Download Long?Term, Stable Differentiation of Human Embryonic Stem Cell

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Long-Term, Stable Differentiation of Human Embryonic Stem
Cell-Derived Neural Precursors Grafted into the Adult
Mammalian Neostriatum
IGOR NASONKIN,a VASILIKI MAHAIRAKI,a LEYAN XU,a GLEN HATFIELD,a BRIAN J. CUMMINGS,d,e CHARLES EBERHART,f
DAVID K. RYUGO,g DRAGAN MARIC,h ELI BAR,a VASSILIS E. KOLIATSOSa,b,c
a
Department of Pathology, Division of Neuropathology, bDepartment of Neurology, and cDepartment of Psychiatry
and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA;
d
Physical Medicine & Rehabilitation and eReeve-Irvine Research Center, University of California Irvine, Irvine
California, USA; fDepartments of Ophthalmology and Oncology and gDepartments of Otolaryngology and
Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; hFlow Cytometry
Core Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda,
Maryland, USA
Key Words. Cellular therapy • Embryonic stem cells • Neural differentiation • Neural induction • Neural stem cells • Pluripotent stem cells
• Stem cell plasticity • Stem cell transplantation
ABSTRACT
Stem cell grafts have been advocated as experimental
treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem
cells (HESCs) are a rich source of neural precursors (NPs)
for grafting, but have been questioned for their tendency
to form tumors. Here we studied the ability of HESCderived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably
differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time
(6 months). NPs were derived from adherent monolayer
cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity
and an avid differentiation into neurons that projected via
major white matter tracts to a variety of forebrain targets.
A third of NP-derived neurons expressed the basal forebrain-neostriatal marker dopamine-regulated and cyclic
AMP-regulated phosphoprotein. Graft-derived neurons
formed mature synapses with host postsynaptic structures,
including dendrite shafts and spines. NPs inoculated in
white matter tracts showed a tendency toward glial (primarily astrocytic) differentiation, whereas NPs inoculated
in the ventricular epithelium persisted as nestin(1) precursors. Our findings demonstrate the long-term ability of
noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while
maintaining some cell fate plasticity that is strongly influenced by particular central nervous system (CNS) niches.
STEM CELLS 2009;27:2414–2426
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Neural stem cells presently considered as cell therapies for
neurological disorders are derived primarily from adult or fetal neural tissue or from in vitro differentiated embryonic
stem cells [1–3]. Although both methods of derivation have
advantages and disadvantages and new methodologies involving somatic cell derivation are being developed [4], the
human embryonic stem cell (HESC) approach continues to
provide a theoretically inexhaustible and very pliable source
of cells [5]. The plasticity and differentiation potential of
HESCs have been demonstrated in recent in vitro studies
where these cells were induced to evolve into specific neuronal and glial lineages to become mesencephalic dopaminergic
neurons [6–8], motor neurons [9–11], and oligodendrocytes
[12]. The traditional ES cell differentiation paradigm involves
the formation of embryoid bodies [13, 14] where cells evolve
in a fashion similar to that in the embryonic gastrula; that is,
they go through the three main embryonic germ layer stages.
These cultures contain high admixtures of non-neuroepithelial
cells [15], a condition that may be a disadvantage for neural
Author contributions: I.N.: design and performance of most experiments, manuscript writing; V.M., L.X., G.H., B.J.C., E.B., D.M., and
D.R.: performance of some experiments, manuscript writing; C.E.: design of some experiments, manuscript writing; V.E.K.: conception
and design, financial support, manuscript writing and editing.
Correspondence: Igor Nasonkin, Ph.D., NIH/NEI, Neurobiology, Neurodegeneration & Repair Laboratory (N-NRL), 9000 Rockville
Pike, Bldg. 6, Rm 341, Bethesda, MD 20892-0610, USA. Telephone: 617-388-4104; Fax: 301-480-1769; e-mail: [email protected].
gov; or Vassilis E. Koliatsos, M.D., The Johns Hopkins University School of Medicine, Division of Neuropathology, 558 Ross Research
Building, 720 Rutland Avenue, Baltimore, Maryland 21205, USA. Telephone: 410-502-5172; Fax: 410-955-9777; e-mail: koliat@jhmi.
edu Received January 28, 2009; accepted for publication June 24, 2009; first published online in STEM CELLS EXPRESS July 16, 2009.
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.177
V
STEM CELLS 2009;27:2414–2426 www.StemCells.com
Nasonkin, Mahairaki, Xu et al.
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replacement therapies that require more defined cell populations for transplantation. A new generation of protocols
designed to avoid embryoid body formation has achieved a
greater degree of in vitro commitment of HESCs to neuroepithelial fates [16, 17]. Further progress was made with the
incorporation of protocols designed to mimic neural induction
events that occur early in development [18], including those
utilizing adherent monolayer conditions in which bone morphogenetic protein (BMP) signaling is blocked by noggin [19,
20]. These cultures allow for the homogeneous exposure of
HESCs to the neural lineage-promoting action of noggin and
can reliably generate neural precursors (NPs) for further
differentiation.
To test the generic potential of noggin-differentiated
HESCs for therapeutic applications in the nervous system, we
evaluated the in vivo outcomes of grafting these cells in the
adult mammalian forebrain. In the present study, we demonstrate the consistent, controlled differentiation of HESCs to
NPs using noggin without additional inducing agents or
trophic factors and the ability of such derived NPs to integrate, to various degrees, into the adult mammalian forebrain.
Within the course of several months explored in this study,
HESC-derived NPs undergo substantial neuronal differentiation without apparent tumorigenesis, project axons to distant
locations, and form structurally mature synapses in their
innervation targets.
METHODS
Propagation and Neuroepithelial Differentiation
of HESCs
The HESC line BG01, included in the NIH Human Embryonic
Stem Cell Registry (http://stemcells.nih.gov/research/registry),
was obtained from BresaGen, Inc., Athens, GA (http://www.
novocell.com/) [5]. HESC colonies were grown on mitotically
inactivated mouse embryonic fibroblasts (MEFs; 2 106 cells
per 35-mm dish) prepared from E12 ICR mice (Taconic, Hudson,
NY, http://www.taconic.com). Culturing conditions were according to company guidelines, except that higher concentrations
of bFGF (10 ng/ml, Sigma-Aldrich, St. Louis, http://www.
sigmaaldrich.com) and 15% knockout replacement serum (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) were used.
Colonies were passaged every 4–7 days by manual dissection.
For neural induction, days 4–6 HESC colonies growing on MEFs
were treated with a medium composed of 50% fresh HESC medium (see above) and 50% Neurobasal (NB) medium (Invitrogen), supplemented with 100 ng/ml noggin (Sigma-Aldrich).
Over the next 2 days, noggin concentration was gradually
increased (day 2, 200 ng/ml; day 3, 250 ng/ml) and, on day 3,
colonies were mechanically dislodged from the MEF layer and
transferred to gelatin-coated tissue culture plates in NB complete
medium. At 4 weeks, cultures were transferred to new gelatincoated dishes (passage 1, P1) in NB complete medium with
bFGF and noggin. NPs were passaged once again (P2) and then
used for transplantation or were frozen. Details are provided in
the supporting information data.
Monitoring Differentiation of HESCs In Vitro
Quantitative
Real-Time
Polymerase
Chain
Reaction.
Undifferentiated HESC colonies (days 4–5 after passaging) growing on MEFs were collected by mechanical dissection and placed
into Buffer RLT (Qiagen, Valencia, CA, http://www1.qiagen.com). Total RNA was prepared according to manufacturer’s
instructions. HESC colonies differentiating on gelatin-coated 35mm dishes were lysed directly on the plates on days 7, 14, 21,
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and 28 and at P2 (1–2 plates per time point) using Buffer RLT.
Total RNA was extracted from the lysates using an RNeasy kit
(Qiagen) and was treated with DNase according to manufacturer’s
protocol. Real-time polymerase chain reaction (RT-PCR) was performed essentially as described [2] (supporting information data).
Immunocytochemistry. NP cultures were rinsed with phosphate-buffered saline and briefly fixed with 4% freshly depolymerized, neutral-buffered paraformaldehyde for 15 minutes. Cultures were processed for the immunocytochemical detection of
neural stem cell and differentiation protein markers as per details
described in supporting information data. Both single and multiple epitope immunocytochemistry (ICC) were performed and
combined with DAPI counterstain. Total number of cells in 10
randomly selected fields was counted with the help of an automated cell-counting program (AxioVision; Carl Zeiss, Jena, Germany, http://www.zeiss.com).
Immunophenotyping of NPs with Flow Cytometry. Cells
were first dispersed into a uniform single-cell suspension using
papain digestion protocol as described elsewhere [21]. The resulting mixture of cells was immunolabeled with the following cocktail of lineage-selective surface markers: rabbit IgG1 anti-human
CD133 and mouse IgM anti-human CD15 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), mouse IgG1
anti-human CD29 (BD Biosciences, San Jose, CA, http://
www.bdbiosciences.com), and a mixture of tetanus toxin fragment C (TnTx)-mouse anti-TnTx IgG2b. Methodological details
are included in supporting information data.
Animals and Surgical Procedures
Nude rats (strain CR: NIH-rnu; male; 6–8 weeks old; n ¼ 22)
were purchased from the National Cancer Institute. All surgical
procedures conformed to protocols approved by the Animal Care
and Use Committee of the Johns Hopkins Medical Institutions
and were carried out by using gas anesthesia (enflurane:oxygen:nitrous oxide ¼ 1:33:66) and aseptic methods. NPs were injected
in suspension (15 103 cells in 1 ll) into the neostriatum of animals mounted on a Kopf stereotaxic unit (David Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com). Injections
were made under microscopic guidance via pulled glass micropipettes controlled by a Nanoinjector device (World Precision
Instruments, Sarasota, FL, http://www.wpiinc.com). Animals were
killed with perfusion-fixation as described elsewhere [2] at 1.5
months (n ¼ 8), 3 months (n ¼ 7), and 6 months (n ¼ 7).
The number of cells grafted was based on a pilot experiment
designed to optimize integration of graft with host tissues and to
monitor tumorigenesis as a function of graft size (see supporting
information data, pilot experiment, and supporting information
Fig. 1A–1E). In this pilot, we varied the number of cells
(between 20 103 and 120 103) grafted in a single inoculation
site into the neostriatum. Variance in the number of injected cells
between 20 103 and 120 103 appeared to have little effect
on the degree of tissue integration or the mitotic activity of
grafts. Human-specific synaptophysin immunoreactivity was seen
in small patches of grafted cells at 1.5 months, but increased substantially by 3 months and was seen throughout the graft at 6
months postgrafting (supporting information Fig. 2). Although the
results of these pilot studies were reassuring with respect to general histopathology and tumorigenesis, the transient appearance of
neuroepithelial phenotypes led us to decrease the number of cells
in the graft to 15 103.
Monitoring Differentiation of HESCs In Vivo:
Histology and ICC
The survival, growth, and phenotypic fate of HESC-derived NPs
in vivo were assessed with immunoperoxidase or dual-label immunofluorescence. Tissues were prepared from animals perfused
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Differentiation of HESC-Derived Neural Precursors
Figure 1. Neural induction/differentiation of
HESCs with noggin in adherent monolayer, from
day 0 to P2. (A): Appearance of undifferentiated
HESCs on MEFs at day 0 by phase contrast. (B):
Induction/differentiation protocol used to generate
NPs used in this study. (C): Day 0 (undifferentiated) HESCs express, by ICC, index pluripotency
markers including Oct3/4, SSEA4, Tra1-60, and
Tra1-81. (D): Oct3/4 immunoreactivity is substantially reduced in differentiating HESCs between
day 9 (left) and day 28 (right) in culture, evidence
of shedding of HESC pluripotency. Scale bars:
20 lm.
with 4% freshly depolymerized paraformaldehyde as described
elsewhere [2]. Brain blocks were sectioned (30 lm) in the coronal or sagittal planes. Primary antibodies were used to disclose
human (graft) versus rat (host) cell identity, mitotic activity, and
neuronal and glial phenotype specification and included a number
of well-characterized monoclonal antibodies and antisera (supporting information Table 1). Normal IgG from the species of origin of primary antibodies served as negative controls.
For immunoperoxidase staining, sections were processed with
peroxidase-antiperoxidase or avidin-biotin-peroxidase [22]. Duallabel immunofluorescence was used to study NP mitotic activity
and differentiation, combined immunoreactivity for human nuclear antigen (HNu) and that for a cell proliferation or differentiation marker, and was performed essentially as described elsewhere [2]. ICC controls were prepared by replacing primary
antibodies with pre-immune serum from the same species of origin and were negative in all cases. To measure rates of differentiation of HESC-derived NPs, we used two representative sections
through the middle of each graft and counted the total number of
HNu(þ) cells, and also the cells dually labeled with HNu and
one of the chosen phenotypic markers on 10 randomly selected
100 fields per case. The total number of HNu(þ) cells and
Nasonkin, Mahairaki, Xu et al.
double-labeled profiles were pooled for each case, and percentages of double-labeled cells were generated per case and then
grouped per time point designation (1.5, 3, or 6 months). Variation in differentiation, as a function of time point after surgery,
was studied with one-way ANOVA followed by Tukey’s multiple
comparison post hoc test. Results are expressed as mean standard error.
Electron Microscopy
Following transcardiac perfusion with 4% freshly depolymerized
paraformaldehyde, brains were removed and embedded in a gelatin-albumin mixture hardened with glutaraldehyde. Vibratome
sections were subjected to synaptophysin ICC, embedded, and
resectioned at the ultrathin level (supporting information data).
RESULTS
Controlled Differentiation of HESCs to NPs In Vitro
HESC colonies were grown on a MEF layer supplemented
with bFGF and differentiated to neuroepithelial fate with noggin (Fig. 1A, 1B). The pluripotency markers Oct3/4, SSEA4,
Tra1-60, and Tra1-81 were amply expressed in these starting
HESC colonies (Fig. 1C, 1D). After HESC colonies were dislodged from the MEF layer, they attached almost immediately
to gelatinized plates and showed robust growth and flat
appearance. Ten days after transfer, multiple early rosettes
appeared at the center of the colonies. The cultures acquired
three-dimensional growth at 15 days after transfer; at this
time point, HESC colonies grew in the form of early rosettes
(i.e., three-dimensional structures comprised of columnar epithelial cells) and proliferated vigorously. In 3 weeks, early rosette structures became less well defined and, by 1 month,
most of them disappeared and cultures acquired two-dimensional neuroepithelial characteristics.
Differentiation of HESCs to NPs was monitored with RTPCR, ICC, and flow cytometry (Figs. 1–3). RT-PCR data
demonstrate that, with ongoing noggin treatment, there is a
gradual upregulation of neural markers and a downregulation
of pluripotency markers such as Oct3/4 (Figs. 1D, 2A), Nanog
(Fig. 2A), and SSEA4 (data not shown). The stem cell marker
Sox2 continues to be expressed in differentiating HESCs.
Neural markers show gradual upregulation, with most of them
rising between day 7 and day 21; further upregulation of neural markers is detected at passage 2 after the replating of NPs
(Fig. 2A). The endodermal marker GATA-4 becomes undetectable by passage 2 (Fig. 2A). The neural crest marker
Msx1 shows gradual upregulation (Fig. 2A), whereas two
additional markers of mesoderm and endoderm (Brachyury
and Pdx1, respectively) are absent during the differentiation
process (data not shown). The expression of a panel of neurotrophic factors including the neurotrophins NGF and BDNF,
GDNF, VEGF, and bFGF increases between day 14 and day
21 and then decreases or reaches a plateau (Fig. 2B). A representative panel of transcription factors conveying anterior,
posterior, and dorsal-ventral position specification in the neural tube failed to demonstrate a predominant pattern in cultured NPs, despite some up- or downregulation of individual
transcripts over time (Fig. 2C).
HESC-derived NPs were profiled by ICC immediately
prior to transplantation (Fig. 3A–3E). More than 80% of the
cells was immunoreactive for nestin (Fig. 3A). Seven percent
of the cells expressed the class III b-tubulin epitope TUJ1,
serving as a marker of immature neurons (Fig. 3B). Six percent of the cells expressed the astrocytic marker GFAP (Fig.
3C), whereas 10% of the cells expressed S100b, a marker of
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ependymal, radial glial, and early astrocytic cells (Fig. 3D).
Multiple-antigen ICC confirmed further the predominant phenotypes of P2 NPs (Fig. 3E). For example, nestin was found
to colocalize extensively with musashi-1 and vimentin, a pattern confirming the neural stemness of our NPs, whereas these
cells were negative for pax6, an early marker of neuroepithelium. The neural stem cell marker CD15 (SSEA-1) was
expressed by approximately 15% of NPs, whereas the common glial precursor marker A2B5 was present in less than 1%
of NPs. Approximately 7% of the cells was mitotically active
by Ki67 immunostaining (data not shown).
Immunophenotyping of P2 NPs by flow cytometry showed
that less than 1% of the total NPs express the surface stem
cell marker CD133 that denotes the presence of symmetrical
division (Fig. 3F). In contrast, the great majority (approximately 79%) of NPs were positive for tetanus toxin (TnTx), a
marker of neuronal progenitors [23, 24]. CD15þTnTx cells
comprised 16.80% of our NP population. A small subset
(4.3%) of NPs was CD15TnTx (Fig. 3F-2) and 14.5% of
these CD15TnTx NPs was CD29þ cells (Fig. 3F-3).
The above findings demonstrate the consistent derivation
of multipotential NPs from HESCs by gradual differentiation.
A combination of methodologies including ICC and immunophenotyping indicate that, at the time of grafting into brain,
the majority of our NPs were at the stage of very early neuronal progenitors [nestin(þ) and TnTx(þ) cells], whereas a sizeable one fifth or so of our NP population was at an earlier
neural stem stage (CD15þTnTx cells), capable of generating
neurons and glial cells.
Survival and Proliferation of HESC-Derived
NPs in the Neostriatum
To investigate the in vivo potential of HESC-derived NPs to
integrate into the adult mammalian brain, we grafted P2 NPs
into the neostriatum of young adult nude rats. HNu-immunostained material demonstrated the successful engraftment and
excellent survival of NPs in this forebrain site (Fig. 4A–4C),
with few scattered apoptotic profiles detected only in 1.5month grafts. In some 1.5-month grafts, clusters of packed
basophilic cells were present, but these were not observed at
later time points. Because of the well-known propensity of
HESC-derived grafts for tumorigenesis [25], we tested grafts
for the expression of the nuclear proliferation marker Ki67 at
1.5, 3, and 6 months after transplantation (Fig. 4D, 4E). The
Ki67 proliferation index within the grafts decreased from
8.3% 2.4% at 1.5 months (n ¼ 8) to 4.2% 0.7% at
3 months (n ¼ 7) and then to 1.2% 0.6% at 6 months (n ¼
7), suggesting a gradual exit from the cell cycle and differentiation of grafted NPs.
Consistent with a pattern of progressive maturation over
time, the distinction of grafts from surrounding normal brain
tissue, although pronounced at 1.5 months, gradually disappeared as the grafts assumed a more homogeneous appearance
(supporting information Fig. 1). No teratomas [26], neural
tumors [27], or clusters of Ki67 immunoreactive cells were
observed within the grafts, findings that indicate the absence
of tumorigenic proliferative masses.
Plasticity, Migration, and Neuronal Differentiation
of HESC-Derived NPs
In the primary grafting location (neostriatum), the majority of
NPs differentiated into TUJ-1-positive neurons (Fig. 5). Close
to 50% (44.6% 3.3%) of HNu(þ) cells were nestin-positive
at 1.5 months, a rate reduced to approximately 10% (8.5% 1.4%) at 3 months and less than 1% (0.2% 0.1%) at
6 months after grafting. A large number of HNu(þ) cells
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Differentiation of HESC-Derived Neural Precursors
Figure 2. Monitoring differentiation of HESCs in vitro by RT-PCR. Relative gene expression is shown as fold change in gene expression using
the comparative Ct (DDCt) method. Expression levels of genes in the initial HESC sample were used as standard (equal to 1) to which all other
expression values were compared and expressed as fold changes. Bars corresponding to data from P2 NPs are shown in darker colors, to distinguish them from data derived from day 7 to day 28 HESC colonies that continuously differentiate within the same plates. (A): Expression of index
pluripotency and neural markers in differentiating HESCs, measured by quantitative RT-PCR at days 7, 14, 21, 28, and 49 (P2). Stem cell markers
are shown in yellow, early neural markers are in green, neuronal and glial markers are in gray, neural crest markers are in blue, and endodermal
markers are in pink. The expression of mesodermal marker Brachyury was not detectable and, therefore, is not shown. (B): Expression of trophic
factors including neurotrophins (NGF, BDNF, GDNF, VEGF, and bFGF) by differentiating HESCs at days 7, 14, 21, 28, and 49. (C): Expression
of selected dorsal-ventral (blue), anterior (pink), and posterior (purple) markers by differentiating NPs at day 28 and P2. Several markers (Otx2,
Emx2, Dlx2, and Nkx2.1) were not detectable and therefore are not shown. Abbreviations: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; GDNF, glial-derived neurotrophic factor; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor.
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Figure 3. Immunocytochemical (ICC) and
immunophenotypic profiling of HESC-derived
NPs at the time of transplantation. (A–D): These
single-epitope ICC preparations illustrate the
expression of selective neural stem, neuronal,
and glial markers. Insets represent magnifications of areas shown with asterisks in the
merged image. Although the vast majority of
cells express the neural stem cell marker nestin
(A), one tenth or less of them express the class
III b-tubulin epitope TUJ1, a marker of immature neurons (B), or the glial (primarily astroglial) markers GFAP (C) and S100b (D). (E):
These four-epitope ICC preparations serve to
further characterize the phenotype of HESCderived NPs at the time of transplantation. Epitope combinations are nestin-vimentin-A2B5pax6 (top) and nestin-vimentin-musashi1-CD15
(bottom). Nestin colocalized extensively with
vimentin (top, bottom) and musashi (bottom).
Pax6, revealed with Alexa Fluor 750, was not
present in these cells and is not shown here. A
subpopulation of NPs expressed the early neuronal precursor marker CD15 (bottom), whereas
very few expressed A2B5 (top). Asterisks indicate areas further enlarged in the insets. (F):
Immunophenotyping of HESC-NPs by flow
cytometric analysis. HESC-derived NPs were
dispersed into a uniform single-cell suspension
and labeled with antibodies targeting CD133,
CD29, CD15, and TnTx. Dead cells were counterstained with DAPI and the resulting vital phenotypes assayed by flow cytometry. Data are
presented as two-parameter dot density plots in
pseudocolor that reveal the relative abundance
(percentiles) of different cell populations by
using either electronically gated regions as in
(1) (Live Cells, Dead Cells, Debris) or quadrants
as in (2, 3). In (1), DAPI was used in conjunction with forward scatter to discriminate
between live (DAPI-negative) and dead (DAPIpositive) cells. Data in (2, 3) are based on live
cells, which represent approximately 80% of the
preparation. In (2), the great majority of live
cells are TnTxþ (approximately 79%, that is, the
sum of 48.21 and 30.73). CD15 labeling further
subdivides these neuronal-restricted precursors
into CD15þ (30.73%) and CD15 (48.21%)
subpopulations. CD15þTnTx NPs comprise
16.80% of total NPs. The CD133 versus CD29
plot in (3) is gated on 4.26% of CD15TnTx
cells (see panel F 2). Only 14.47% of these cells
are CD29þ, which amounts to approximately
1% of total live cells (14.47 4.26/100).
CD133þ cells represent less than 1% (0.75%) of
the total population of live cells sorted. Abbreviations: Scale bars: 50 lm.
within the graft expressed doublecortin, a marker for migrating neuroblasts and early neurons [28, 29] (Fig. 5A). More
than one half of HNu(þ) cells were doublecortin(þ) at 1.5
months (52.1% 3.4%) and 3 months (69.6% 5.3%); a
similar percentage of HNu(þ) cells also expressed the doublecortin-like kinase epitope DCAMKL-1 [72.0% 3.4% of
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HNu(þ) cells] at 3 months after grafting. Doublecortin
expression was reduced to less than 5% (4.7% 0.7%) of
HNu(þ) cells at 6 months after grafting (Fig. 5C). On the basis of a sharp rise in TUJ-1 colocalization of HNu(þ) between
1.5 months (20.1% 3.9%) and 3 months (70.8% 6.8%)
after grafting (Fig. 5B, 5C), we assume that neuronal fate was
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Differentiation of HESC-Derived Neural Precursors
Figure 4. Transplantation strategy and survival of human NPs in rat neostriatum. (A): This is a composite sagittal map of individual striatal
grafts at maximal inoculation size. Contours of each individual graft are shown in black. Contours of rat brain and the ventricular lining are in
bold gray. The map shows a reliable coverage of a large section of anterior caudate-putamen with rare invasion into the adjacent globus pallidus.
Major anatomical landmarks are depicted for orientation purposes (see abbreviations below). (B): This coronal section through the anterior septum shows a typical striatal graft of human NPs 3 months after inoculation. (C): This is an HNu-stained sagittal section through the middle of
HESC-derived NP graft 3 months after grafting. The section was counterstained with cresyl violet. Most HNu(þ) cells have large round nuclei
consistent with mature neurons. The inset represents the magnification of framed area. (D, E): Mitotic activity within HESC-derived NP grafts as
shown by dual staining for HNu and Ki67. (D): Mitotic profiles of NPs in grafts measured at 1.5, 3, and 6 months. Bars indicate group averages
SEM. Variance in mitotic profiles between the three cohorts was significant (ANOVA, p ¼ .014). Significant post hoc differences are indicated
with brackets. (E): A representative area within a graft 3 months after transplantation, dually stained with HNu (red nuclear marker) and Ki67
(green nuclear marker). The three images illustrate the low rate of mitotic activity in HESC-derived grafts. The single double-stained nuclear profile (bottom) is indicated with an asterisk. Abbreviations: Acb, nucleus accumbens; AOD, anterior olfactory nucleus, dorsal; AOV, anterior olfactory nucleus, ventral; Cx, cortex; cc, corpus callosum; GP, globus pallidus; Hy, hypothalamus; STh, subthalamic nucleus; Str, corpus striatum
(neostriatum); th, thalamus; VP, ventral pallidum. *, p < .05. Scale bars: (B, C): 100 lm (main panel), 10 lm (inset); (E): 10 lm.
Nasonkin, Mahairaki, Xu et al.
Figure 5. Neuronal differentiation of HESC-derived NPs in rat neostriatum. Photomicrographs represent cases of neuronal differentiation
of HNu(þ) cells by confocal microscopy from subjects prepared
3 months after grafting. (A): Dual ICC for HNu and doublecortin
shows many HNu(þ) cells that are also immunoreactive for doublecortin (DCX). (B): Double ICC for HNu and TUJ1 demonstrates the
abundance of double-stained cells in the grafts. (C): Bar graphs
depicting neuronal cell fate choice in striatal grafts of HESC-derived
NPs at 1.5, 3, and 6 months represented by percentages of DCX-labeled (left panel) and TUJ1-labeled (right panel) HNu(þ) cells. Bars
indicate group averages SEM. Variance between the three cohorts
was significant for both DCX (ANOVA, p ¼ .0003) and TUJ1
(ANOVA, p ¼ .0004). Significant post hoc differences are indicated
with brackets. *, p|<|.05; **, p|<|.001. Scale bars: 10 lm.
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established, for the majority of grafted NPs, prior to 3 months
after grafting. Further increase of TUJ-1 expression between 3
and 6 months (to 86.6% 1.8%) was not significant.
Human-specific synaptophysin immunostaining was seen
in islands of grafted cells at 1.5 months, but increased substantially by 3 months and was seen throughout the graft at
6 months after grafting (supporting information Fig. 2). There
was no GAD or ChAT colocalization with HNu(þ) profiles or
human-specific synaptophysin(þ) terminals in 6-month grafts.
Only sparse colocalization of GluR2/3 and HNu was seen in
grafts and there was no visible VGLUT1 or VGLUT2 immunoreactivity in human-specific synaptophysin(þ) terminals. A
number of markers of basal forebrain and basal ganglia neurons were expressed by differentiating NPs. DARPP-32 was
not detected in 3-month grafts, but was expressed by nearly
one third (30.1% 3.2%) of HNu(þ) cells at 6 months after
grafting (supporting information Fig. 3A–3C). The calciumbinding protein calretinin was expressed by less than 1% of
HNu(þ) cells at 3 months, but was present in 6.5% 1.6% of
HNu(þ) cells at 6 months after grafting (supporting information Fig. 3D, 3D0 ). Parvalbumin, another calcium-binding protein, was undetectable in 1.5- or 3-month grafts and was found
in less than 1% of HNu(þ) cells at 6 months after grafting.
In a few cases in which grafting inadvertently involved
large white matter tracts of the forebrain, that is, the fornix,
the internal capsule, or the genu of the corpus callosum, a
majority of NPs had astroglial phenotypes as judged by confocal analysis of GFAP immunoreactivity in HNu(þ) profiles
and also with human-specific SC121 immunoreactivity
(Fig. 6). In these cases, dual ICC for HNu and the oligodendrocyte markers O4 or RIP also revealed small numbers (less than
5%) of oligodendrocytes differentiating from grafted NPs.
Very few HNu(þ) cells were found in parenchymal sites
away from the graft. The only exception was the ventral forebrain pia which, in virtually all cases examined, was densely
populated by HNu(þ) bipolar cells; all these cells expressed
doublecortin (supporting information Fig. 4), but were negative for nestin and did not display neuronal configurations;
that is, they had no discernible axons by SC121 ICC. However, when HESC-derived NPs were spuriously deposited
along the needle track and became part of the host ventricular
epithelium, they all maintained a nestin-positive state months
after the injection and none expressed doublecortin (supporting information Fig. 5).
In concert, our dual ICC experiments with graft-specific
and differentiation markers indicate a predominantly neuronal
differentiation of HESC-derived NPs in the neostriatal grafting sites. When cells were inoculated into white matter tracts,
fate choice was astrocytic and, in some cases, oligodendrocytic. However, when human NPs were deposited in the ependymal region, they persisted in a neural stem cell state. The
previous patterns demonstrate a fate-choice plasticity of
grafted human NPs that may depend on host microenvironment. Parenchymal migration of those NPs was difficult to establish; pial neuroblast profiles consistently observed in all
grafted cases could have arisen either from pial or via microvessel migration.
Evidence for Circuit Integration of HESC-Derived
NPs in the Rat Forebrain: Axons and Synapses
Axonal projections from the graft were visualized as early as
1.5 months with doublecortin and DCAMKL-1 antibodies and
at 3 and 6 months after grafting with human synaptophysin
and human cytosol-specific SC121 antibodies (Fig. 7A, 7B).
Human NF70(þ) axons were evident only at 6 months after
grafting (data not shown). These projections appeared as
2422
Differentiation of HESC-Derived Neural Precursors
Figure 6. Astroglial differentiation of HESC-derived NPs in fornix (A, B), genu of corpus callosum (C), and internal capsule/globus pallidus
(C, D). Sections were processed for ICC with GFAP (A) or human cytosol-specific SC121 (B–D) antibodies. The section in (A) was processed
with immunofluorescence; sections in other panels were processed with immunoperoxidase. (A, B): In this case of spurious engraftment of human
NPs into the fornix bundle, the vast majority of HNu(þ) or SC121(þ) cells displayed astrocytic cytologies [both (A) and (B)] and GFAP expression (A). Insets in (A) represent a confocal microscopic analysis of the profile indicated by connecting lines; top inset is a traditional confocal
appearance of the index profile with a subsequent virtual resectioning at the x- and y-axis; bottom inset is a compressed z-stack image of the
index profile. Inset in (B) shows a low-power image of the fornix from which the main panel was derived. (C, D): In this case of a peripheral
involvement of the callosal genu (C) or globus pallidus (D) in the graft, there was a predominant differentiation of human NPS into astrocytes.
Insets in (C) and (D) are magnifications of framed areas in main panels. Scale bars: (A, C, D): 100 lm; (B): 10 lm.
dense bundles of axons traversing most white matter tracts in
the broad vicinity of the graft including the striatopallidal
pencils, stria terminalis, internal capsule, anterior commissure
(olfactory and temporal limbs), the forceps minor and body of
the corpus callosum, and furthermore, the cerebral peduncle
and callosal radiations.
Numerous human synaptophysin(þ) boutons were found
in the surroundings of the graft as early as 3 months after
grafting; some of these structures made contact with other differentiated NPs and these graft-derived cell-to-cell contacts
became even denser at 6 months (supporting information
Figs. 2, 3). Synaptophysin(þ) boutons away from the graft
were evident at 6 months after grafting in layer I of frontal
cortex, the olfactory bulb (internal plexiform layer), anterior
olfactory nucleus, bed nucleus-central amygdala inserts in the
substantia innominata, the globus pallidus, substantia nigrapars reticulata, and the subthalamic nucleus (Fig. 7C).
Ultrastructural analysis of tissues from animals at 3 and
6 months after grafting stained for human synaptophysin and
SC121 in a pre-embedding fashion confirmed the presence of
synaptic structures originating from differentiated human NPs.
Human synaptophysin(þ) terminals were especially dense
around the graft, in substantia innominata and in the bed nucleus of stria terminalis. Labeled terminals were also evident
in frontal, motor, somatosensory, and auditory cortex and in
the olfactory bulb. Many labeled structures were en passant
swellings with round (38–50 nm in diameter) vesicles, suggestive of synaptic vesicles, but did not exhibit pre- and postsynaptic membrane specializations (not shown). Numerous labeled terminal swellings were also observed. In a few of
these terminals, synaptic vesicles and membrane specializations were visible at the same plane, allowing for their unambiguous classification as synapses (Fig. 7D–7F). In all these
synapses, the unlabeled postsynaptic elements had the
Nasonkin, Mahairaki, Xu et al.
2423
Figure 7. Evidence for some local and remote integration of differentiated human NPs in rat forebrain circuitry manifested by early (A) and late (B)
pathway formation, remote synaptic configurations (C), and the formation of mature synapses as evidenced by immuno-EM labeling for human synaptophysin and the human cytosolic marker SC121 (D–F). (A): Early projections from the 1.5-month graft visualized by doublecortin (arrowheads indicate bundles of pioneer axons). Inset in (A), top, represents a magnification of the axonal front marked with an asterisk in main panel. Inset in (A),
bottom, represents a confocal microscopic analysis, with a subsequent virtual resectioning at the x- and y-axis, of the framed area in main graft. (B):
Extensive formation of axon bundles by mature (6-month old) differentiated grafts occurs along most established forebrain pathways including the
striato-pallidal pencils and the anterior commissure (ac and sppen, top left), thalamic striae and the mamillothalamic tract (tstriae and mtt, top right),
the rostral migratory stream (RMS, bottom left), and the genu-forceps minor of the corpus callosum (fm/cc, bottom right). In this panel, graft-derived
axon bundles are visualized with SC121 ICC. Sections are counterstained with fast green. (C): Synaptophysin immunoreactivity in terminal fields in
6-month-old grafts. These panels illustrate representative synaptic boutons in the olfactory bulb (OB) (internal plexiform and outer granule cell layer),
layers I and II of the frontal cortex (Cx), the globus pallidus (GP), and the islands of Calleja (IC). Insets represent magnifications of areas indicated
with asterisks. (D–F): Electron microscopic images of synapses with human signature [(D, E): human synaptophysin labeling; (F): SC121 labeling]
taken close to the striatal graft (D), in the nucleus accumbens (B), and in the glomerular layer of the olfactory bulb (C). Sections in (D) and (E) were
prepared 3 months after grafting; section in (F) was prepared 6 months after grafting. Note postsynaptic densities (arrows) and synaptic vesicles in all
labeled terminals. Abbreviations: ac, anterior commissure; cc, corpus callosum; fm, forceps minor of corpus callosum; GR, graft; mtt, mammillothalamic tract; RMS, rostral migratory stream; sppen, striato-pallidal pencils; tstriae, thalamic striae. Scale bars: (A, B): 100 lm; (C): 10 lm.
structure of dendritic shafts or spines. Unlabeled postsynaptic
structures were inferred to be of host (rat) origin due to their
distance from the graft and the paucity of parenchymal migration of differentiated NPs. SC121 immunoreactivity was very
www.StemCells.com
similar in ultrastructural appearance to that of human
synaptophysin.
In concert, differentiated grafts of human NPs show an
early elaboration of axons that form bundles and traverse
Differentiation of HESC-Derived Neural Precursors
2424
along established pathways in the host forebrain. Later grafts
show an abundance of synaptophysin- or SC121-immunoreactive boutons in terminal fields; a number of these boutons
demonstrate ultrastructural features of mature synapses.
DISCUSSION
General Conclusions
Our findings show an extensive neuronal differentiation of
HESC-derived NPs and considerable structural integration
into the adult rodent forebrain. The long time points used in
this paper (up to 6 months) serve to show the stable, tumorfree differentiation that is key for the safety and therapeutic
potential of HESC-derived NP grafts [30]. The long survival
times used here also show the ongoing differentiation of these
HESC-derived cells several months after grafting, that is, time
frames that are unusual in stem cell transplantation studies
[31], but may be necessary for establishing the full grafting
potential of the slowly differentiating human cells. The number of NPs used for transplantation appears to be an important
variable that, when optimized to the appropriate level (15 103 cells per animal), may eliminate neuroepithelial tube formation and safeguard the host from tumorigenesis and graft
overgrowth [32].
NPs used in this paper have been derived by exposing adherent monolayers of HESCs to high concentrations of noggin
which, by blocking BMP, arrests non-neural fate choices [33]
and, further downstream, may also inhibit astroglial differentiation [34]. The pluripotent markers Oct3/4 and Nanog are
almost eliminated by day 28 of the noggin-based protocol due
to the efficient neuralization of HESCs, thus drastically reducing the tumorigenic potential of grafted cells. Despite a predominant neuronal differentiation, such derived NPs were capable of making astroglial fate choices when inoculated into
white matter tracks, a finding that supports the identity of a
portion of these NPs as neural stem cells at the time of transplantation. Further support of their plastic nature at the time
of transplantation is the arrest of these NPs in a nestin(þ)
state in the case of inoculation in the ependymal region.
Further Comments on Culturing Methodology
The culture conditions used here to prepare NPs from HESCs
and expand them prior to grafting were established in a previous report [20] in which we have also shown the karyotypic
stability and lack of Neu5Gc contamination of these precursors. The present paper demonstrates further the advantage of
the adherent monolayer system in effecting a synchronized
downregulation of pluripotency genes such as Nanog and
Oct3/4 [35] and the upregulation of neural markers. Sox2, a
genetic marker of both HESCs and neural stem cells [36], is
expressed throughout differentiation. P2 proved to be a good
time choice for transplantation of cells treated with our noggin-based differentiation protocol. At this time point, cells
appear to be plastic enough to acquire either neuronal or
astroglial fates in vivo, yet also mature enough to avoid excessive proliferation. Similar differentiation patterns were
seen when the HESC lines BG02 TE03 ES04 were used in
lieu of BG01 under the exact same culture conditions (I.
Nasonkin, unpublished observations).
Most NPs at the time of grafting were early neuronal progenitors, although one fifth or so (the cell population exhibiting the CD15þTnTx immunophenotyping profile) may exhibit a broader fate plasticity and generate both neurons and
glial cells [37]. Another NP subpopulation defined by the
CD15TnTx CD29þ profile may give rise to radial glia and/
or astrocytes.
Optimizing between proliferation propensity and neural
‘‘stemness’’ of precursors is a challenging task that has been
addressed and managed before [38]. A prompt reduction of
mitotic index has been consistent across grafting experiments
and is an especially encouraging finding of the present study.
Reduction in mitoses was eventually also encountered in pilot
grafts with much higher numbers of cells than the ones used
in the main study, despite the tendency of such grafts to go
through an initial period of neuroepithelial proliferation (supporting information Fig. 1). In contrast to forebrain transplantation, NP grafting into the brainstem and spinal cord showed
a propensity for tumorigenesis and was not pursued further.
Migration and Differentiation Choices
of Human NPs
On one hand, as we observed previously in the case of spinal
cord grafts [2], the host microenvironment plays a decisive
role in determining differentiation choices of grafted NPs. On
the other hand, the large population of neuronal progenitors in
our NP preparation is certainly consistent with the predominant neuronal differentiation of these NPs in the rat forebrain.
Astrocytes present in mature grafts originated either in neural
stem cells or in the small number of astroglial precursors
present in the inoculum. Besides cell-intrinsic signals, such as
POU, Notch, and bHLH, extrinsic molecular cues including
noggin, BMPs, bFGF, EGF, LIF, and neurotrophins are likely
to play key roles in promoting differentiation choices faithful
to the cellular composition of each one of these diverse
microenvironments [39–44]. The neuronal bias in differentiation of striatal grafts was evident in vivo with the prompt
expression of doublecortin and then TUJ1 and synaptophysin
by 3 months after grafting, although the expression of neurofilament markers and axonal elongation/pathfinding and elaboration of terminals for most forebrain targets would take
another 3 months. This long time course of in vivo differentiation may be appropriate for the longer differentiation cycle
of human NPs [45] and argues for the extension of survival
times in human NP grafting experiments beyond the conventional 10–12 weeks. Despite advanced neuronal differentiation, including the late acquisition of DARPP-32 by one third
of human NPs, these cells did not appear to achieve definitive
neurotransmitter specialization, that is, become GABAergic
neurons.
DARPP-32 is an early marker of lateral ganglionic eminence (LGE)-derived basal forebrain neurons, including medium spiny striatal projection neurons [46], and has been
found in at least one other occasion in neurons differentiating
from primate ES-derived progenitors grafted into striatum
[47]. Expression of DARPP-32 in graft-derived neurons indicates that a substantial number of grafted NPs correctly
acquired basal forebrain identity, yet failed to achieve the
expected GABAergic phenotype of striatal projection neurons
by 6 months after grafting. It is possible that whereas striatal
progenitor cells acquire GABAergic and DARPP-32 identities
under the influence of synchronized inductive signals in the
developing LGE, in artificial systems combining initial in
vitro differentiation followed by further maturation in the
adult striatal environment NPs may acquire the one (i.e.,
DARPP-32) but not the other (i.e., GABA) differentiation
marker [46, 48–52]. The importance of temporally and positionally appropriate inductive signaling is evident in studies
using ex vivo derived NSCs as spinal cord grafts [1, 2], where
spinal cord-derived NPs may be better able to undergo neuronal differentiation to GABAergic neurons compared to brain-
Nasonkin, Mahairaki, Xu et al.
derived precursors [53]. Another possibility is that human
NPs require more time in vivo to achieve full GABAergic
differentiation.
Axons, Synapses, and Structural Integration
in the Host Forebrain
SC121 ICC labels all axons projecting from differentiated
NPs and shows that these axons take established pathways in
their vicinity but do not grow through the gray matter; in
doing so, these axons reach both the appropriate innervation
targets of the striatum such as globus pallidus and subthalamic nucleus but also nonconstitutive targets such as neocortex. This pattern is consistent with previous findings that
intact white matter tracts organize the axonal trajectories of
transplanted developing [54, 55] and adult [56] neurons in a
parallel molding fashion that is mediated, among other things,
by a neurite-inhibitory effect of myelin [57].
The synaptic nature of terminal or en passant swellings labeled with human synaptophysin and SC121 was ascertained
here by the presence of synaptic vesicles accumulating next
to dense material on the cytoplasmic side of the terminal
membrane [58], a synaptic cleft 20–30-nm wide, and an accumulation of dense cytoplasmic material on the postsynaptic
membrane. The identity of such profiles as synapses between
graft-derived and host neurons was based on either a combination of human synaptophysin immunoreactivity of the synapse and a postsynaptic structure remote from the graft or the
combination of SC121 immunoreactivity of the synapse and
an unlabeled postsynaptic structure in any location. Synaptic
specializations between graft and host neurons were not visible at 3 months but were apparent at 6 months, that is, took
several months before these human neurons established synaptic contacts with host postsynaptic structures.
2425
conditions used here, these cells maintain their cell fate plasticity and continue to respond to differentiation cues from particular CNS microenvironments.
The substantial degree of neuronal differentiation of our
striatal NP grafts raises the issue of their potential as replacement therapies for neurodegenerative diseases of the basal ganglia, such as Huntington’s disease. Such enthusiasm, however,
should be tempered by the fact that these cells do not show
complete differentiation into the main projection neurons of
the striatum, that is, medium spiny neurons. With progress in
our understanding of molecular cues specifying developmental
fate choices in the basal ganglia-basal forebrain, one can foresee modifications of the in vitro protocol reported here or
ongoing treatment of these grafts in vivo. These additions will
hopefully optimize the differentiation of human NPs and determine their potential as cell replacement therapies for diseases
of the basal ganglia. In the mean time, their experimental therapeutic value lies primarily in their ability to make synaptic contacts with host neurons and, via these contacts, support injured
host neurons with released trophic signals [41].
ACKNOWLEDGMENTS
We thank Alex Kecojevic, Annie Welsh, Tan Pongstaporn, and
Christa Baker for their technical help and StemCells Inc. and Dr.
Christopher Walsh for their generous gifts of the SC121 and
DCAMKL1 antibodies, respectively. This work was supported
by the National Institutes of Health grants NS45140-03, NIDCD
DC000232, NIDCD P30 DC005211, and EY01765, the Robert
Packard Center for ALS Research at Johns Hopkins, and the
Muscular Dystrophy Association.
Relevance for Experimental Therapeutics
Our findings demonstrate the generic ability of NPs derived
from noggin-treated human HESCs and optimized for cell
number and maturation stage, to survive and differentiate in
the adult CNS without evident tumorigenesis. Despite a predominantly neuronal bias of grafted NPs based on the culture
REFERENCES
1
2
3
4
5
6
7
8
9
Cummings BJ, Uchida N, Tamaki SJ et al. Human neural stem cells
differentiate and promote locomotor recovery in spinal cord-injured
mice. Proc Natl Acad Sci U S A 2005;102(39):14069–14074.
Yan J, Xu L, Welsh AM et al. Extensive neuronal differentiation of
human neural stem cell grafts in adult rat spinal cord. PLoS Med
2007;4(2):e39.
Tabar V, Panagiotakos G, Greenberg ED et al. Migration and differentiation of neural precursors derived from human embryonic stem cells
in the rat brain. Nat Biotechnol 2005;23(5):601–606.
Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent
stem cells from adult human fibroblasts by defined factors. Cell 2007;
131(5):861–872.
Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell
lines derived from discarded embryos. Stem Cells 2003;21(5):
521–526.
Perrier AL, Tabar V, Barberi T et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci
U S A 2004;101(34):12543–12548.
Chung S, Shin BS, Hwang M et al. Neural precursors derived from
embryonic stem cells, but not those from fetal ventral mesencephalon,
maintain the potential to differentiate into dopaminergic neurons after
expansion in vitro. Stem Cells 2006;24(6):1583–1593.
Zeng X, Cai J, Chen J et al. Dopaminergic differentiation of human
embryonic stem cells. Stem Cells 2004;22(6):925–940.
Shin S, Dalton S, Stice SL. Human motor neuron differentiation from
human embryonic stem cells. Stem Cells Dev 2005;14(3):266–269.
www.StemCells.com
DISCLOSURE
OF
OF
POTENTIAL CONFLICTS
INTEREST
The authors indicate no potential conflicts of interest.
10 Lee H, Shamy GA, Elkabetz Y et al. Directed differentiation and
transplantation of human embryonic stem cell-derived motoneurons.
Stem Cells 2007;25(8):1931–1939.
11 Li XJ, Du ZW, Zarnowska ED et al. Specification of motoneurons from
human embryonic stem cells. Nat Biotechnol 2005;23(2):215–221.
12 Keirstead HS, Nistor G, Bernal G et al. Human embryonic stem cellderived oligodendrocyte progenitor cell transplants remyelinate and
restore locomotion after spinal cord injury. J Neurosci 2005;25(19):
4694–4705.
13 Bain G, Kitchens D, Yao M et al. Embryonic stem cells express neuronal properties in vitro. Dev Biol 1995;168(2):342–357.
14 Okabe S, Forsberg-Nilsson K, Spiro AC et al. Development of neuronal precursor cells and functional postmitotic neurons from embryonic
stem cells in vitro. Mech Dev 1996;59(1):89–102.
15 Schulz TC, Palmarini GM, Noggle SA et al. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci 2003;4:27.
16 Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of
transplantable neural precursors from human embryonic stem cells.
Nat Biotechnol 2001;19(12):1129–1133.
17 Shin S, Mitalipova M, Noggle S et al. Long-term proliferation of
human embryonic stem cell-derived neuroepithelial cells using defined
adherent culture conditions. Stem Cells 2006;24(1):125–138.
18 McMahon JA, Takada S, Zimmerman LB et al. Noggin-mediated antagonism of BMP signaling is required for growth and patterning of
the neural tube and somite. Genes Dev 1998;12(10):1438–1452.
19 Gerrard L, Rodgers L, Cui W. Differentiation of human embryonic
stem cells to neural lineages in adherent culture by blocking bone
morphogenetic protein signaling. Stem Cells 2005;23(9):1234–1241.
20 Nasonkin IO, Koliatsos VE. Nonhuman sialic acid Neu5Gc is very
low in human embryonic stem cell-derived neural precursors
Differentiation of HESC-Derived Neural Precursors
2426
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
differentiated with B27/N2 and noggin: Implications for transplantation. Exp Neurol 2006;201(2):525–529.
Maric D, Barker JL. Fluorescence-based sorting of neural stem cells
and progenitors. Curr Protoc Neurosci 2005;Chapter 3:Unit 3.18.
Xu L, Yan J, Chen D et al. Human neural stem cell grafts ameliorate
motor neuron disease in SOD-1 transgenic rats. Transplantation 2006;
82(7):865–875.
Maric D, Maric I, Chang YH et al. Stereotypical physiological properties emerge during early neuronal and glial lineage development in
the embryonic rat neocortex. Cereb Cortex 2000;10(8):729–747.
Maric D, Fiorio Pla A, Chang YH et al. Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated
by basic fibroblast growth factor (FGF) signaling via specific FGF
receptors. J Neurosci 2007;27(8):1836–1852.
Przyborski SA. Differentiation of human embryonic stem cells after
transplantation in immune-deficient mice. Stem Cells 2005;23(9):
1242–1250.
Cooke MJ, Stojkovic M, Przyborski SA. Growth of teratomas derived
from human pluripotent stem cells is influenced by the graft site.
Stem Cells Dev 2006;15(2):254–259.
Eberhart CG. In search of the medulloblast: Neural stem cells and
embryonal brain tumors. Neurosurg Clin N Am 2007;18(1):59–69,
viii–ix.
Deuel TA, Liu JS, Corbo JC et al. Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon
outgrowth. Neuron 2006;49(1):41–53.
Koizumi H, Tanaka T, Gleeson JG. Doublecortin-like kinase functions
with doublecortin to mediate fiber tract decussation and neuronal
migration. Neuron 2006;49(1):55–66.
Isacson O. The production and use of cells as therapeutic agents in
neurodegenerative diseases. Lancet Neurol 2003;2(7):417–424.
Sonntag KC, Pruszak J, Yoshizaki T et al. Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from
human embryonic stem cells using the bone morphogenic protein antagonist noggin. Stem Cells 2007;25(2):411–418.
Aubry L, Bugi A, Lefort N et al. Striatal progenitors derived from
human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci U S A 2008;105(43):
16707–16712.
Mũnoz-Sanjúan I, Brivanlou AH. Neural induction, the default model
and embryonic stem cells. Nat Rev Neurosci 2002;3(4):271–280.
Rajan P, Panchision DM, Newell LF et al. BMPs signal alternately
through a SMAD or FRAP-STAT pathway to regulate fate choice in
CNS stem cells. J Cell Biol 2003;161(5):911–921.
Boyer LA, Mathur D, Jaenisch R. Molecular control of pluripotency.
Curr Opin Genet Dev 2006;16(5):455–462.
Kuroda T, Tada M, Kubota H et al. Octamer and Sox elements are
required for transcriptional cis regulation of Nanog gene expression.
Mol Cell Biol 2005;25(6):2475–2485.
Capela A, Temple S. LeX/ssea-1 is expressed by adult mouse CNS
stem cells, identifying them as nonependymal. Neuron 2002;35(5):
865–875.
Brederlau A, Correia AS, Anisimov SV et al. Transplantation of
human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: Effect of in vitro differentiation on graft survival and
teratoma formation. Stem Cells 2006;24(6):1433–1440.
Tomita K, Moriyoshi K, Nakanishi S et al. Mammalian achaete-scute
and atonal homologs regulate neuronal versus glial fate determination
in the central nervous system. EMBO J 2000;19(20):5460–5472.
40 Chen H, Tung YC, Li B et al. Trophic factors counteract elevated
FGF-2-induced inhibition of adult neurogenesis. Neurobiol Aging
2007;28(8):1148–1162.
41 Lim DA, Tramontin AD, Trevejo JM et al. Noggin antagonizes BMP
signaling to create a niche for adult neurogenesis. Neuron 2000;28(3):
713–726.
42 Weible MW II, Chan-Ling T. Phenotypic characterization of neural
stem cells from human fetal spinal cord: Synergistic effect of LIF and
BMP4 to generate astrocytes. Glia 2007;55(11):1156–1168.
43 Castro DS, Skowronska-Krawczyk D, Armant O et al. Proneural
bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif. Dev Cell 2006;11(6):
831–844.
44 Ge W, He F, Kim KJ et al. Coupling of cell migration with neurogenesis by proneural bHLH factors. Proc Natl Acad Sci U S A 2006;
103(5):1319–1324.
45 Naimi S, Jeny R, Hantraye P et al. Ontogeny of human striatal
DARPP-32 neurons in fetuses and following xenografting to the adult
rat brain. Exp Neurol 1996;137(1):15–25.
46 Deacon TW, Pakzaban P, Isacson O. The lateral ganglionic eminence is
the origin of cells committed to striatal phenotypes: Neural transplantation and developmental evidence. Brain Res 1994;668(1–2):211–219.
47 Ferrari D, Sanchez-Pernaute R, Lee H et al. Transplanted dopamine
neurons derived from primate ES cells preferentially innervate
DARPP-32 striatal progenitors within the graft. Eur J Neurosci 2006;
24(7):1885–1896.
48 Lauder JM, Han VK, Henderson P et al. Prenatal ontogeny of the
GABAergic system in the rat brain: An immunocytochemical study.
Neuroscience 1986;19(2):465–493.
49 Stenman J, Toresson H, Campbell K. Identification of two distinct
progenitor populations in the lateral ganglionic eminence: Implications
for striatal and olfactory bulb neurogenesis. J Neurosci 2003;23(1):
167–174.
50 Allain AE, Meyrand P, Branchereau P. Ontogenic changes of the spinal GABAergic cell population are controlled by the serotonin (5-HT)
system: Implication of 5-HT1 receptor family. J Neurosci 2005;
25(38):8714–8724.
51 Wichterle H, Turnbull DH, Nery S et al. In utero fate mapping reveals
distinct migratory pathways and fates of neurons born in the mammalian basal forebrain. Development 2001;128(19):3759–3771.
52 Lin PY, Hinterneder JM, Rollor SR et al. Non-cell-autonomous regulation of GABAergic neuron development by neurotrophins and the
p75 receptor. J Neurosci 2007;27(47):12787–12796.
53 Koliatsos VE, Xu L, Yan J. Human stem cell grafts as therapies for
motor neuron disease. Expert Opin Biol Ther 2008;8(2):137–141.
54 Wictorin K, Brundin P, Gustavii B et al. Reformation of long axon
pathways in adult rat central nervous system by human forebrain neuroblasts. Nature 1990;347(6293):556–558.
55 Wictorin K, Clarke DJ, Bolam JP et al. Extensive efferent projections
of intra-striatally transplanted striatal neurons as revealed by a species-specific neurofilament marker and anterograde axonal tracing.
Prog Brain Res 1990;82:391–399.
56 Davies SJ, Fitch MT, Memberg SP et al. Regeneration of adult axons
in white matter tracts of the central nervous system. Nature 1997;
390(6661):680–683.
57 Pettigrew DB, Crutcher KA. Myelin contributes to the parallel orientation of axonal growth on white matter in vitro. BMC Neurosci 2001;2:9.
58 Schikorski T, Stevens CF. Quantitative ultrastructural analysis of hippocampal excitatory synapses. J Neurosci 1997;17(15):5858–5867.
See www.StemCells.com for supporting information available online.