Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells.
ABSTRACT Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.
- SourceAvailable from: Alireza Shoae Hassani[Show abstract] [Hide abstract]
ABSTRACT: Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was significantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These findings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.Neural Regeneration Research 04/2014; 9(8):845-850. · 0.14 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The promise of human pluripotent stem cell (hPSC) therapies is ever-increasing, with the rapid proliferation of reports over the past several years demonstrating homogeneous and efficient differentiation of hPSCs to clinically relevant cell types (1–3). Such discoveries have renewed the enthusiasm and motivation for the acceleration of clinical trials using hPSC-derived cells to treat nervous system injuries and ocular degeneration, as well as diabetes and cardiac diseases in the near future. On the path to clinical implementation of hPSC therapies, the need for scalable culture technologies capable of producing sufficient quantities of cells becomes of paramount importance, as the proposed therapies demonstrate efficacy in human patients. However, despite several significant advances in recent years establishing defined, xeno-free conditions for hPSC culture (4–6), numerous challenges still remain to be over-come, including advanced 3D platforms capable of efficient, facile, and robust expansion, differentiation, and retrieval of hPSCs.Proceedings of the National Academy of Sciences 12/2013; · 9.81 Impact Factor
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ABSTRACT: Seven years have passed since the initial report of the generation of induced pluripotent stem cells (iPSCs) from adult human somatic cells, and in the intervening time the field of neuroscience has developed numerous disease models using this technology. Here, we review progress in the field and describe both the advantages and potential pitfalls of modeling neurodegenerative and neurodevelopmental diseases using this technology. We include tables with information on neural differentiation protocols and studies that developed human iPSC lines to model neurological diseases. We also discuss how one can: investigate effects of genetic mutations with iPSCs, examine cell fate-specific phenotypes, best determine the specificity of a phenotype, and bring in vivo relevance to this in vitro technique.Journal of neurogenetics 03/2014; · 0.73 Impact Factor
Wnt signaling and a Smad pathway blockade direct the
differentiation of human pluripotent stem cells to
multipotent neural crest cells
Laura Menendeza, Tatiana A. Yatskievychb, Parker B. Antinb, and Stephen Daltona,1
aDepartment of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, GA 30602;
andbDepartment of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ 85724
Edited by Floyd Bloom, The Scripps Research Institute, La Jolla, CA, and approved October 4, 2011 (received for review August 23, 2011)
Neural crest stem cells can be isolated from differentiated cultures
of human pluripotent stem cells, but the process is inefficient and
requires cell sorting to obtain a highly enriched population. No
specific method for directed differentiation of human pluripotent
cells toward neural crest stem cells has yet been reported. This
severely restricts the utility of these cells as a model for disease and
development and for more applied purposes such as cell therapy
and tissue engineering. In this report, we use small-molecule
compounds in a single-step method for the efficient generation of
self-renewing neural crest-like stem cells in chemically defined
media. This approach is accomplished directly from human pluripo-
tent cells without the need for coculture on feeder layers or cell
sorting to obtain a highly enriched population. Critical to this
suppression of the Activin A/Nodal pathway. Over 12–14 d, plurip-
otent cells are efficiently specified along the neuroectoderm lineage
toward p75+Hnk1+Ap2+neural crest-like cells with little or no con-
tamination by Pax6+neural progenitors. This cell population can be
clonally amplified and maintained for >25 passages (>100 d) while
retaining the capacity to differentiate into peripheral neurons,
smooth muscle cells, and mesenchymal precursor cells. Neural crest-
like stem cell-derived mesenchymal precursors have the capacity for
differentiation into osteocytes, chondrocytes, and adipocytes. In
sum, we have developed methods for the efficient generation of
self-renewing neural crest stem cells that greatly enhance their po-
tential utility in disease modeling and regenerative medicine.
developmental biology|embryonic stem cells|human induced pluripotent
neural plate and the nonneural ectoderm during vertebrate em-
bryogenesis (1, 2). As neural crest cells delaminate from the roof
plate upon closing of the neural tube, they migrate throughout the
body where they contribute to the peripheral nervous system,
connective and skeletal cranial tissues, melanocytes, and valves of
the heart. Specification of ectoderm into neural, neural plate
border, and epidermal cells is directed by overlapping but distinct
combinations of signaling molecules centering around Wnt, BMP,
and Fgf pathways (3–10). Knowledge of these pathways has been
instrumental in establishing conditions for differentiation of hu-
man pluripotent cells in culture along a neuroectoderm pathway
for the generation of neural progenitor cells (NPCs) and a wide
range of neuronal subtypes (11–13).
Efficient methods for generation of NPCs from human plurip-
otent cells have recently been made possible by the use of specific
inhibitors, such as Noggin and SB 431542, that function by
blocking BMP and Activin A/Nodal signaling, respectively (12).
Simultaneous inhibition of these pathways is sufficient to drive
pluripotent cells in culture down the neuroectoderm pathway,
generating a population of Pax6+Sox1+Sox2+NPCs that can
fromneural rosettesinculture,NPCscan beamplifiedduetotheir
self-renewing capacity and further differentiated into a wide range
eural crest stem cells are a multipotent cell population arising
at theneural plateborder oftheneural ectodermbetween the
of neural cell types (13, 14). Neural crest cells are typically found
interspersed with neural rosettes in such cultures and can be
obtained only as a highly enriched cell population by cell-sorting
techniques (15, 16). Alternative methods for generating neural
coculture on feeder layers (16, 17), are relatively inefficient, and
also require cell sorting to generate highly enriched populations.
These methods all involve complex, multistep procedures that
produce relatively low yields of p75+Hnk1+neural crest cells.
These issues highlight the limitations of current approaches and,
tissue engineering, regenerative medicine, and drug-screening
applications. An efficient, single-step method for generation of
neural crest cells from pluripotent cells would therefore represent
a significant advancement in understanding neural crest cell bi-
ology and its biomedical application.
The heterogeneity of neuroectoderm cultures from which neu-
ral crestcells are currentlyisolated led ustoreevaluate thegeneral
approach from a cell-signaling perspective. Although low levels of
BMP and Activin A signaling are considered prerequisites for the
generation of NPCs from human embryonic stem cells (hESCs)
and human induced pluripotent stem cells (hiPSCs), the potential
role of Wnt in specifying neural crest cells has not been evaluated.
This is somewhat surprising considering the well-established role
5). In this report, we describe a highly efficient, one-step method
for efficient generation of neural crest-like cells (NCSCs) from
Wnt signaling under conditions of low global Smad signaling.
Cultures arising under these conditions are composed of highly
enriched NCSCs that are devoid of other contaminating neuro-
ectoderm cell types. NCSCs can be maintained over extended
periods in culture while retaining developmental potential for
peripheral neurons and mesenchymal cell-derived osteocytes,
chondrocytes, and adipocytes. These findings significantly increase
the opportunities for the use of neural crest cells and their deriv-
atives in tissue engineering, regenerative medicine, and drug-
Activation of the Wnt Pathway Redirects Neural Progenitors Toward
a Neural Crest Fate. Human pluripotent cells can be efficiently
differentiated into Pax6+NPCs by simultaneous inhibition of
Activin A/Nodal and BMP signaling with SB 431542 and Noggin,
respectively (Fig. 1 A and B) (12). Although Pax6+Sox1+Sox2+
NPCs predominate in cultures where Smad signaling is blocked,
relatively minor amounts of p75+neural crest cells are also
Author contributions: L.M., P.B.A., and S.D. designed research; L.M., T.A.Y., and P.B.A.
performed research; P.B.A and S.D. analyzed data; and L.M. and S.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 29, 2011
| vol. 108
| no. 48www.pnas.org/cgi/doi/10.1073/pnas.1113746108
generated under these conditions (Fig. 1B and Fig. S1), which is
consistent with previous findings (12).
The signaling requirements that determine a NCSC versus a
NPC fate have not been previously defined in culture. In the ab-
sence of a specific method that allows for the generation of highly
enriched cultures of NCSCs, FACS isolation of Hnk1+p75+cells
fromNPC cultures hasbeenthemethodofchoice toobtainhESC-
derived neural crest cells (18). Because canonical Wnt signaling
performs known roles in promoting neural crest formation in
vertebrate development (3–5), we asked whether concomitant
activation of Wnt signaling combined with global Smad inhibition
would more efficiently divert early neuroectoderm away from a
NPC fate toward a neural crest-like identity. This was initially
tested by addition of (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO),
a small-molecule inhibitor of glycogen synthase kinase 3 (GSK3)
that acts as a Wnt mimetic in a variety of contexts (19, 20). Ad-
dition of BIO to hESC cultures efficiently activates the canonical
Wnt pathway, as indicated by the activation of a β-catenin–de-
pendent luciferase reporter (Fig. S2). Concurrent Smad inhibition
combined with activation of Wnt signaling (+BIO) after 12 d
SB + Noggin
SB + Noggin
+ SB 431542
SB + 1 ng/ml Wnt
SB + 5 ng/ml Wnt
SB + 50 ng/ml Wnt
SB + 10 ng/ml Wnt
SB + 0.5 µM BIO
SB + 0.1 µM BIO
SB + 2 µM BIO
SB + 1 µM BIO
SB + 0 µM BIO
into neural progenitor cells and neural crest cells together with markers for the two cell types. (B) Treatment of hESCs with SB 431542 (20 μM) and Noggin
(500 ng/mL) promotes differentiation into Pax6+cells but concurrent treatment with BIO suppresses this and generates p75+Pax6−neural crest-like cells.
(Scale bar, 100 μm.) (C) Flow cytometry showing that Dickkopf (Dkk) decreases the p75brightpopulation in NPC cultures generated by treatment with SB
431542 and Noggin. (D) Real-time PCR data for Ap2 and Pax6 from p75dimor p75brightsorted cells (from C). Activation of the canonical Wnt pathway by (E)
GSK3 inhibition with BIO (0.1–2 μM) or by (F) addition of Wnt3a (1–50 ng/mL) promotes the formation of p75brightHnk1brightcells in a dose-dependent
manner. Isotype controls are shown in red, and positive cells are shown in blue. The percentage of double p75+ Hnk1+ cells is shown in each graph in E and F.
hESC (WA09) differentiation to neuroprogenitor cells is inhibited by Wnt signaling. (A) Schematic summarizing differentiation of pluripotent cells
Menendez et al.PNAS
| November 29, 2011
| vol. 108
| no. 48
severely inhibited the formation of Pax6+cells while markedly
increasing the percentage of p75+cells (Fig. 1B). These cultures
lost markers for pluripotent cells, such as Nanog and Oct4 (Fig.
S3). Addition of Dickkopf (Dkk), a Wnt antagonist, severely re-
duced the low level of p75brightcells present in NPC cultures
obtained by treatment with Noggin and SB 431542 (Fig. 1C).
p75brightcells expressed highlevels ofthe neural crest markerAp2
but low levels of the NPC marker Pax6 (Fig. 1D). p75dimcells, on
the other hand, expressed high levels of Pax6 transcript but low
levels of Ap2, indicating that p75dimand p75brightcells represent
NPC and neural crest-like populations, respectively (Fig. 2).
These data indicate that the minor population of p75brightneural
crest-like cells in NPC cultures has a requirement for Wnt sig-
naling (discussed in further detail below).
To establish a role for Wnt signaling in the specification of
hESC-derived neural crest-like cells, weperformed dose–response
experiments where the GSK3 inhibitor BIO or Wnt3a were added
to cultures treated with SB 431542 and Noggin. Increasing the
amount of BIO (0–2 μM) or recombinant Wnt3a (0–50 ng/mL)
increased the proportion of Hnk1brightp75brightcells in a dose-
dependent manner (Fig. 1 E and F). As the concentration of BIO
was reduced, the proportion of p75dimcells increased, indicating
was confirmed by comparing p75dimand p75brightcells that were
generated following treatment with an intermediate concentration
of BIO (0.5 μM). QuantitativeRT-PCR analysis shows that p75dim
cells expressed elevated levels of Pax6 and Sox2 but low levels of
Ap2, p75, and Slug transcripts (Fig. S4A). Conversely, p75bright
cells show elevated levels of Ap2, p75, and Slug transcripts but low
levels for Sox2 and Pax6. Immunostaining also confirmed that
Sox2 expression showed a dose-dependent response to BIO when
away from a neural progenitor fate toward a neural crest-like
identity at higher concentrations (Fig. S4B). As expected, the
pluripotent cell markers Nanog and Oct4 were lost over a broad
range of BIO concentrations in the presence of SB 431542 (Fig
S4B). Elevating Wnt signaling in the context of low Smad activity
therefore directs cells away from a Pax6+p75dimpopulation to a
neural crest-like fate.
Pax3 Brna3 Zic1
Relative transcript levels
isotype control +BIO +SB +Noggin +BIO +SB +Wnt3a +SB
+BIO +SB +BMP4 +SB +BIO
WA09 hESCs treated as indicated for 15 d. Cells were analyzed by probing with antibodies for p75 and Hnk1. The percentage of double negative and positive
cells is indicated in the bottom left and top right, respectively, of each graph. (B) Immunocytochemistry and bright field (Lower Right) of WA09 hESCs treated
with BIO and SB 431542 for 12 d. Cells were probed with antibodies as indicated: p75, Pax6, Ap2, Hnk1, and DAPI (DNA). (Scale bar, 100 μM.) (C) RT-PCR
transcript analysis of hESCs and NCSCs (passage 10) treated with BIO and SB 431542. Transcript levels were normalized to Gapdh control. Assays were per-
formed in triplicate and are shown as ±SD. (D) Schematic illustration of the signaling requirements for neural crest differentiation from hESCs.
hESC differentiation to neural crest cells requires Wnt signaling and is antagonized by Activin A and BMP pathways. (A) Flow cytometry analysis of
| www.pnas.org/cgi/doi/10.1073/pnas.1113746108Menendez et al.
Next, we performed further analysis to define the signaling
requirements required to efficiently specify hESC-derived Hnk1+
p75+neural crest-like cells. Addition of SB 431542, BIO, and
Noggin (SBioN) generated a highly enriched Hnk1+p75+pop-
ulation but, unexpectedly, omission of Noggin had no major im-
pact (Fig. 2A), indicating that active suppression of BMP signaling
is not required under our conditions. This can be explained by the
low level of basal BMP-dependent Smad1,5,8activity in these cells
(Fig. S5). Addition of BMP4 to SBio-treated cultures, however,
suppressed the transition to an Hnk1brightp75brightstate showing
that BMP signaling antagonizes this pathway (Fig. 2A). Recom-
binant Wnt3a can substitute for BIO when combined with SB
431542, but BIO and SB 431542 by themselves are ineffective.
To characterize the Hnk1brightp75brightcell population in fur-
ther detail, immunostaining was performed using antibodies for
neural crest markers p75, Ap2, Hnk1, and the NPC marker Pax6.
were positive for neural crest markers, but <5% expressed Pax6
(Fig. 2B). Elevated levels of Ap2, p75, Sox9, Sox10, Pax3, Brna3,
and Zic1 transcripts further show that the p75+population gen-
erated with SBio is closely related to authentic neural crest cells
(Fig. 2C). hESC-derived NCSCs could be maintained as a stable,
containing media (>25 consecutive passages). Similar results were
obtained when different hESC lines and hiPSCs were treated with
SBio-containing media (Figs. S6 and S7). We conclude that acti-
vation of canonical Wnt signaling combined with low Smad2,3 and
Smad1,5,8 activity are strict requirements for the efficient gener-
ation of neural crest-like cells from hESCs (Fig. 2D).
Multilineage Differentiation of hESC-Derived Neural Crest Cells. The
ectoderm in vertebrate embryos, capable of forming a diverse ar-
hESC-derived NCSCs described in this report, we asked if these
cells could differentiate into lineages previously shown to be
generated by hESC-derived neural crest-like cells (15). The ex-
periments designed to answer this question were performed on
WA09-derived NCSCs that had been self-renewed for >10 pas-
sages to establish that the developmental potential of these cells
was retained over time. First, we confirmed that NCSCs have the
capacity for neural differentiation as previously described (12).
-tubulin peripherin -tubulin peripherin -tubulin peripherin
WA09 WA09 WA09
derived (B) neural crest-like stem cells. BIO and SB 431542-treated NCSCs were
differentiated to peripherin+β-tubulin+cells for 14 d after switching to N2-
based neural differentiation media. Fixed cells were then probed with anti-
bodies for peripherin and β-tubulin. DNA was detected by staining with DAPI.
(Scale bar, 100 μm.)
CD44 CD90 CD13
9.79 0 1.85 98.1
96.2 0 0.25 3.17
rived NCSCs into mesenchymal progen-
itors. (A) Schematic illustrating possible
differentiation pathways for NCSCs. (B)
Bright-field view of mesenchymal cells
generated from NCSCs after treatment
for 4 d with 10% FBS-containing media.
(Scale bar, 100 μm.) (C) Loss of p75 ex-
pression detected by flow cytometry as
NCSCs are converted to mesenchymal
cells. (D) Flow cytometry analysis show-
ing marker expression (blue) in NCSCs
and mesenchymal cells. Red, isotype
control. The percentage of positive cells
for each antigen is shown in each
quadrant of each graph.
Differentiation of WA09-de-
Menendez et al.PNAS
| November 29, 2011
| vol. 108
| no. 48
After culture in media containing a mixture of factors (BDNF,
GDNF, NGF, neurotrophin-3, and dbcAMP) that promote neu-
ral differentiation (18), ∼75% of cells expressed β-tubulin and a
similar number expressed peripherin/neurofilament 4 (Fig. 3A),
indicative of peripheral neurons. Similar results were obtained
with two other hESC lines (RUES1 and RUES2) and hiPSCs
(Fig. 3 B–D).
Neural crest cells can also form mesenchymal precursor cells
in vitro (15, 21). By culturing cells in media containing 10% FBS
(21, 22), we confirmed that SBio-generated NCSCs can be effi-
4-d period (Fig. 4 A and B). Mesenchymal cells produced were
highly enriched for mesenchymal stem cell markers such as CD73,
CD44, CD105, and CD13 but lost expression of p75 (Fig. 4 C and
D). We also showed that hESC-derived mesenchymal cells could
be converted into smooth muscle cells, chondrocytes, osteocytes,
and adipocytes (Fig. 5). Many of these observations were repeated
using NCSCs derived from other hESC lines (RUES1, RUES2)
and from hiPSCs (Figs. S8 and S9). In summary, neural crest-like
cells generated by our highly efficient one-step method using
small-molecule compounds is capable of multilineage differenti-
ation. This is comparable to the developmental potential of neural
crest cells isolated from NPC cultures by FACS sorting reported
previously (15, 18).
In Vivo Potential of hESC-Derived NCSCs. To assess the in vivo ac-
tivity of SBio-derived neural crest, cell aggregates labeled with the
DiO cell tracer were implanted into Hamburger-Hamilton (HH)
stage 8–10 chicken embryos (n = 26) along the boundary between
the neural and nonneural ectoderm at the level of the forming
forebrain and midbrain (Fig. 6A). Of the 19 embryos in which
aggregates remained in place, migrating cells were observed in 13.
Seventy-two hours after injection, fluorescently labeled cells were
observed in the head and pharyngeal regions (Fig. 6B), including
the cranial ganglion (Fig. 6 C–J). The identity of cells was con-
firmed by staining with the human-specific nuclear antigen anti-
body (hNA; Fig. 6E). To confirm the developmental potential of
injected NCSCs and their ability to generate peripheral neurons
in vivo, we assessed hNA-positive cells forexpression of the neural
markers Tuj1 or peripherin. Double hNA/Tuj1-positive cells were
observed in small clusters throughout the head mesenchyme (Fig.
6 G–J). hNA/peripherin-positive cells were also found in the
mesenchyme and incorporated into host cranial ganglia (Fig. 6 C–
F). The injected cells therefore migrate and differentiate into
peripheral neurons in vivo, which is consistent with the expected
characteristics of neural crest cells.
Several reports have described the generation of neural crest
progenitor cells from human pluripotent cells. These involve co-
culture on PA6 or M5 feeder layers (15, 17), differentiation
through an embryoid body stage (23), and differentiation along a
neuroectoderm pathway using inhibitors of the Smad pathway
(18). The latter represents a culture system primarily designed to
generate Pax6+NPCs. Minor amounts of p75+neural crest cells
produced in this system are likely to be a consequence of signaling
heterogeneities in the culture dish. None of these approaches
represents a guided approach to specifically generate neural crest
cellsand, as a major downside, require a cell-sorting step to isolate
highly enriched neural crest cell populations. This is obviously a
significant obstacle that must be circumvented for the utility of
neural crest cells to be fully realized in an experimental and cell
therapy setting. This report describes a guided differentiation
strategy specifically for the purpose of generating neural crestcells
without significant amounts of other ectoderm-derived lineages.
At the molecular level, neural crest cells generated by our method
(A) DiO-labeled cells at time of injection and (B) 48 h later showing cell mi-
gration. (C–F) Immunocytochemistry and bright-field images of the same mi-
croscopic field 72 h after injection showing cells that had incorporated into a
cranial ganglion area and differentiated to peripheral neurons. Cells were
probed with antibodies for peripherin and hNA and counterstained with DAPI
(DNA). (Scale bar, 50μm.)(G–J) Images ofthe same microscopic field showing a
cluster of human NCSCs (hNA-positive) in the head mesenchyme adjacent to
the neural tube. Many of the cells are also Tuj1-positive. (Scale bar, 20 μm.)
In vivo migration and differentiation of WA09 hESC-derived NCSCs.
Oil red O
showing the lineages capable of being formed from neural crest-derived
mesenchymalcellsinculture.(B)Bright-field picture (Left)afterdifferentiation
into calponin+smooth muscle actin+(SMA) smooth muscle cells. (C) Oil red O-
stained adipocytes and (Right) a bright-field image of adipocytes showing oil
droplets. (D) Osteocytes produced by differentiation of neural crest-derived
mesenchymal cells, detected by staining with Alizarin Red and alkaline phos-
phatase (AP) staining. (E) Differentiation of mesenchymal cells to chon-
drocytes, as detected by staining with Alcian Blue. (Scale bar, 100 μm.)
Differentiation of NCSC-derived mesenchymal cells. (A) Schematic
| www.pnas.org/cgi/doi/10.1073/pnas.1113746108Menendez et al.
is indistinguishable from that generated by other methods (15, 18)
and, importantly, displays a similar differentiation potential.
The rationale for our directed-differentiation approach is based
on the known roles of canonical Wnt signaling in neural crest
formation during vertebrate development (3–5). The signaling
conditions for neural crest progenitor specification from hESCs
and hiPSCs involve inhibition of GSK3, an antagonist of Wnt
signaling, and inhibition of Activin A/Smad signaling with SB
431542. BMP4 antagonized neural crest specification, but inhib-
itorssuch asNoggin werenot required due tothe lowbasallevel of
Smad1,5,8 signaling in our system. The activation of Wnt signaling
crest cell fate, both of which require low levels of global Smad sig-
naling. Wnt therefore controls a molecular switch that determines
differential ectoderm fates arising from human pluripotent cells
in culture. These results indicate that suppression of Wnt signaling
with Dkk, for example, may be a useful approach to reduce the
number of contaminating neural crest cells from NPC cultures.
In summary, we describe a method for directed differentiation
of human pluripotent cells toward a neural crest fate. The method
is highly efficient and cost-effective and precludes formation of
contaminating Pax6+NPCs. Removing the need for FACS-assis-
ted purification provides a platform from which the basic biology
ofneural crest cellscanbe better understood and better applied to
disease modeling. This approach also establishes a starting point
for thegeneration ofneuralcrestcellsata scalethatcanbeusedin
applications in tissue engineering and regenerative medicine.
Materials and Methods
Stem Cell Culture. WA09 (WiCell), RUES1, RUES2 (A. Brivanlou, The Rockefeller
University, New York) hESCs, and the hiPSC lines Fib2-iPS4 and Fib2-iPS5
(George Daley, Children’s Hospital, Boston) were cultured on Geltrex-coated
Activin A (10 ng/mL), LR-Igf (200 ng/mL), and Fgf2 (8 ng/mL) as described
Neuroprogenitor Cell, Neural Crest, and Mesenchymal Cell Differentiation. NPC
differentiation was performed as described (18). Briefly, cells were plated on
Geltrex-coated plates in defined media without Activin A, supplemented with
20μM SB431542(Tocris)and500ng/mLNoggin(R&D Systems) for11d with or
without 2 μM of BIO (GSK3 Inhibitor IX; Calbiochem) or 15 ng/mL Dkk (R&D
Systems). For direct neural crest differentiation, cells were plated at a density
of 1 × 105cells/cm2in defined media lacking Activin A and supplemented with
2 μM BIO and 20 μM SB 431542 (SB media). Media was replaced every day.
Additional experiments were performed by adding 25 ng/mL Wnt3a, 500 ng/
mL Noggin or 5–50 ng/mL of BMP4 (R&D Systems) to SB media. For peripheral
neuron differentiation, neural crest cells were plated in poly-L-ornithine/
laminin or Geltrex-coated four-well chamber slides. The following day, SB
media was switched to DMEM/F12 N2 supplemented media with BDNF (10 ng/
mL), GDNF (10 ng/mL), NGF (10 ng/mL), neurotrophin-3 (10 ng/mL), ascorbic
acid (200 μM), and dbcAMP (0.5 mM). Cells were grown for 10–14 d and
assayed by immunocytochemisty. For mesenchymal differentiation, neural
crest cells were cultured in media containing 10% FBS and passed every 4–5 d.
Osteocyte, adipocyte, and chondrocyte differentiation was performed
according to manufacturer directions using StemPro Osteogenesis Kit, Stem-
Pro Chondrogenesis Differentiation Kit, and StemPro Adipogenesis Differen-
tiation Kit (Invitrogen), respectively.
Immunocytochemistry and Flow Cytometry. Cells were fixed in 4% para-
formaldehyde in PBS and stained with the primary antibodies listed in Table
S1. Secondary antibodies were Alexa Fluor 488- and Alexa Fluor 555-conju-
gated (Invitrogen). DNA was visualized by staining with DAPI. For flow
cytometry, 1 million live cells dissociated with Accutase were resuspended in
PBS and incubated for 30 min on ice with primary conjugated antibodies
(Table S1). Unconjugated p75 (neurotrophin receptor) and Hnk1 antibodies
were detected with Alexa Fluor 633- and Alexa Fluor 488-conjugated sec-
ondary antibodies, respectively. Cells were analyzed using the CyAN ADP
(Beckman Coulter) and FlowJo software.
Real-Time PCR and Western Blot Analysis. RNA was extracted using the RNeasy
Mini Kit (Qiagen). One microgram of total RNA was used for cDNA synthesis
using the iScript cDNA kit (BioRad). Ten nanograms of cDNA were used for
real-time PCR with Taqman assays (Applied Biosystems) on a MyiQ real-time
PCR iCycler (BioRad). Each sample was analyzed in duplicate, and gene ex-
pression was normalized to GAPDH. For Western blot analysis, hESCs were
plated in defined media and then switched to SB media alone or with ad-
dition of 5–50 ng/mL BMP4 (R&D Systems) or 500 ng/mL Noggin for 3 or 6 h.
After protein extraction with RIPA buffer, ∼30 μg of total protein extract
were loaded in 12% SDS gels, transferred to nitrocellulose membranes, and
probed with antibodies for pSmad1,5,8, Smad1 (Cell Signaling Technolo-
gies), and Cdk2 (Santa Cruz Biotechnology).
InOvo Transplantation. NeuralcrestcellswerepassagedwithAccutaseandthen
seeded on agarose-coated plates to form small-sized aggregates. Three days
later, cell aggregates were labeled with the fluorescent dye DiO (3,3'-dio-
ladecyloxacarbocyanine perchlorate) (Invitrogen) as described (24). A slit was
ectodermand theformingneural tube,anda smallDiO-labeled cellaggregate
was inserted and positioned under a fluorescence stereomicroscope. For fur-
ther details, see SI Materials and Methods.
ACKNOWLEDGMENTS. This work was supported by Grant HD049647 from the
National Institute of Child Health and Human Development (to S.D.), by Grant
GM75334 from the National Institute for General Medical Sciences (to S.D.), and
byGrant P41RR018502fromtheNational Center for ResearchResources(to S.D.).
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| November 29, 2011
| vol. 108
| no. 48
Correction for “Wnt signaling and a Smad pathway blockade
direct the differentiation of human pluripotent stem cells to
multipotent neural crest cells,” by Laura Menendez, Tatiana A.
Yatskievych, Parker B. Antin, and Stephen Dalton, which ap-
peared in issue 48, November 29, 2011, of Proc Natl Acad Sci USA
(108:19240–19245; first published November 14, 2011; 10.1073/
The authors note that they omitted a reference to an article by
Wang et al. The complete reference appears below.
Additionally, the authors note that on page 19245, left column,
third full paragraph, lines 1–6, “WA09 (WiCell), RUES1, RUES2
(A. Brivanlou, The Rockefeller University, New York) hESCs,
and the hiPSC lines Fib2-iPS4 and Fib2-iPS5 (George Daley,
Children’s Hospital, Boston) were cultured on Geltrex-coated
plates (Invitrogen) in chemically defined media containing
Heregulin β (10 ng/mL), Activin A (10 ng/mL), LR-Igf (200 ng/
mL), and Fgf2 (8 ng/mL) as described previously (24)” should
instead appear as “WA09 (WiCell), RUES1, RUES2 (A. Brivanlou,
The Rockefeller University, New York) hESCs, and the hiPSC
lines Fib2-iPS4 and Fib2-iPS5 (George Daley, Children’s Hos-
pital, Boston) were cultured on Geltrex-coated plates (In-
vitrogen) in chemically defined media containing Heregulin β
(10 ng/mL), Activin A (10 ng/mL), LR-Igf (200 ng/mL), and Fgf2
(8 ng/mL) as described previously (25).”
25. Wang L, et al. (2007) Self-renewal of human embryonic stem cells requires insulin-like
growth factor-1 receptor and ERBB2 receptor signaling. Blood 110:4111–4119.
| June 5, 2012
| vol. 109
| no. 23www.pnas.org