Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells.
ABSTRACT To understand the range of competence of embryonic stem (ES) cell-derived neural precursors, we have examined in vitro differentiation of mouse and primate ES cells into the dorsal- (neural crest) and ventralmost (floor plate) cells of the neural axis. Stromal cell-derived inducing activity (SDIA; accumulated on PA6 stromal cells) induces cocultured ES cells to differentiate into rostral CNS tissues containing both ventral and dorsal cells. Although early exposure of SDIA-treated ES cells to bone morphogenetic protein (BMP)4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the fourth day of coculture causes differentiation of neural crest cells and dorsalmost CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons. Notably, high concentrations of Shh efficiently promote differentiation of HNF3beta(+) floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naive precursors that have the competence of differentiating into the "full" dorsal-ventral range of neuroectodermal derivatives in response to patterning signals.
- SourceAvailable from: Masami Nozaki[Show abstract] [Hide abstract]
ABSTRACT: The developmental fate of the multipotent neural crest (NC) is determined along with the neural axis in which NC cells are generated. Only the cranial NC can differentiate to mesectodermal derivatives such as osteoblasts, chondrocytes, and adipocytes in vivo. Here, we attempted to selectively differentiate mouse embryonic stem (ES) cells into cranial NC stem cells and propagate them to explore their developmental potential to differentiate into mesectodermal derivatives. Using aggregation cultures in feeder- and serum-free neural induction medium (NIM) without serum replacement and L-glutamine, we obtained NIM neurospheres composed of neuroepithelium. The NIM neurospheres expressed the rostral markers Otx1 and Otx2, but not non-rostral markers Hoxb4, Hoxb9, Lbx1, and TH, which characterize cranial neurospheres. Subsequently, AP2α, Sox9, p75, Snail, Slug, and Twist-positive NC cells were differentiated in 4-day adhesion cultures of cranial neurospheres. Additionally, sphere clusters in adhesion cultures were differentiated into osteoblast while migrating cells were not. By taking advantage of the sphere-formation capability, we isolated and propagated NC stem cells from the sphere clusters and confirmed their multipotency. NC stem cells expressed NC and stem cell markers, and maintained differentiation potency in the NC derivatives. These results show that cranial NC stem cells were obtained reproducibly and efficiently without special inducing factors, gene transfection or fluorescence-activated cell sorting (FACS) selection.Stem Cells and Development 08/2014; · 4.20 Impact Factor
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ABSTRACT: Members of the transforming growth factor-β (TGF-β) family have been implicated in embryogenesis as well as inthe determination of the cell fatesofmouse and human embryonic stem (ES) cells, which are characterized by their self-renewal and pluripotency.The cellular responses to TGF-βfamilysignals are divergent depending on the cellular context and local environment. TGF-β family signals play critical roles both in the maintenance of the pluripotent state of ES cells by inducing the expression of Nanog, Oct4, and Sox2, and in their differentiation into various cell types by regulating the expression of master regulatory genes. Moreover, multiple lines of evidence have suggested the importance of TGF-βfamilysignalsin establishing induced pluripotent stem (iPS) cells. Since ES and iPScells have great potential for applications in regenerative medicine, it is criticalto figure out the mechanisms underlying their self-renewal, pluripotency, and differentiation.Here, wediscuss the roles of TGF-β familyligands and their downstreamsignaling molecules,Smad proteins, in the maintenance of thepluripotency and lineage specification of mouse and human ES and iPScells.Seminars in Cell and Developmental Biology 06/2014; · 6.20 Impact Factor
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ABSTRACT: The neural crest is the name given to the strip of cells at the junction between neural and epidermal ectoderm in neurula-stage vertebrate embryos, which is later brought to the dorsal neural tube as the neural folds elevate. The neural crest is a heterogeneous and multipotent progenitor cell population whose cells undergo EMT then extensively and accurately migrate throughout the embryo. Neural crest cells contribute to nearly every organ system in the body, with derivatives of neuronal, glial, neuroendocrine, pigment, and also mesodermal lineages. This breadth of developmental capacity has led to the neural crest being termed the fourth germ layer. The neural crest has occupied a prominent place in developmental biology, due to its exaggerated migratory morphogenesis and its remarkably wide developmental potential. As such, neural crest cells have become an attractive model for developmental biologists for studying these processes. Problems in neural crest development cause a number of human syndromes and birth defects known collectively as neurocristopathies; these include Treacher Collins syndrome, Hirschsprung disease, and 22q11.2 deletion syndromes. Tumors in the neural crest lineage are also of clinical importance, including the aggressive melanoma and neuroblastoma types. These clinical aspects have drawn attention to the selection or creation of neural crest progenitor cells, particularly of human origin, for studying pathologies of the neural crest at the cellular level, and also for possible cell therapeutics. The versatility of the neural crest lends itself to interlinked research, spanning basic developmental biology, birth defect research, oncology, and stem/progenitor cell biology and therapy. Birth Defects Research (Part C), 2014. © 2014 Wiley Periodicals, Inc.Birth Defects Research Part C Embryo Today Reviews 09/2014; · 4.44 Impact Factor
Generation of neural crest-derived peripheral neurons
and floor plate cells from mouse and primate
embryonic stem cells
Kenji Mizuseki*, Tatsunori Sakamoto†‡, Kiichi Watanabe*†, Keiko Muguruma§, Makoto Ikeya*, Ayaka Nishiyama*,
Akiko Arakawa†¶, Hirofumi Suemori?, Norio Nakatsuji?, Hiroshi Kawasaki†, Fujio Murakami§, and Yoshiki Sasai*†?**
*Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047 Japan; Departments of†Medical Embryology and
Neurobiology and?Development and Differentiation, Institute for Frontier Medical Sciences and Departments of‡Otolaryngology–Head and Neck
Surgery, and¶Dermatology, Kyoto University, Kyoto 606-8507 Japan; and§Laboratory for Neuroscience, Graduate School of Frontier Biosciences,
Osaka University, Toyonaka 560-8531, Japan
Edited by Igor B. Dawid, National Institutes of Health, Bethesda, MD, and approved March 24, 2003 (received for review December 2, 2002)
To understand the range of competence of embryonic stem (ES)
cell-derived neural precursors, we have examined in vitro differ-
entiation of mouse and primate ES cells into the dorsal- (neural
crest) and ventralmost (floor plate) cells of the neural axis. Stromal
cell-derived inducing activity (SDIA; accumulated on PA6 stromal
cells) induces cocultured ES cells to differentiate into rostral CNS
tissues containing both ventral and dorsal cells. Although early
exposure of SDIA-treated ES cells to bone morphogenetic protein
(BMP)4 suppresses neural differentiation and promotes epidermo-
genesis, late BMP4 exposure after the fourth day of coculture
causes differentiation of neural crest cells and dorsalmost CNS
cells, with autonomic system and sensory lineages induced pref-
erentially by high and low BMP4 concentrations, respectively. In
contrast, Sonic hedgehog (Shh) suppresses differentiation of neu-
ral crest lineages and promotes that of ventral CNS tissues such as
motor neurons. Notably, high concentrations of Shh efficiently
promote differentiation of HNF3??floor plate cells with axonal
guidance activities. Thus, SDIA-treated ES cells generate naı ¨ve
precursors that have the competence of differentiating into the
‘‘full’’ dorsal–ventral range of neuroectodermal derivatives in re-
sponse to patterning signals.
by using PA6 stromal cells as an inducer source (1, 2). When
cultured on PA6 cells under serum-free conditions, mouse ES
cells differentiate into neural cells at a ?90% efficiency. After
3–5 days of coculture on PA6 cells, the majority of ES cells
differentiate into nestin?neuroectodermal precursor cells.
TuJ1?postmitotic neurons appear on coculture days 5–6, and
tyrosine hydroxylase (TH)?dopaminergic neurons accumulate
during days 7–9. The inducing activity present on the surface of
PA6 cells was named stromal cell-derived inducing activity
(SDIA; ref. 1).
Recent reports on ES cell differentiation have suggested the
possibility that information on in vivo neurogenesis might be
systematically linked to stem cell technology (3, 4). However, it
remains to be known whether ES cell-derived neural precursors
generated in vitro can produce the full dorsal–ventral range of
neuroectodermal derivatives in response to embryonic posi-
tional information. To address this question, we have tested in
this study whether SDIA-treated ES cells have the competence
of differentiating into the dorsal- (neural crest) and ventralmost
(floor plate) cells under embryologically relevant conditions.
e have recently established conditions that induce effi-
cient neural differentiation of embryonic stem (ES) cells
Materials and Methods
Cell Culture and Treatment with Patterning Factors. Mouse ES cells
(EB5), primate ES cells (cynomolgus monkey-derived; pur-
chased from Asahi Technoglass, Funabashi, Japan), and PA6
cells were maintained and used for induction as described (1, 2,
5). Human bone morphogenetic protein (BMP)4 and mouse
Shh-N (25–198) were purchased from R&D Systems and freshly
added at each medium change. The day on which ES cells are
seeded on PA6 is defined as day 0.
Immunocytochemistry, Statistics, and RT-PCR. Cells were fixed with
secondary antibodies conjugated with FITC, cy3, or cy5. For
statistics, ?100 colonies were observed in each experiment, and
three or more experiments were performed. P values for statis-
tical significance (t test) are described in the corresponding
figure legends. The values shown in graphs represent the mean ?
SD. RT-PCR was performed with ES cell colonies detached
from feeder cells as described (1). The primary antibodies and
primers used are described in Supporting Materials and Methods,
which are published as supporting information on the PNAS web
Colony Isolation and Axon Guidance Assays. The 3D collagen gel
assay for axon guidance was performed by using isolated ES cell
colonies (day 8; ref. 1) and the cerebellar plate region excised
from embryonic day 13 Wistar rats as a responder (6).
Positional Identity of Neural Tissues Induced from ES Cells by SDIA.
by RT-PCR (Fig. 1A). SDIA-treated mouse ES cells express the
forebrain markers Otx2 and Six3, the ventral diencephalon
marker Rx, the ventral forebrain marker Nkx2.1, the midbrain–
hindbrain border marker En2, and the hindbrain marker Gbx2,
but not the spinal cord markers Hoxb4 and b9 (lane 2). These
results show that a majority of neural cells induced by SDIA
express rostral neural markers. This idea is consistent with our
previous report that dopaminergic neurons generated by the
SDIA method are those of the midbrain type (1).
We next attempted to alter the rostral–caudal identity of
SDIA-induced neural cells by the caudalizing factor retinoic acid
(RA; ref. 7). RA treatment (0.2 ?M all-trans RA; Fig. 1A, lane
3) suppressed the forebrain markers Otx2, Six3, Rx, and Nkx2.1,
whereas it induced the hindbrain marker Gbx2 and the spinal
cord markers Hoxb4 and b9. RA treatment did not significantly
affect the level of neural cell adhesion molecule (NCAM)
expression (Fig. 1A). This demonstrates that the differentiation
potential of SDIA-treated ES cells is not limited to the rostral
brain region but can also be modified in the caudal direction.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: BMP, bone morphogenetic protein; ES cells, embryonic stem cells; Peri,
peripherin; PNS, peripheral nervous system; RA, retinoic acid; SDIA, stromal cell-derived
inducing activity; Shh, Sonic hedgehog; TH, tyrosine hydroxylase; NCAM, neural cell adhe-
**To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
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axis. SDIA-treated ES cells contained cells expressing a variety
of dorsal-ventral neural markers (Fig. 1B), including HNF3??
(floor plate), Nkx2.2?(ventral CNS cells flanking the floor
plate), TH??Peripherin (Peri)?(8) (CNS dopaminergic), Pax6?
(ventral to dorsal), and Pax7?(alar plate; i.e., dorsal CNS) cells.
Interestingly, few cells expressed a phenotype of Math1?(the
dorsalmost interneuron progenitor; ref. 9) or TH??Peri?(pe-
ripheral autonomic neuron lineage), suggesting that SDIA-
treated ES cells do not efficiently generate cells arising from the
dorsalmost region of the neural tube, including neural crest
Induction of Neural Crest and Dorsalmost CNS Differentiation in
SDIA-Treated ES Cells by Late Exposure to BMP4. The neural crest
arises from the juncture of dorsal CNS and nonneural ectoderm
(10). A number of BMP family factors are expressed in this
dorsal region (10–12). Although BMP signals inhibit neural
induction at the early gastrula stage, the same signals promote
neural crest determination when applied at later developmental
stages (10–12). We therefore examined the effects of BMP4 by
focusing on the temporal aspect. Without BMP treatment, a high
percentage of ES cell colonies contained TuJ1?neurons after
being cocultured on PA6 cells for 9 days (Fig. 1C, lane 1).
Consistent with a previous study (1), TuJ1?colonies decreased
drastically when the cells were treated with BMP4 on and after
day 0, 1, or 2 (Fig. 1C, lanes 2–4), whereas E-cadherin?
epidermal precursors increased (ref. 1; not shown). Similar
results were obtained for the number of colonies containing
TH??Peri?CNS dopaminergic neurons (Fig. 1D, lanes 1–4).
Under these conditions, few colonies contained TH??Peri?
peripheral autonomic cells (Fig. 1E, lanes 1–4). In contrast, a
number of TH??Peri?cells appeared in SDIA-treated ES cell
5–9; also see Fig. 2 E and F). Especially when SDIA-treated ES
cells were incubated with BMP4 during days 5–9, an efficient
induction of the TH??Peri?population was observed (51 ? 4%
colonies, n ? 264, four independent experiments; Fig. 1E, lane
7; also see Fig. 2C for percent of neurons), whereas TH??Peri?
CNS neurons were rarely seen (Fig. 1D, lane 7). These results
suggest that late BMP exposure promotes differentiation of at
least one type of the neural crest-derived peripheral nervous
system (PNS) lineages.
RT-PCR analysis showed that BMP treatment (from day 5
onward; 5 nM) induced the neural crest markers (10) Ncx (13),
Snail, Slug, dHand, and Msx1 (Fig. 1F) in SDIA-treated ES cells
(lane 3; lane 2 shows control SDIA-treated ES cells without
BMP4). The CNS markers Lbx-1 (14) and Pax7 were suppressed,
whereas the panneural marker NCAM was largely unaffected
(lanes 2 and 3). In situ hybridization analyses showed that a
significant portion of SDIA?BMP-treated ES cell colonies ex-
pressed the neural crest marker Ncx-1 (Fig. 1 G, H, and J), but
not in control SDIA-treated ES cell colonies (n ? 100; Fig. 1 I
day (E)10.5 embryo; lanes 2 and 3, SDIA-treated ES cells without and with 0.2
?M RA treatment during days 4–8, respectively. (B) Percent immunoreactive
cells?total cells are shown. (C–E) BMP4 (0.5 nM) treatment during the days
indicated above. Positive colonies were defined as ones containing TuJ1?(C),
RT-PCR analysis with neural crest and neural markers. Lane 1, E10.5 whole
embryos; lanes 2 and 3, SDIA-treated ES cells without or with 5 nM BMP4
treatment during days 5–8, respectively. (G) Ncx expression in E11.5 mouse by
in situ hybridization. Dorsal root (arrow) and sympathetic (arrowhead) gan-
glia. (H) SDIA?BMP-treated ES cell colonies (day 8). (I) SDIA-treated ES cell
colonies. (J) Percentages of Ncx?colonies at 0, 0.5, and 5 nM BMP4 (n ? 100,
200, and 200, respectively).*, P ? 0.005 vs. control;**, P ? 0.005 vs. control.
Mizuseki et al. PNAS ?
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and J). Neither SDIA?BMP- nor SDIA-treated ES cells ex-
pressed a detectable level of the mesodermal markers Brachyury
or of the endodermal marker Sox17 (17) (not shown). These
results suggest that BMP treatment at the right time can promote
We next examined the dose-dependent effect of BMP4 (from
day 5 onward) quantitatively on the fates of SDIA-treated ES
cells (Fig. 2 A and B). Immunostaining showed that BMP4
significantly decreased the number of HNF3??, Nkx2.2?, and
biphasic induction of Math1 and the roof plate marker Gdf7 (18)
?0.5 nM was also observed by RT-PCR analysis (Fig. 2B).
One current obstacle in the study of mammalian neural crest
determination is the lack of appropriate panneural crest mark-
ers. Therefore, we first counted the number of AP2?cells (19),
which are expressed in both neural crest cells (NCAM?; Fig. 6
A–F, which is published as supporting information on the PNAS
web site) and in epidermal cells (NCAM?). BMP4 increased the
number of AP2??NCAM?cells in a dose-dependent manner
(Fig. 2A). The sensory ganglion neurons are the major popula-
tion of cells that are Brn3a??Peri?in early neurogenesis (refs.
8, 20, and 21; Fig. 6 G–J). Dose-response analysis (Fig. 2C)
showed that BMP4 induced the differentiation of Brn3a??Peri?
neurons (Fig. 2D) at a lower concentration (0.5 nM) and less
efficiently at 5 nM. In contrast, TH??Peri?cells were induced
more efficiently at 5 nM (Fig. 2C). TH??Peri?cells have a
round-shaped cell body and are positive for Phox2b (Fig. 2 E–G;
characteristic for the autonomic lineage; ref. 22). Consistently,
the autonomic lineage marker eHand (10) was preferentially
induced at 5 nM (Fig. 2B). This differential requirement of BMP
concentrations for sensory and autonomic cells is reminiscent of
a previous report that high BMP concentrations preferentially
promote differentiation of autonomic neurons from rat-
dissociated neural tube explants, whereas low concentrations
induce differentiation of sensory neurons (21).
At both high and low BMP concentrations, no cells positive for
the smooth muscle cell marker SMA (23), the pigment cell
marker Trp-2 (24), or the Schwann cell marker P0 (25) were
observed (n ? 100 colonies each; not shown). To ask whether
SDIA?BMP-treated ES cells also have the competence of dif-
ferentiating into nonneural derivatives, we performed an in vitro
study using culture on a matrix substrate. SDIA?BMP-treated
ES cell colonies were isolated on day 8 by papain digestion,
replated on fibronectin-coated dishes, and cultured without
BMP4 in a medium containing chicken embryo extracts (26).
Neuronal (Peri, TuJ, Brn3a) and glial (glial fibrillary acidic
protein; positive in ?10% colonies) markers were found only
inside of the colonies or in their vicinities (not shown). In
contrast, numerous SMA?cells were found within the popula-
tion of cells migrating from the ES cell colonies (Fig. 7, which is
published as supporting information on the PNAS web site;
SMA?cells were found around 85 ? 7% of 158 colonies, four
independent experiments; In such colonies, SMA?cells occu-
pied 84 ? 6% of the migratory cells). Control SDIA-treated ES
cells cultured under the same conditions generated SMA?cells
in only 4.5 ? 3.5% colonies (n ? 120, four independent
experiments). In contrast, Trp-2?cells or P0?cells were not
detected in SDIA?BMP-treated ES cells under any of the
conditions tested above or in medium containing ET-3 and SCF
(27) or GGF (26), even after long culture up to 24 days (not
Collectively, these results demonstrate that BMP treatment, as
it does in vivo (11, 12), causes dorsalization of ES cell-derived
which differentiate into, at least, the autonomic, sensory, and
smooth muscle cell lineages in vitro.
Shh Suppresses Neural Crest Differentiation and Induces Ventral CNS
Differentiation. Shh suppresses the development of dorsal tissues
and promotes the differentiation of ventral CNS tissues (11).
Consistent with this activity, when applied to SDIA-treated ES
cells, Shh treatment (days 4–9) suppressed the appearance of
AP2??NCAM?and Brn3a??Peri?cells in a dose-dependent
manner (Fig. 3A). Nkx2.2?and HNF3??ventral cells were
significantly increased by Shh treatment, whereas the alar plate
marker Pax7 was suppressed. In addition, a high proportion of
Islet1?2?neurons (28) were found among TuJ1?neurons (26 ?
2%, n ? 227 colonies, 30 nM Shh during days 4–9, four
independent experiments; Fig. 3B). They exhibited a marker
phenotype of HB9?, Phox2b?(Fig. 3 B–D), choline acetyle-
transferase?(ChAT), Peri?, Lim3?, AP2?, and Brn3a?(not
shown). This is consistent with the marker characteristics for
somatic motor neurons of the midbrain–rostral hindbrain and
branchiomotor?visceral motor neurons, which are located in the
brainstem basal plate (i.e., brainstem-type motor neurons in the
cranial nerve nuclei III, IV, V, VII, IX, X, and XI; refs. 22 and
28–31). Consistent with this idea, differentiation of HB9??
Neurons were defined as TuJ1? cells.*, P ? 0.001 vs. 0 nM;**, P ? 0.05 vs. 0.5
nM;***, P ? 0.001 vs. 0 nM;****, P ? 0.005 vs. 0.5 nM. (B) RT-PCR analysis of
BMP4 concentration [0.5 nM; immunostained with anti-Brn3a (red) and anti-
Peri (green) antibodies]. (E–G) Cells with autonomic-lineage markers induced
at a high BMP4 concentration (5 nM). Anti-TH (E–G, green); anti-Peri (F, red);
anti-Phox2b (G, blue) antibodies.
www.pnas.org?cgi?doi?10.1073?pnas.1037282100Mizuseki et al.
Phox2b??Islet1?2?neurons was suppressed by additional RA
treatment, whereas it induced efficient differentiation of neu-
rons displaying the characteristics of somatic motor neurons of
the spinal cord and caudal hindbrain (Islet1?2?, HB9?,
Phox2b?; Lim3?, ChAT?; refs. 22 and 28–31; Fig. 8 A–L, which
is published as supporting information on the PNAS web site,
and data not shown). These findings demonstrate that Shh
promotes differentiation of ventral CNS tissues and suppresses
that of dorsal CNS and neural crest cells.
Directed Differentiation of Functional Floor Plate Cells. Shh exerted
its ventralizing activity on ES cell-derived neural precursors. A
similar conclusion has been reported in the study using the
Shh?RA-treated embryoid bodies (EBs) (4). Interestingly, there
is a clear qualitative difference between the observations in two
studies. In the study using the Shh?RA treatment of EBs, the
ventralmost CNS markers such as HNF3? were not significantly
induced even at a very high concentration of Shh (4). Thus, it
can be only partially ventralized even by strong Shh signaling. In
contrast, Shh treatment (300 nM) showed strong ventralizing
effects and resulted in production of numerous Nkx2.2?and
3 A, E, and F) in SDIA-treated ES cells.
This difference led us to ask whether HNF3??cells generated
in SDIA?Shh-treated ES cells had additional features of floor
plate cells. Most of the HNF3??cells were NCAM?and were
found in tightly packed cell clusters (Fig. 3 E–G). RT-PCR
analyses showed that SDIA?Shh-treated ES cells expressed
significant levels of the floor plate factors Shh and Netrin1 (Fig.
3H, lane 3), whereas SDIA?BMP-treated cells did not (lane 2).
We next tested whether SDIA?Shh-induced tissues possessed
axon guidance activities similar to floor plate tissues by using a
3D coculture assay (Fig. 3I). In accordance with previous reports
(6, 32), when metencephalic alar plate [cerebellar plate (CP)]
explants were cultured alone in collagen gels, virtually no
neurites were induced from dorsal surface (Fig. 3J); however,
when cocultured with floor plate explants at a distance, neurites
were induced to grow dorsally toward the floor plate explants (in
the right direction, Fig. 3K). Similarly, when cocultured with
SDIA?Shh-treated ES cells, many neurites extended from CP
explants toward the ES cell colony located dorsally (Fig. 3 L and
N). SDIA?BMP-treated ES cell colonies did not show significant
axon growth inducing activities on CP explants (Fig. 3 M and N).
These results indicate that Shh treatment induces differentiation
of functional floor plate cells in SDIA-treated ES cells.
Differentiation of Neural Crest Derivatives, CNS Neurons, and Floor
Plate Cells from Primate ES Cells. Finally, we examined the differ-
entiation competence of SDIA-treated primate ES cells (2, 5).
Primate ES cells differentiated into Brn3a??Peri?(sensory
lineage) and TH??Phox2b??Peri?(autonomic lineage) neurons
(Fig. 4 A–C) under the same conditions used in the mouse cell
experiments (Fig. 2), i.e., at low and high BMP doses, respec-
tively. SDIA?Shh-treated primate ES cells differentiated into
cells with the brainstem-type motor neuron phenotype (Islet1?
2?, HB9?, Phox2b?, choline acetyletransferase (ChAT)?, and
markers. (B–D) HB9??Phox2b??Islet1?2?motor neurons induced by Shh (30
nM, days 4–9). (E and F) Double staining of Nkx2.2 (green) and HNF3? (red) in
an SDIA?Shh-treated ES cell colony. The NCAM staining in E indicates the
colony area. (G) A high-magnification view. NCAM (green) and HNF3? (red).
2, SDIA?BMP-treated; and lane 3, SDIA?Shh-treated ES cells. (I) A flat whole-
showing chemoattraction activities on CP. (L) CP cocultured with SDIA?Shh-
treated ES cells (Shh 300 nM). (M) CP cocultured with SDIA?BMP-treated ES
from CP explants. Mean lengths of dorsal neurite outgrowth (micrometers)
with CP alone (none, n ? 12), FP (n ? 12), undifferentiated ES (ES, n ? 14),
SDIA?BMP-treated ES (ES?BMP, n ? 24), and SDIA?Shh-treated ES cells (ES?
Shh, n ? 28).*, P ? 0.001 vs. none, ES, and ES?BMP.
Mizuseki et al. PNAS ?
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Lim3?; Fig. 4 D and E and not shown), whereas SDIA?Shh?RA
treatment induced neurons with caudal somatic motor neuron
markers (Islet1?2?, HB9?, Phox2b?, ChAT?, and Lim3?; Fig. 9,
which is published as supporting information on the PNAS web
site). SDIA?Shh treatment also induced a significant number of
Nkx2.2?and HNF3??cells from primate ES cells (Fig. 4 F and
G). These results show that SDIA-treated primate ES cells can
generate the full range of dorsal and ventral neural tissues, as
In contrast, quantitative analysis with dorsal–ventral markers
previous study, primate ES cells differentiated into neural cells
about half as efficiently as mouse cells on PA6 cells (NCAM?at
45% efficiency and TuJ1?at 25%; ref. 2). More importantly,
without BMP or Shh treatment, SDIA-treated primate ES cell
colonies expressed only dorsal markers (AP2?NCAM, Brn3a,
and Pax7; Fig. 4G), whereas mouse ES cell colonies cultured on
PA6 expressed both ventral (HNF3?, Nkx2.2) and dorsal (Pax7)
neural markers (Fig. 1B). Primate ES cells expressed the ventral
markers HNF3? and Nkx2.2 only in the presence of Shh (Fig.
4G). Thus, SDIA-treated primate ES cells (without Shh or BMP
treatment) have a positional identity that is more dorsally
deviated than SDIA-treated mouse ES cells. This may partly
explain why primate ES cells seemed to require a higher Shh
concentration (300 nM) for ventral marker induction than did
mouse cells (30 nM) (Figs. 3A and 4G), and why a number of
Brn3a??Peri?cells were generated even without BMP4 treat-
ment (Fig. 4C) from primate ES cells.
Together with our previous report (1), the present study dem-
onstrates that systematic generation of major ectodermal cate-
gories (epidermis, neural crest, roof plate, CNS neurons, and
floor plate) from ES cells in vitro is now made possible by
manipulating the timing, concentration, and combination of
exogenous factors. Our induction strategy and working hypoth-
esis are summarized in Fig. 5. This systematic induction involves
at least two critical phases of fate determination. In phase I (days
0–3), SDIA treatment promotes ectodermal differentiation.
SDIA suppresses mesodermal differentiation even in the pres-
ence of BMP4 (1), which has been shown to have a mesoderm-
inducing activity on ES cells in vitro (33, 34). Instead, when
treatment of exogenous BMP4 is started during phase I (early
out early BMP exposure, SDIA-treated ES cells acquire a
differentiation competence similar to naı ¨ve neuroectodermal
Phase II (days 4–7) is critical for the acquisition of the
dorsal–ventral identity of the neuroectodermal derivatives. In
the absence of exogenous BMP4, SDIA-treated ES cells have the
competence of differentiating into a variety of dorsal and ventral
cells of the rostral CNS (e.g., midbrain dopaminergic neurons),
except for the dorsalmost cells of the neural tube. Addition of
Shh promotes differentiation of ventral CNS cells, including
floor plate cells, Nkx2.2?cells, and brainstem-type motor neu-
rons, as well as caudal motor neurons with additional RA
treatment. In particular, a high Shh concentration induces
efficient differentiation of functional floor plate cells (Fig. 3).
Motor neurons generated with SDIA?Shh are also functional;
innervating C2C12-derived myotubes and causing nicotinic ace-
tylcholine receptor-dependent muscular contraction in vitro
(Fig. 8 G–L and Movies 1–3, which are published as supporting
information on the PNAS web site).
Treatment of exogenous BMP4 during phase II (late BMP
exposure) results in suppression of ventral neural differentiation
and promotion of dorsal differentiation. SDIA-treated ES cells
with late BMP4 exposure have the competence of differentiating
into neural crest derivatives and dorsalmost CNS cells (Math1?
cells and roof plate) (Figs. 1 and 2). Among neural crest-derived
cells, a high BMP4 concentration preferentially induces differ-
entiation of autonomic neuron lineages, whereas a low concen-
neurons and autonomic neurons, respectively. Cells were treated with 0, 0.5,
and 5 nM BMP4 during days 7–13 and analyzed on day 13 for Brn3a?Peri and
TH?Peri staining, respectively. Neurons were defined as TuJ1?cells.*, P ?
0.001 vs. control;**, P ? 0.005 vs. BMP4 0.5 nM;***, P ? 0.001 vs. control. (D
and E) Islet1?2??Phox2b?motor neurons generated by SDIA?Shh treatment.
ventral markers. BMP4 and Shh were added during days 7–13 and 3–13,
(A–C) At low and high concentrations, BMP4 induced sensory bipolar
ventralmost CNS differentiation by timely exposure to patterning factors.
www.pnas.org?cgi?doi?10.1073?pnas.1037282100Mizuseki et al.
tration more efficiently promotes generation of sensory neurons
(Fig. 2C). When SDIA?BMP4-treated ES cells are replated and
cultured without exogenous BMP4 on fibronectin after phase II,
smooth muscle cells are efficiently produced.
Interestingly, the timing of phases I and II would correspond
well with the embryonic days of pre-?early gastrulation and
neurulation, if 3.5 days (the embryonic day of the inner cell mass
from which ES cells are derived) are added to the induction
Questions for Future Studies
An important question to be addressed in future studies is the
molecular nature of SDIA. Its elucidation is crucial for the
establishment of a feeder-free induction system. As reported in
our previous study (1), the SDIA activity is not detected in
conditioned medium of PA6 cells but on the cell surface. This
makes biochemical approaches difficult for molecular identifi-
cation. The candidate approach has been unsuccessful so far (1).
Although early neural crest markers are induced by SDIA?
BMP4 treatment (Fig. 1), this study shows the generation of PNS
neurons and smooth muscle cells but not that of other neural
crest lineages. Modifying culture conditions should allow us to
ask whether SDIA?BMP-induced progenitors can generate
other neural crest derivatives. A challenging future study is to
purify neural crest progenitors from SDIA?BMP-treated ES
cells by FACS (26) and examine the differentiation capacity into
neuronal and nonneuronal lineages. For instance, it is worth
testing whether the crest progenitors can differentiate into such
cells as sensory and enteric neurons and form functional con-
nections in vivo. It is also intriguing to test whether induced
progenitors can differentiate into connective tissues, which
consist of a major but less understood lineage of the head neural
crest. It remains to be clarified how other signaling factors
implicated in neural crest differentiation (35) may work in the
SDIA system. Our preliminary study shows that neural crest
differentiation is not significantly induced in BMP4-treated ES
cells simply by adding fibroblast growth factor, Wnt, or RA
signals without SDIA treatment.
Collectively, SDIA-based differentiation should serve as a
system to bridge a gap between knowledge of neural develop-
ment and application in regenerative medicine of the nervous
system. The method may be used to even broader application as
our recent reports indicate that SDIA-treated ES cells can
differentiate into ectoderm-derived sensory tissues, such as
retinal pigment epithelium (2) and lens cells (36).
We are grateful to R. Ladher, H. Enomoto, N. Osumi, and N. Rao for
comments on this work; to J.-F. Brunet (Centre National de la Recher-
che Scientifique Unite ´ Mixte de Recherche 8542 Ecole Normale Su-
pe ´rieure, France), J. E. Johnson (University of Texas Southwestern
Medical Center, Dallas), J. J. Archelos (Karl-Franzens-Universita ¨t,
Graz, Austria), and V. J. Hearing (National Cancer Institute, Bethesda)
for antisera; to H. Niwa (RIKEN, Kobe, Japan) for GFP-expressing ES
cells; to A. Sehara and Y. Tanabe for helpful discussion; to N. Sasai for
help in floor plate marker analysis; and to Y. Nakano and T. Katayama
for excellent technical assistance. This work was supported by grants
from the Organization of Pharmaceutical Safety and Research (to Y.S.),
the Japan Society for the Promotion of Science (to N.N. and K.M.), the
Ministry of Education, Culture, Sports, Science, and Technology (to
Y.S., N.N., and K.M.), the Ministry of Health, Labor, and Welfare (to
Y.S.), and the Human Frontier Science Program Organization (to Y.S.).
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May 13, 2003 ?
vol. 100 ?
no. 10 ?