Adherent Self-Renewable Human Embryonic Stem Cell-
Derived Neural Stem Cell Line: Functional Engraftment in
Experimental Stroke Model
Marcel M. Daadi*, Anne-Lise Maag, Gary K. Steinberg
Department of Neurosurgery and Stanford Stroke Center, Stanford University School of Medicine, Stanford, California, United States of America
Background: Human embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in
neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating
specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated
that the direct enrichment method generates a heterogeneous population of cells that may contain residual
undifferentiated stem cells that could lead to tumor formation in vivo.
Methods/Principal Findings: We isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs
using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and
leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and
uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not
express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages.
The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56
hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after
transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited
no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naı ¨ve nude rat
brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain
parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.
Conclusions/Significance: The SD56 human neural stem cells derived under the reported conditions are stable, do not form
tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a
renewable, homogenous source of neural cells that will be valuable for basic and translational research.
Citation: Daadi MM, Maag A-L, Steinberg GK (2008) Adherent Self-Renewable Human Embryonic Stem Cell-Derived Neural Stem Cell Line: Functional
Engraftment in Experimental Stroke Model. PLoS ONE 3(2): e1644. doi:10.1371/journal.pone.0001644
Editor: Tailoi Chan-Ling, University of Sydney, Australia
Received September 28, 2007; Accepted January 23, 2008; Published February 20, 2008
Copyright: ? 2008 Daadi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by Russell and Elizabeth Siegelman, Bernard and Ronni Lacroute, the William Randolph Hearst Foundation, John and
Dodie Rosekrans, the Edward E. Hills Fund, Gerald and Marjorie Burnett, and NIH NINDS grants RO1 NS27292 and P01 NS37520.
Competing Interests: Stanford University has filed a patent application on this method of derivation and expansion of neural stem cells.
To date there have been no effective treatments for improving
residual structural and functional deficits resulting from stroke.
Current therapeutic approaches, such as the use of thrombolytics,
benefit only 1 to 4% of patients . Consequently, the majority of
stroke patients experience progression of ischemia associated with
debilitating neurological deficits. Recent evidence has suggested
that the transplantation of cells derived from cord blood, bone
marrow or brain tissue (fetal and adult) enhances sensorimotor
function in experimental models of stroke [2,3]. However, the
normal human-derived somatic stem cells used in these studies
have a limited capacity to differentiate into the diverse neural cell
types optimal for structural and physiological tissue repair and are
not amenable for large-scale cell production.
Unlike other sources of stem cells, hESC lines possess a nearly
unlimited self-renewal capacity and the developmental potential to
differentiateinto virtuallyanycelltype ofthe organism.Assuch,they
constitute an ideal source of cells for regenerative medicine. The
successful derivation of hESC lines from the inner cell mass of
preimplantation embryos and their long-term maintenance in vitro
over multiple passages has been demonstrated  and standardized.
Differentiation and enrichment processes that direct hESCs towards
a neural lineage have also been achieved. To promote neuralization,
ESCs were cultured in a defined media supplemented with
morphogens or growth factors [5,6,7] or cultured under conditions
that promote ‘‘rosettes’’, structures morphologically similar to the
developing neural tube [8,9]. This neuralization process has proven
invaluable in understanding the specification of hESC-derived
neural tissue [10,11,12]. However, the enriched neural progeny
derived displayed overgrowth and limited migration after grafting
into normal newborn mice  and lesioned adult rat striatum
[12,14,15,16]. The inner cores of these grafts contained tumorigenic
precursor cells (reviewed in ). These findings suggest that neural
cells generated by acute exposure to growth factors and/or
morphogens may still be heterogeneous and potentially tumorigenic.
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We report an alternative method for the isolation and the
perpetuation of a multipotent hNSC line from the hESCs with a
primitive mode of self-renewal. We also demonstrate their long-
term expansion, non-tumorigenic properties and functional
engraftability in an experimental model of stroke.
1. In vitro isolation, growth and differentiation of hESC-
The hESCs were maintained and expanded on mouse feeder
layer in media supplemented with bFGF (Figure 1A). After cell
dissociation, a portion of the hESCs was cultured in serum free
medium containing EGF, bFGF and LIF. These factors are known
to stimulate the proliferation of human fetal-derived NSCs
[18,19]. After 3 days in vitro (DIV), there was selective survival
and growth of cells that aggregated in clusters or spheres
(Figure 1B). These primary spheres were harvested and replated
in fresh media. During the following week, the spheres attached to
the flask and a fibroblast-like cell population began to migrate out
(Figure 1C). Secondary spheres (2u spheres) were generated from
these cultures and lifted off by the end of the week leaving a hollow
in the middle of the attached cells (Figure 1D). The floating 2u
spheres were collected and replated in fresh growth medium for
2 weeks. The cultures were then passaged by collagenase cell
dissociation every 7 DIV for an additional 4 passages (Figure S1).
At the 5thand 6thpassages, spheres were dissociated into a single-
cell suspension using trypsin-EDTA. At this stage there was a
change in the hNSCs’ adherent properties, and the cells began to
grow as a monolayer with multiple foci of cells throughout the
Figure 1. Isolation and purification of hNSCs from the hESCs. The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B)
were isolated and replated to eliminate other non-neural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow
cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2u spheres were then
passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. (G, H) The hNSCs were passaged
every 5–7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated
at each passage as the fold of increase6the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G)
and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (I) RT-PCR analysis showing the down regulation
of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (J) Cytogenetic evaluation of
the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female
chromosome complement (46,XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial
glial cell marker 3CB2 (M) in virtually all the progeny. (N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single
(N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and
nestin in the progeny. Bars: (A, B, C, D) 200 mm; (E, F) 100 mm; (K–M) 20 mm; (N–P) 10 mm.
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culture (Figure 1F). The adherent hNSC culture stained uniformly
for nestin (Figure 1K), vimentin (Figure 1L) and with the radial
glial marker 3CB2 (Figure 1M) indicating their homogeneity and
NSC identity. Under these culture conditions, it is noteworthy that
we did not observe the formation of rosettes which has been
previously reported to occur under certain conditions during
neuralization of hESCs [8,20,21]. RT-PCR analysis confirmed
that these hNSCs did not express the pluripotency transcripts Oct-
4 and Nanog (Figure 1I). Furthermore, the hNSCs did not express
transcripts for brachyury and foxa2, marker genes for mesoderm and
endoderm respectively (negative result, data not shown).
To ascertain self-renewal ability under clonal conditions, a
single cell suspension was plated at clonal density (1–2 cell/10 ml).
To determine if the hNSCs divide symmetrically, we pulsed
cultures with the thymidine analog, bromodeoxyuridine (BrdU),
after plating and looked for the expression of nestin in the progeny.
Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P). After
2 days, plated single cells first underwent a symmetric cell division
and gave rise to daughter cells that were both positive for BrdU
and nestin. The clone of cells continued to grow over the 3 DIV
and all the progeny expressed nestin. BrdU labeling demonstrated
that it was rare for only one daughter cell to inherit the BrdU and
thus had undergone asymmetric segregation of the chromatids
(Figure S2). G-band karyotyping of these hNSCs confirmed the
normal, non-transformed nature of cells after 12 passages
(Figure 1J). We named the derived hNSCs SD56 (intermittently
referred to as SD56 hNSCs or hNSCs).
Under these defined growth conditions, the hNSCs showed
stable growth with a 2.760.2 fold increase every 5 to 7 days
(Figure 1G). The population doubling at each passage averaged at
1.460.1 (Figure 1H). The viability of hNSCs at each passage was
consistent at the approximate value of 98%. The projected
cumulative cell numbers demonstrated that trillions of cells could
be generated over a period of 5 months (Figure 1G). We expanded
the isolated hNSCs lines for over 20 passages with a stable
phenotype. An initial cell bank of 75 vials containing 2 to 5 million
cells each was generated and cryopreserved.
Upon removal of the mitogenic factors and plating on a
coverslip pre-coated with poly-L-ornithine (PLO) substrate, the
hNSCs spontaneously differentiated into neurons, astrocytes and
oligodendrocytes, a property that is consistent with normal
multipotent hNSCs (Figure 2). After 2 DIV, hNSCs expressed
transcripts for the neural-specific genes nestin, Notch1 and neural
cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage
specific markers b-tubulin class III, medium-size neurofilament
(NF-M) and microtubule-associated protein 2 (MAP-2) for
neurons, GFAP for astrocytes and myelin basic protein (MBP)
for oligodendrocytes (Figure 2A). Transcripts for the GABAergic
cell marker glutamic acid decarboxylase (GAD) were expressed,
but transcripts for the tyrosine hydroxylase (TH), a marker for
Figure 2. hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types. Dissociated hNSCs
were washed free of growth factors and plated on poly-L-onithine coated glass coverslips. Differentiated cultures were either harvested after 2 DIV for
total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (A) Differentiated hNSCs expressed
the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (b-tubulin class III, MAP-2, NCAM and medium-size neurofilament,
NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP). The hNSCs expressed transcripts for GAD, but not for TH. B, C & D, morphology of NSC-
derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell
nuclei. (E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (F) Quantitative analysis of immunostained
10 day-old cultures for the 3 neural cell types. Results are mean6s.e.m. of three independent experiments, each performed in duplicate. Bars: (c)
20 mm; (d, e) 10 mm.
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dopaminergic neurons, were undetectable. Immunocytochemical
analysis (Figure 2B–F) of 10 day-old cultures demonstrated that
the proportion of nestin+ cells was 36.662.7%, 62.562.8%
expressed the neuronal marker TuJ1, 1.960.3% expressed the
astrocytic marker GFAP and 7.160.4% were oligodendrocytes
and expressed galactocerebrocide (GC) (Figure 2F). A subset
(9.861.6%) of the GFAP+ astrocytes co-expressed nestin.
2. The isolated hNSCs are normal and do not form
tumors in normal nude rats
The self-renewal and pluripotent abilities of the hESCs are also
associated with tumorigenic properties. Therefore, the first critical
step toward developing therapeutic hNSCs is to verify that they
are non-tumorigenic. The monolayer culture of SD56 hNSCs
clearly demonstrated contact inhibition of growth, a normal
karyotype and did not express the pluripotency transcripts Oct-4
and Nanog. Removal of mitogenic factors and continued culture
on plastic resulted in cell senescence that is characteristic of non-
transformed cells. To determine whether SD56 hNSCs form
tumors in vivo, we transplanted them at high density (see Methods)
into the forebrain and subcutaneously into the flank of nude rats.
The animals were kept for a two-month post-transplant survival
period. To label mitotically active cells in vivo during S-phase, the
rats were injected IP with BrdU (50 mg/kg) every 8 hours during
the last 24 hours before euthanasia. The transit amplifying
endogenous precursors located in the subventricular zone (SVZ)
were labeled (Figure S3); however, we were unable to detect
grafted SD56 hNSCs co-localizing the human-specific nuclear
marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs
were detected in the flank of the transplanted animals suggesting
that the grafted cells are not tumorigenic.
3. Transplanted cells survived, migrated toward and
engrafted into the stroke-damaged host tissue
To investigate the survival and functional engraftment in an
injury environment, hNSCs (46105) were transplanted into the
ischemic boundary zone in the rat striatum one week after the
middle cerebral artery occlusion (MCAO) was performed. Animals
were euthanized two months later and the brains processed for
histo-pathology and immunocytochemistry. Grafted SD56 hNSCs,
identified with hNuc, demonstrated a 37.0615.8% survival rate
and a remarkable dispersion toward the stroke-damaged tissue
with no sign of overgrowth or tumorigenesis. The majority of
grafted cells (61.264.7%) migrated at least 200 mm away from the
injection site andpenetrated
806.4649.3 mm into the stroke-damaged tissue (Figure 3A–C).
Immunostaining with the blood vessel marker, GluT1, revealed
dilated vessels in the infarcted striatum and a close association
between vessels and the grafted hNSCs (Figure 3B, 3C). The
grafted cells rarely expressed the proliferation marker Ki67
(Figure 3D), 29.864.4% expressed nestin (Figure 3E), 6.560.9%
expressed doublecortin (DCX) and 60.868.1% were TuJ1+
(Figure 3F, G). Grafted cells rarely co-expressed the astroglial
marker GFAP (Figure 3H) or differentiated into CNPase-
expressing oligodendrocytes (Figure 3I). Immunostaining for
GAD demonstrated that 25.162.3% of grafted hNSCs differen-
tiated into GABAergic neurons while less than 2% were positive
for glutamate (Figure 3J, K).
an averagedistance of
4. Transplanted cells improve sensorimotor function of
the stroke-disabled forelimb
We asked whether transplanted SD56 hNSCs could enhance
the recovery of sensorimotor function that is compromised in the
stroke-injured rats. We used the cylinder test to measure the
sensorimotor asymmetry in forelimb use during spontaneous
exploration . To establish the baseline of the stroke-induced
sensorimotor deficit, spontaneous behavior of rats in a transparent
cylinder was videotaped one week after stroke (pre-transplant,
Figure 4). Tests were then conducted 4 and 8 weeks after vehicle
and SD56 hNSCs transplantation. Stable asymmetry in forelimb
use was observed 7 days post-stroke (pre-transplant, Figure 4).
Ischemic rats used their impaired forelimbs (contralateral to lesion)
during lateral exploration less than they did before stroke.
Transplantation of SD56 hNSCs significantly enhanced the
independent use of the impaired contralateral forelimb 4 weeks
post transplantation (P,0.05 vs pre-transplant). Eight weeks after
transplantation the improvement in the use of the impaired
forelimb was stable and significant when compared to the pre-
transplant group and significantly improved in comparison to
vehicle treated group at 8 weeks (Figure 4). In the vehicle treated
group, the independent use of the contralateral forelimb remained
impaired 4 and 8 weeks post-injection. In an independent study
and using the same MCAO rat animal model, we found that
transplantation of dermal fibroblasts did not improve the stroke-
induced motor deficits (unpublished data).
Our results indicate that a self-renewable and homogenous
population of hNSCs, SD56, was derived from hESCs using
defined media supplemented with a specific combination of
growth factors. The SD56 hNSCs grew as an adherent monolayer
culture, uniformly expressed molecular features of hNSCs
including nestin, vimentin and the radial glial marker 3CB2,
and did not express the pluripotency markers Oct4 or Nanog. The
self-renewal property of the hNSCs was characterized by a
predominant symmetrical mode of cell division. They exhibited no
chromosomal abnormalities and demonstrated non-tumorigenic
properties after implantation into ischemic brains and into naı ¨ve
nude rat brains and flanks. Furthermore, the transplanted SD56
hNSCs migrated toward the stroke-damaged adult brain paren-
chyma, engrafted and improved the independent use of the stroke-
Maintenance of stem cells requires symmetrical and asymmet-
rical cell divisions to both expand and to give rise to specialized
progeny of a specific tissue (reviewed in ). In vivo, a complex
cellular micro-environment or niche ensures the self-maintenance
property of NSCs [24,25,26,27]. In vitro, defined growth factors
and extracellular matrices support stem cell self-renewal [28,29].
The embryonic stem cells can propagate in a predominantly
proliferative symmetrical mode, leading to homogeneous cell
cultures growing relatively quickly with minimal cell differentiation
[30,31,32,33,34]. Tissue specific stem cells, however, self-renew in
a predominant asymmetric mode to maintain them selves and
compensate for the loss of differentiated cells due to disease or
injury. Thus, NSCs isolated from developing or adult brain grow
as a mixture of undifferentiated and differentiated cells due the
predominant asymmetrical mode of cell division [35,36,37,
38,39,40,41]. A recent study has reported that a murine ESC-
derived NSC line (LC1) is propagated as an adherent homogenous
culture with a dominant mode of symmetrical self-renewal . A
combination of EGF and FGF2 was sufficient to propagate these
NSCs as an adherent monolayer. However, the SD56 hNSC line
described here required the combination of EGF, bFGF and LIF
for self-maintenance. Although there are morphological and
molecular similarities between our hNSCs and the NSCs
previously described , the methods of isolation and growth
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are different. In addition to the different combination of growth
factors used, the hNSC line we have isolated did not go through
the rosette-structure stage. The in vitro analysis of BrdU
incorporation and nestin expression indicated that our hNSCs
divide predominantly symmetrically. This type of growth pattern is
characteristic of primitive normal stem cells undergoing mostly
symmetric cell division to increase the stem cell pool at the early
stage of development or during tissue regeneration after injury
. RT-PCR and immunocytochemistry analysis demonstrated
that these undifferentiated SD56 cells did not express any
pluripotency, endodermal or mesodermal markers. Furthermore,
the SD56 hNSCs described here exhibited the multipotential
characteristic to differentiate into neurons, astrocytes and
oligodendrocytes both in vitro and upon transplantation. Together
these findings suggest that the hNSC line we isolated are
appropriately programmed and share similar characteristics with
the definitive NSCs of the developing brain.
The SD56 hNSCs demonstrated a remarkable ability to migrate
toward the stroke-damaged parenchyma of the adult rat brain.
This directed migration by the majority of the grafted cells could
be due to their uniform cellular composition, which results in an
equal response to the host microenvironment.
In previous studies, subpopulations of transplanted hESCs that
were enriched in neural cells migrated in host microenvironments
conducive to cell migration, such as the developing brain or in
structures such as the rostral migratory stream [13,20]. In the
adult lesioned brain, the grafted hESC-derived neural cells
proliferated and formed rosettes , teratomas [12,15] or a
Figure 3. Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals. (A) Schematic drawing of a frontal
section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (B, C) Photos
show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1
(red, B & C) showing blood vessels and dispersed hNSCs in the graft zone. C: higher magnification of the inset in B. (D–I) Photos taken from frontal
sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (D) Note the endogenous Ki67+
cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (E) Examples of grafted SD56 hNSCs
showing co-expression of hNuc (green) and nestin (red). (F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs
(arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (G) Examples show the majority of grafted NSC progeny co-expressing hNuc
(red) and the neuronal marker TuJ1 (green). Grafted NSCs rarely differentiate into GFAP+ astrocytes (H). In I, rare example of grafted NSC progeny
becoming an oligodendrocyte identified by the expression of CNPase (green). Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely
expressed glutamate (K). (Abbreviations: Cx: cortex, Str: striatum). Bars: (B, C) 100 mm; (D, F) 20 mm; (E, G–K) 10 mm.
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cellular mass that induced a gliotic host response whereby local
astrocytes demarcated the grafts . Enriched neural cultures
derived from mouse  and monkey ESCs  have produced
behavioral improvements when transplanted into animal models
of stroke and brain injury. However, in these cases, the
transplanted non-human ESCs also formed a mass with signs of
overgrowth in the core, as well as deformations [44,45,46]. ESCs
plated at low density acquire neural identity within few hours after
plating . Interestingly, nearly all viable cells expressed nestin,
the early neural fate marker Sox1, and the pluripotency marker
Oct4. Together, these studies are seminal and suggest that
complete neuralization may not be achieved through certain
enrichment processes, consequently the neural cells could revert to
a pluripotent stage . The dispersion of the grafted hNSCs
within host parenchyma may allow for more graft-host interactions
that could stabilize differentiation, inhibit growth and prevent
gliotic host response.
In the present study, SD56 hNSC-transplanted animals
demonstrated a stable improvement in the sensorimotor function
when evaluated for spontaneous exploratory activity in the
cylinder test that detects long-lasting deficits in forelimb use in
the experimental models of stroke . The transplantation of
hNSCs significantly enhanced the independent use of the impaired
contralateral forelimb 8 weeks post transplantation. This is the first
report demonstrating that the transplantation of hNSCs derived
from hESCs can improve neurologic behavior after experimental
stroke. Together, these findings are encouraging and suggest that
these cells are promising for future development. In addition to
their therapeutic application, the hNSCs isolated under the
reported conditions offer a means to interrogate host environments
and unravel mechanistic features of self-renewal, non-tumorige-
nicity and functional engraftability in animal models of neurolog-
Materials and Methods
hESC and NSC Cultures
The hESC line H9 (WiCell Research Institute) was propagated
every 5 to 7 days on irradiated mouse embryonic fibroblasts. The
cell culture media consisted of a 1:1 mixture of Dulbecco’s
modified Eagle’s medium (DMEM) and F12 nutrient, 20% serum
replacement (Invitrogen), 0.1 mM b-mercaptoethanol, 2 mg/ml
heparin and 4 ng/ml bFGF (R&D Systems). To generate the
NSCs, dissociated hESCs were cultured in a chemically defined
medium composed of DMEM/F12 (1:1) including glucose (0.6%),
glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES
buffer (5 mM) [all from Sigma except glutamine (Invitrogen)]. A
defined hormone mix and salt mixture (Sigma), including insulin
(25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM),
putrescine (60 mM), and selenium chloride (30 nM) was used in
place of serum. The medium was supplemented with EGF (20 ng/
ml), bFGF (10 ng/ml) and LIF (10 ng/ml). Dissociated hNSCs
were plated at a density of 100,000 cell/ml in Corning T75
(Invitrogen) culture flasks in the defined media together with the
growth factors. After 5–7 DIV, the adherent culture was incubated
in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the
addition of trypsin inhibitor (Invitrogen) then gently triturated to
achieve single cell suspension. The cells were then washed twice
with fresh medium and reseeded in fresh growth factor-containing
media at 100,000 cells/ml. This procedure was performed for 21
passages and the fold of increase and population doubling were
calculated at each passage. For clonal analysis, single spheres or
confluent hNSC cultures were single cell dissociated and serially
diluted to yield 1–2 cell/10 ml. A 10-ml-cell suspension was then
added to each of 96 or 24 well plates containing 200–300 ml of
growth media. Wells containing one viable cell were marked the
next day and re-scored 5 to 7 days later for cell proliferation. The
differentiation of the hNSCs was performed as previously
described . Dissociated hNSCs were plated at a density of
106cells/ml in control (media/hormone mix) medium devoid of
any growth factors and supplemented with 1% fetal bovine serum
(FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass cover-
slips in 24-well Nunclon culture dishes with 0.5 ml/well. After 2,
7–15 DIV cultures were fixed and processed for immunocyto-
chemistry or used for RT-PCR analysis.
Long-term cultures of hNSCs were incubated at 37uC and
harvested for metaphase chromosomes when the cultures were
75% confluent. Metaphase chromosomes were obtained by
standard chromosome harvest methods by exposure to Colcemid
at 0.1 mg/ml for 1 hour at 37uC, a 2-minute exposure to trypsin/
EDTA, hypotonized with 0.057 M KCl and fixed with 3:1
methanol:acetic acid. Slide preparations were made by dropping
the fixed cell pellet onto cold, wet slides and air-dried. After
incubating the slides at 90uC for 30 minutes, chromosomes were
trypsin banded and then Wright/Giemsa stained for G-banding
analysis. Twenty metaphase cells were completely analyzed and a
normal female chromosome complement was found (46,XX).
Tumorigenicity in nude rats
All animal experiments were conducted according to the
National Institute of Health (NIH) guidelines and approved by
the IACUC. Normal adult NIH-Nude rats (n=5, 8 week-old,
Figure 4. Transplantation of NSCs improves sensorimotor
function of the stroke-disabled forelimb. Forelimb use during
spontaneous lateral exploration was measured in the cylinder test (see
Method and Results sections for details). Groups of vehicle injected (n=7)
and hNSCs (n=10) transplanted are represented. The animals were tested
as described in Method section. Note the significant increase in the
independent use of the impaired contralateral forelimb at 4 and 8 weeks
post transplantation (P,0.05 vs pre-transplant group). The contralateral
forelimb remained impaired inthe vehicletreatedgroupat4 and 8 weeks
post-injection. Bars represent percentages6s.e.m. of steps taken by the
ipsilateral, contralateral and both forelimbs simultaneously. *P,0.05 vs
pre-transplant group;#P,0.05 vs vehicle groups.
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Taconic, Germantown, New York, United States) were used to
test the tumorigenic potential of the SD56 hNSCs. Undifferenti-
ated hNSCs from passage 9 were single cell dissociated using
trypsin-EDTA and suspended at the concentration of 125,000
cell/ml in preparation for cell transplantation. Two ml of the cell
suspension were stereotaxically transplanted into 4 sites within the
striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm,
DV: 25.0; AP:+0.5 mm, ML: +3.0 mm, DV: 25.0; AP:20.5 mm,
ML: +3.0 mm, DV: 25.0; AP: 21.0 mm, ML: +3.5 mm, DV:
25.0 mm with the incisor bar set at 3.4 mm. The injection rate was
1 ml/min, and the cannula was left in place for an additional 5 min
before retraction. For the flank tumor assay, 26106cells (125,000
cell/ml) were injected subcutaneously to the side of the adult nude
rats. To label mitotically active cells in vivo during S-phase, the rats
were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours
during the last 24 hours before euthanasia. After 2-month survival
time, ratswereeuthanized anda postmortemexamination for tumor
formation was performed.
Induction of Focal Ischemia and Cell Transplantation
All animal experimentations were conducted according to the
National Institute of Health (NIH) guidelines and approved by the
IACUC. Sprague Dawley adult male rats (n=17, 275g–310g,
Charles River Laboratories, Wilmington, Massachusetts, United
States) were subjected to one and a half hour suture occlusion of
the middle cerebral artery (MCAO), as previously described 
and immunosuppressed 2 days before cell transplantation and
daily thereafter for one week with i.p. injections of cyclosporine A
(20 mg/ml, Sandimmune, Novartis Pharmaceuticals). Thereafter
oral cyclosporine was used at 210 mg/ml in drinking water until
euthanasia. Undifferentiated SD56 hNSCs from passages between
P9 and P13 were single cell dissociated using trypsin-EDTA in
preparation for cell transplantation. One week after the stroke
lesion, 2 ml of the hNSCs, at a concentration of 50,000 cell/ml,
were stereotaxically transplanted into 4 sites within the lesioned
striatum (n=10) at the following coordinates: AP: +1.0 mm, ML:
+3.2 mm, DV: 25.0; AP: +0.5 mm, ML: +3.0 mm, DV: 25.0;
AP: 20.5 mm, ML: +3.0 mm, DV: 25.0; AP: 21.0 mm, ML:
+3.5 mm, DV: 25.0 mm with the incisor bar set at 3.4 mm. The
injection rate was 1 ml/min, and the cannula was left in place for
an additional 5 min before retraction. As a control group, we used
rats subjected to ischemia and injected with the vehicle (n=7). All
animals underwent baseline motor behavioral assessment before
and after the ischemic lesion, and 4 & 8 weeks after cell
transplantation. The animals were killed after 2-month survival
time by transcardial perfusion with phosphate buffered saline
(PBS) followed by 4% paraformaldehyde. The brains were
cryoprotected in an increasing gradient of 10, 20 and 30%
sucrose solution and cryostat sectioned at 40 mm and processed for
Cultures were fixed with 4% paraformaldehyde for 15 min.
Both cells and brain sections were rinsed in PBS for 365 min then
incubated for 2 hrs (cultures) or overnight (brain sections) with the
appropriate primary antibodies for multiple labeling. Secondary
antibodies raised in the appropriate hosts and conjugated to FITC,
RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoR-
esearch) were used. Cells and sections were counterstained with
the nuclear marker 49,6-diamidine-29-phenylindole dihydrochlo-
ride (DAPI). Positive and negative controls were included in each
run. Immunostained sections were coverslipped using fluorsave
(Calbiochem) as the mounting medium. The following antibodies
were used: Anti-human Nuclei (hNuc, monoclonal 1:100,
Chemicon), Anti-TuJ1 (monoclonal 1:100, Covance; Polyclonal
1:200, Aves Labs); anti-GAD65/67 (polyclonal 1:1000, Chemi-
con); Anti-glial fibrillary acidic protein (GFAP, monoclonal
1:1000, Chemicon; polyclonal 1:200, Aves Labs); Anti-galactocer-
ebrocide (GC, monoclonal 1:250, Chemicon); Anti-CNPase
(polyclonal 1:200, Aves Labs); Anti-Glucose Transporter type 1
(Glut-1 polyclonal, 1:500, Chemicon); Anti-Nestin (polyclonal
1:1000, Chemicon); Anti-vimentin (monoclonal 1:500, Calbio-
chem); Anti-3CB2 (monoclonal 1:500, Developmental Studies
Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1:250,
SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1:250, Santa-
Cruz Biotechnology). Fluorescence was detected, analyzed and
photographed with a Zeiss LSM550 laser scanning confocal
photomicroscope. For each animal, quantitative estimates of the
total number of grafted cells were stereologically determined using
the optical fractionator procedure . A computer-assisted image
analysis system was performed using Stereo Investigator software
(MicroBrightField, Inc.). The rostral and caudal limits of the
reference volume were determined by first and last frontal sections
containing grafted cells. The striatum and cortex were accurately
outlined at low magnification (2.56 objective). The optical
fractionator probe was selected to perform systematic sampling of
the immunoreactive cell population distributed within the serial
sections to estimate the population number in the volume of tissue.
The counting frame of the optical fractionator was defined at
50650 mm squares and the systematic sampling was performed by
translating a grid with 2006200 mm squares onto the sections of
interest using the Stereo Investigator software. The sample sites were
systematically and automatically generated by the computer and
examined using a 606 objective of a Nikon Eclipse TE 300
microscope. The counting frame displayed inclusion and exclusion
lines and only immunoreactive cell bodies falling withinthe counting
dispersion was measured by counting the number of cells within
200 mm distancefrom thegraftsite. The numberand distanceinmm
of cells dispersed beyond 200 mm was also measured. An average of
2,000 cells was counted per animal. Double labeling was determined
using the confocal laser scanning microscope by random sampling of
100 or more cells per marker for each animal, scoring first for
hNuc+, followed by DAPI+ nuclei and then the marker of choice.
The double labeling was always confirmed in x-z and y-z cross-
sections produced by the orthogonal projections of z-series.
Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from cultured cells using RNAeasy kit
(Quiagen). Aliquots (1 mg) of total RNA from the cells were reverse
transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM
KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTPs, and 0.5 mg
oligo-dT(12–18) with 200 U Superscript RNase H-Reverse
Transcriptase (Invitrogen). PCR amplification was performed
using standard procedure with Taq Polymerase. Aliquots of cDNA
equivalent to 50 ng of total RNA were amplified in 25 ml reactions
containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM
MgCl2 , 50 pmol of each primer, 400 mM dNTPs, and 0.5 U
AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed
using the following thermal profile: 4 min at 94uC; 1 min at 94uC,
1 min at 60uC, 1.5 min at 72uC, for 30–40 cycles; 7 min at 72uC,
and finally a soak at 4uC overnight. The following day, 10 ml
aliquots of the amplified products were run on a 2% agarose Tris–
acetate gel containing 0.5 mg/ml ethidium bromide. The products
were visualized through a UV transilluminator, captured in a
digital format using Quantify One Gel Analysis software (Bio-Rad
Laboratories) on a Macintosh G4 computer.
Stem Cell Therapy for Stroke
PLoS ONE | www.plosone.org7February 2008 | Volume 3 | Issue 2 | e1644
The PCR primers specific to each transcript were as follows:
GFAP, forward (F), 59-TCATCGCTCAGGAGGTCCTT–39
Reverse (R), 59-CTG TTGCCAGAGATGGAGGTT–39; MAP2
(F) 59-GAAGACTCGCATCCGAATGG–39, (R) 59-CGCAGGA-
TAGGAGGAAGAGACT–39; MBP (F) 59-TTAGCTGAATTC
GCGTGTGG–39, (R) 59-GAGGAAGTGAATGAGCCGGTTA-
39 were deigned using the Primer Designer software, Version 2.0
(Scientific and Educational Software) . 18S, b-tubulin class III,
N-CAM, Nestin, NF-M, Notch-1 primers . Oct4, Nanog
primers . FOXa2 (HNF3B), Brachyury primers .
The cylinder test was used to assess the spontaneous forelimb use
during lateral exploration movement . Rats were placed in a
transparent acrylic cylinder (20 cm diameter) for 5 minutes. The
cylinder encourages use of the forelimbs for vertical exploration. A
mirror was placed behind the cylinder so that the forelimbs could be
viewed at all times. Testing sessions were videotaped and forelimb
use was scored by a blinded operator. Movements scored were the
independent use of the left or right forelimb or simultaneous use of
both the left and right forelimb to contact the wall of the cylinder
during a fullrear, to initiate a weight-shifting movement, ortoregain
wall. Animals were tested for their baselines after stroke and 4 and
8 weeks after cell transplantation.
Outcome measurement for each experiment was reported as
mean6SEM. All data were analyzed using SPSS 11 for Mac OS X
applying Student’s t-test where appropriate. The One-Way
ANOVA analysis was used to compare group differences for the
forelimb use as the dependant variable and groups as the single
independent factor variable. Differences between the groups were
determined using Bonferroni’s post hoc test. A P-value of less than
0.05 was considered to be statistically significant.
perpetuation processes of the SD56 hNSCs. Neural stem cells
Schematic representation of the isolation and
were derived from hESCs and propagated using defined media
supplemented with EGF, bFGF and LIF (see Results section for
details). The developmental progression of the in vitro neural
specification and patterning was monitored by the expression of
lineage markers as indicated at each stage.
Found at: doi:10.1371/journal.pone.0001644.s001 (0.38 MB TIF)
expression of Nestin. Dissociated hNSCs were plated at clonal
density (1–2 cell/10 ml), pulsed with BrdU and immuno-processed
for nestin expression by the progeny. After 5 DIV, BrdU labeling
demonstrated that asymmetric segregation of the chromatids rarely
occurs (arrow shows one example) in clonally derived cells. Bars:
20 mm. asymmetric segregation of the chromatids rarely occurs
(arrow shows one example) in clonally derived cells. Bars: 20 mm.
Found at: doi:10.1371/journal.pone.0001644.s002 (1.96 MB TIF)
Asymmetric segregation of BrdU and symmetric
forebrain of naı ¨ve nude rats. Photos show frontal sections through
the graft (A) and the subventricular zone (SVZ) (B) in the striatum
immunostained with the human specific anti-hNuc (green in A)
and anti-BrdU (red, A & B) showing 2 BrdU+ cells in graft zone
(A) and the host BrdU+ endogenous SVZ transit amplifying neural
precursors (B). Bars: 20 mm.
Found at: doi:10.1371/journal.pone.0001644.s003 (6.76 MB TIF)
BrdU incorporation by proliferating cells in the
We are indebted to Drs. Theo Palmer, Julie Baker, Eric Chiao and Pak
Chan. We thank members of Drs. Steinberg, Palmer and Chan
laboratories for their help, support and constructive comments, Dr.
Athena Cherry for the karyotype analysis of the cells, Dr. Bruce Schaar for
comments on the manuscript, David Kunis for laboratory support, Guo
Hua Sun and Dr. Sang Hyung Lee for their outstanding technical expertise
in the stroke animal model and help with the cyclosporine injections and
brain sectioning and Beth Hoyte for preparation of the figures.
Conceived and designed the experiments: MD GS. Performed the
experiments: MD AM. Analyzed the data: MD AM GS. Wrote the paper:
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