. B, In vitro imaging analysis of genetically
The ⌬Ct for each candidate was calculated as ⌬Ct of [Ct (gene of engineered hNSCs show increasing fLuc activity with cell den-
sity and a linear correlation (R2⫽0.98). C, Data are representa-
interest)⫺Ct (18S)] and the ⌬⌬Ct was the difference between the Ct
tive of 3 independent experiments performed in triplicate. Rep-
of the treated sample (transplanted animals) and the Ct of the control
resentative bioluminescence imaging of a neonate rat
vehicle sample. The relative expression was calculated as the 2⌬⌬Ct
ˆ
transplanted with the hNSCs and monitored for 4 weeks. D,
according to the methods31 and plotted as relative levels of gene Color scale bar is in photon/s/cm2/sr.
expression. The rat primers were designed using the Primer3
software and are available on request.
 518 Stroke March 2010
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Biotinylated Dextran Amine Injections
The last week before euthanasia of the animals, 3 randomly selected
animals from both animal groups were anesthetized and placed in the
stereotaxic apparatus. After craniotomy, 0.5 ␮L of biotinylated
dextran amine (BDA; 10 000 molecular weight; Molecular Probes;
10% wt/vol solution in sterile phosphate-buffered saline) was in-
jected stereotaxically into the sensorimotor cortex opposite to the HI
lesion site at the stereotaxic coordinates: anteroposterior: ⫹0.5 m,
mediolateral: 2.5 mm, and DV: ⫺1.5 mm. The scalp was then closed
and the animal returned to its cage.
Histopathology, Immunocytochemistry, and
Microscopical Analysis
Immunocytochemistry was performed as we previously reported.26
The following primary antibodies were used: antihuman Nuclei
(hNuc), anti-NeuN, anti-GAD65/67, antiglial fibrillary acidic pro-
Figure 2. Characterization of hNSC grafts.
A, Example of triple labeling of trans-
planted animal with the human nuclear
specific marker hNuc (purple) and the
neuronal markers doublecortin (red) and
TuJ1 (green). B, Higher magnification
shows the dispersion of the grafted
hNuc⫹ (purple) hNSCs in the striatum.
Photomicrographs show coexpression of
hNuc (purple) and the neuronal markers:
TuJ1 (green; C), doublecortin (green; D),
and NeuN (green; E). F, Colocalization of
hNuc and the astroglial marker glial fibril-
lary acidic protein, and (G) example of
grafted cell expressing the GABAergic
neurotransmitter marker GAD. Bars: (A–B)
100 ␮m; (B, far right, E) 20 ␮m; (C, D, F,
G) 10 ␮m.
tein, antigalactocerebrocide, anti-Nestin (Chemicon), anti-TuJ1 (Co-
vance), anti-CNPase (Aves Labs), and antidoublecortin (SantaCruz
Biotechnology). Secondary antibodies raised in the appropriate hosts
and conjugated to fluorescein isothiocyanate, RITC, AMCA, CY3,
or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells
and sections were counterstained with the nuclear marker 4⬘,6-
diamidine-2⬘-phenylindole dihydrochloride. Fluorescence was de-
tected, analyzed, and photographed with a Zeiss LSM550 laser
scanning confocal photomicroscope (Carl Zeiss).
For the rat, anti-Iba-1 (ionized calcium binding adapter molecule;
Wako Bioproducts) and BDA immunohistochemistry was carried
out, as we previously described.29 For each animal, quantitative
estimates of the number of Iba-1⫹ cells were stereologically
determined using the optical fractionator procedure,32 as we previ-
ously described in detail.29 The quantitative analysis of BDA-labeled
fibers was performed, as we previously reported.29 The infarct
 Daadi et al Neural Stem Cells in Neonatal Hypoxia Ischemia 519
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Figure 3. Transplanted hNSCs enhanced axonal sprouting. Confocal image analysis and quantification of BDA-labeled axons and axon
terminals in the sensorimotor cortex (A), corpus callosum (B), striatum (C), and in the thalamus (D) show significant increase on the ipsi-
lateral HI-lesioned and hNSC-transplanted side (n⫽3 per group). Data are expressed as mean⫾SEM, *P⬍0.05 versus vehicle group.

volume was estimated by summing up the infarcted areas in all
animals of each group. To eliminate the effects of edema, infarct size
was calculated as the contralateral hemisphere⫺ipsilateral nonische-
mic hemisphere/contralateral hemisphere and expressed as percent-
age (⫻100%) of infarcted hemisphere.
Statistics
Outcome measurement for each experiment was reported as
mean⫾SEM. Data were analyzed using SPSS 11 for Mac OS X
(SPSS Inc). Significance of intergroup differences was performed by
applying Student t test where appropriate. One-way analysis of
variance was used to compare groups. Differences between the
groups were determined using Bonferroni post hoc test. A probabil-
ity value of ⬍0.05 was considered statistically significant.
Results
Real-Time Imaging of hNSC Transplants
The hNSCs grew as an adherent monolayer culture and all
progeny expressed double fusion construct carrying fLuc and
eGFP reporter genes (Figure 1A).
 520 Stroke March 2010

Image analysis (Figure 1B) demonstrated a linear correla-

tion between plating density and the fLuc activity (Figure
1C). This stable and efficient expression of fLuc gene enabled
us to noninvasively image, in real time, the survival of the
grafted hNSCs at different time points (Figure 1D).

Characterization of the HI Infarct Size and the

hNSC Graft
Microscopic examination of cresyl violet-stained sections
showed significant loss of brain tissue in HI animals in the
neocortex, the striatum, and extending caudally to the hip-
pocampus. This atrophy was accompanied sometimes by the
formation of a porencephalic cyst. The estimation of the
infracted region revealed 17.8%⫾8.6% of the hemisphere
(n⫽6) for the transplanted group and 20.66%⫾12.5% (n⫽6)
for the vehicle group. Although hNSC-grafted animals
showed smaller infarct volume, the difference from the
vehicle group was not significant (n⫽6). Grafted hNSCs
identified with hNuc demonstrated good survival dispersion
and integration into the HI-damaged tissue (Figure 2A–B).
Transplanted cells expressed the neuroepithelial stem cell
marker nestin in 12.8%⫾3.4% and differentiated into neurons
expressing NeuN, TuJ1⫹ (42.9%⫾3.5%), doublecortin
(4.8%⫾1.1%; Figure 2C–E), and the GABAergic marker
GAD (47.6%⫾4.3%; Figure 2G). Grafted cells also differen-
tiated into glial fibrillary acidic protein-expressing astrocytes
(Figure 2F).
Figure 4. Microarray analysis and quantitative real-time reverse
hNSC Grafts Enhance Axonal Sprouting transcribed–polymerase chain reaction measurement of selected
To investigate whether the transplanted hNSCs influenced the genes upregulated in transplanted animals. Genes from the
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rewiring of the HI-damaged tissue, we used BDA to antero-
grade label the sensorimotor cortical projection originating
from the contralateral side. We measured the density of
BDA⫹ crossing fibers in the corpus callosum and of BDA⫹
terminals in the ipsilateral sensorimotor cortex, the striatum,
and thalamic nuclei. The quantitative analysis of BDA-
labeled fibers and terminals, normalized to the total number
of labeled somas at the injections site,29 revealed an increase
in both the number of fibers crossing toward the ipsilateral
hemisphere (Figure 3B) and in the number of terminals in the
ipsilateral sensorimotor cortex (Figure 3A), the striatum
(Figure 3C), and thalamic nuclei (Figure 3D).
Transcriptome Analysis
Gene microarray analysis revealed, in comparison to the
vehicle group, a significant increase in central nervous system
rat endogenous genes involved: (1) in neurogenesis and cell
migration (doublecortin, chemokine [C-X-C motif] receptor 4
[CXCR4], oligodendrocyte lineage transcription factor 2, and
FGF2); (2) in myelination (myelin basic protein); and (3) in
genes involved in cell survival and neurite outgrowth (glial
derived neurotrophic factor, Neurturin, and insulin growth
factor-1; Figure 4A).33 Subsequently, we confirmed the mi-
croarray expression patterns of selected genes using real-time
quantitative reverse transcribed–polymerase chain reaction
(Figure 4B).
Grafts of hNSCs Modulate Microglial Presence in
the HI-Damaged Area
Immunostaining with the pan-microglial marker Iba1 demon-
strated that microglia were homogenously distributed
expression profile with probability values ⬍0.01 were selected
for analysis. A, Fold of increase of genes involved in neurogen-
esis, gliogenesis, and neurotrophic support in HI and HI trans-
plant groups relative to the naïve group. B, Quantitative reverse
transcribed–polymerase chain reaction of the selected genes
confirming the expression patterns seen in the microarray data.
throughout the brain in both vehicle and hNSC-grafted
animals without adverse infiltration or reaction against the
grafts (Figure 5A–B). Stereological quantitative analysis of
the Iba1-positive cells demonstrated a significant increase in
the transplanted striatum (Figure 5D).
Improvement of Motor Behavior in Rats That
Received the hNSC Grafts
To determine the ability of hNSCs to functionally engraft, 1
month after transplantation, the sensorimotor skills of animals
were evaluated using 2 neurobehavioral tests. Our results
showed that during the fourth week, HI transplanted animals
significantly improved in their use of the contralateral impeded
forelimb (Figure 6A–B). The hNSC grafts significantly amelio-
rated the locomotor deficits in the Rotorod test (Figure 6C).
Discussion
Genetically engineered hNSCs were efficiently and noninva-
sively imaged in real time after transplantation into the HI
model of newborn rats and up to 5 weeks of age. We report that
hNSCs engrafted into the ischemic brain enhanced axonal
sprouting and the expression of genes involved in neurogenesis,
gliogenesis, and neurotrophic support; modulated microglial
response; and improved motor function of the animals.
 Daadi et al Neural Stem Cells in Neonatal Hypoxia Ischemia 521

Figure 5. Stereological analysis of Iba1-expressing microglia in vehicle and transplanted groups. Brain sections from vehicle (A) and
hNSC-grafted (B) animals were processed for the pan-microglial marker Iba1 immunohistochemistry. Stereological quantification of the
Iba1⫹ cells in the cortex (C) and striatum (D) of grafted animals. Tx indicates transplant. Bars: (A, B) 20 ␮m.
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It is generally believed that transplanted nonneural cells provide cell replacement and neurotrophic support.29,35,36
such as those derived from bone marrow or cord blood exert This neurotrophic support may be responsible for the signif-
neurotrophic effects on ischemia-injured tissue and may not icant axonal sprouting that we measured in the cortex, the
survive long term,34 whereas neural stem cells are thought to striatum, and the thalamus. The number of BDA-labeled axon

Figure 6. Transplantation of hNSCs improves sen-

sorimotor function. The independent use of the
impaired contralateral forelimb significantly
increased in the transplant group (B; n⫽3) on Days
28 and 30 posttransplantation (*P⬍0.05 versus
vehicle group. A, Bars represent percentages⫾SEM of
steps taken by the ipsilateral, contralateral, and both
forelimbs simultaneously. On Days 29 and 30, the
transplant group (n⫽9) showed significant improvement
(*P⬍0.05) on the Rotorod test.
 522 Stroke March 2010
terminals may vary with the size of the injection site and the
number of BDA⫹ cell bodies. The BDA infusion led to small
(3 to 5 mm2) and circumscribed injection sites in the
sensorimotor cortex. There was no significant difference in
the size of the injection site and the total number of labeled
cells between vehicle and transplanted groups. In addition,
the data were normalized to the total number of labeled cells.
Thus, it is unlikely that the increase of axonal sprouting we
observed is due to differences in the size or location of the
injection site.
After ischemia, regions of the contralateral hemispheres
become activated during the early phase of partial regain of
function or spontaneous recovery in experimental models37
and in patients with stroke.38 – 40 In neonate HI rats, contrale-
sional sprouting could give rise to alternative motor descend-
ing pathways from the motor cortex relaying in subcortical
structures such as the red nucleus and the pontine formation
or direct corticospinal projections may be formed to compen-
sate for functional loss.41 The newly formed pathways could
be generated by multiple mechanisms, including sprouting
from the surviving neurons, unmasking of existing pathways
that are functionally inactive, or the compensatory descend-
ing control channels through alternative functionally active
but redundant pathways.42,43
The intracerebral or intravenous delivery of multipotent
adult progenitor cells provided protection to HI-damaged
tissue in neonates and improved motor and neurological
scores compared with the vehicle group.44 Using retrograde
and anterograde neuroanatomical tracing, Park et al36 dem-
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onstrated that neural stem cells grafted into HI-lesioned
neonates, along with a polyglycolic acid-based biodegradable
polymer scaffold, re-established long-distance cross-callosal
neuronal connections. Recent genomic analysis studies dem-
onstrated that in addition to these growth factors, axonal
sprouting is activity-dependent in constraint-induced move-
ment therapy.45 Together, these data suggest that grafted
hNSCs could exert neurotrophic and/or activity-mediated
effects on a HI-damaged local network and enhance axonal
sprouting. Based on these observations, it seems reasonable
to propose that the newly innervated and recruited area of the
contralesional hemisphere is becoming part of a reorganized
network promoting motor recovery.
Our data are in line with previous studies that show an
increase in the resident microglia/macrophage cells (CD11b)
in ischemic animals that received neurosphere-derived cells.46
Capone et al suggest that microglia activation is required for
neurosphere graft neuroprotective action through secretion of
growth factors, including insulin growth factor-1, vascular
endothelial growth factor-A, transforming growth factor-␤1,
and brain-derived neurotrophic factor.46 Indeed, brain-
derived neurotrophic factor treatment of HI neonates im-
proves spatial memory47 and selective ablation of microglia
through the mutant thymidine kinase gene driven by myeloid-
specific CD11b promoter exacerbated ischemic injury.48
Thus, these findings support the notion that microglia play a
dual role, proinflammatory or anti-inflammatory/neurotro-
phic, depending on their state of activation and functional
phenotype.24
In conclusion, we provide evidence that growth factor-
isolated and perpetuated hNSCs from human embryonic stem
cells are amenable to genetic modification for real time in
vivo imaging and potentially for other therapeutic genes. We
showed that these hNSCs are able to modify the host
microenvironment and enhance neuroanatomical plasticity
after HI in neonates and improve sensorimotor skills.
Acknowledgments
We thank Beth Hoyte for preparation of the figures.
Sources of Funding
This work was supported in part by Russell and Elizabeth Siegelman,
Bernard and Ronni Lacroute, the William Randolph Hearst Founda-
tion, CIRM RS1-00322, the Edward G. Hills Fund, and National
Institutes of Health National Institute of Neurological Diseases and
Stroke grants RO1 NS27292, P01 NS37520, and R01 NS058784.
Disclosures
None.
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