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Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells Laura Menendez a , Tatiana A. Yatskievych b , Parker B. Antin b , and Stephen Dalton a,1 a Department of Biochemistry and Molecular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, GA 30602; and b Department 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 inefcient and requires cell sorting to obtain a highly enriched population. No specic 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 efcient generation of self-renewing neural crest-like stem cells in chemically dened 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 approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 1214 d, plurip- otent cells are efciently specied 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 amplied 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 efcient 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 stem cells N eural crest stem cells are a multipotent cell population arising at the neural plate border of the neural ectoderm between the 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. Specication 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 (310). 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 (1113). Efcient methods for generation of NPCs from human plurip- otent cells have recently been made possible by the use of specic 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 sufcient to drive pluripotent cells in culture down the neuroectoderm pathway, generating a population of Pax6 + Sox1 + Sox2 + NPCs that can assemble into neural rosettes as columnar epithelia. When isolated from neural rosettes in culture, NPCs can be amplied due to their self-renewing capacity and further differentiated into a wide range 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 crest cells from pluripotent cells have been described, but these use coculture on feeder layers (16, 17), are relatively inefcient, 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, consequently, severely limit their utility in scale-up applications for tissue engineering, regenerative medicine, and drug-screening applications. An efcient, single-step method for generation of neural crest cells from pluripotent cells would therefore represent a signicant advancement in understanding neural crest cell bi- ology and its biomedical application. The heterogeneity of neuroectoderm cultures from which neu- ral crest cells are currently isolated led us to reevaluate the general 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 for canonical Wnt signaling in neural crest development in vivo (35). In this report, we describe a highly efcient, one-step method for efcient generation of neural crest-like cells (NCSCs) from hESCs and hiPSCs that involves concurrent activation of canonical 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 ndings signicantly increase the opportunities for the use of neural crest cells and their deriv- atives in tissue engineering, regenerative medicine, and drug- screening applications. Results Activation of the Wnt Pathway Redirects Neural Progenitors Toward a Neural Crest Fate. Human pluripotent cells can be efciently 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 conict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1113746108/-/DCSupplemental. 1924019245 | PNAS | November 29, 2011 | vol. 108 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1113746108 Downloaded by guest on February 13, 2020 Downloaded by guest on February 13, 2020 Downloaded by guest on February 13, 2020

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Page 1: Correction - PNAS · toward p75 +Hnk1 Ap2 neural crest-like cells with little or no con-tamination by Pax6+ neural progenitors. This cell population can be ... Addition of Dickkopf

Wnt signaling and a Smad pathway blockade direct thedifferentiation of human pluripotent stem cells tomultipotent neural crest cellsLaura 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;and bDepartment 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 culturesof human pluripotent stem cells, but the process is inefficient andrequires cell sorting to obtain a highly enriched population. Nospecific method for directed differentiation of human pluripotentcells toward neural crest stem cells has yet been reported. Thisseverely restricts the utility of these cells as a model for disease anddevelopment and for more applied purposes such as cell therapyand tissue engineering. In this report, we use small-moleculecompounds in a single-step method for the efficient generation ofself-renewing neural crest-like stem cells in chemically definedmedia. This approach is accomplished directly from human pluripo-tent cells without the need for coculture on feeder layers or cellsorting to obtain a highly enriched population. Critical to thisapproach is the activation of canonicalWnt signaling and concurrentsuppression of the Activin A/Nodal pathway. Over 12–14 d, plurip-otent cells are efficiently specified along the neuroectoderm lineagetoward p75+ Hnk1+ Ap2+ neural crest-like cells with little or no con-tamination by Pax6+ neural progenitors. This cell population can beclonally amplified and maintained for >25 passages (>100 d) whileretaining 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 fordifferentiation into osteocytes, chondrocytes, and adipocytes. Insum, we have developed methods for the efficient generation ofself-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 pluripotentstem cells

Neural crest stem cells are a multipotent cell population arisingat the neural plate border of the neural ectoderm between the

neural plate and the nonneural ectoderm during vertebrate em-bryogenesis (1, 2). As neural crest cells delaminate from the roofplate upon closing of the neural tube, they migrate throughout thebody where they contribute to the peripheral nervous system,connective and skeletal cranial tissues, melanocytes, and valves ofthe heart. Specification of ectoderm into neural, neural plateborder, and epidermal cells is directed by overlapping but distinctcombinations of signaling molecules centering around Wnt, BMP,and Fgf pathways (3–10). Knowledge of these pathways has beeninstrumental in establishing conditions for differentiation of hu-man pluripotent cells in culture along a neuroectoderm pathwayfor the generation of neural progenitor cells (NPCs) and a widerange 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 specificinhibitors, such as Noggin and SB 431542, that function byblocking BMP and Activin A/Nodal signaling, respectively (12).Simultaneous inhibition of these pathways is sufficient to drivepluripotent cells in culture down the neuroectoderm pathway,generating a population of Pax6+ Sox1+ Sox2+ NPCs that canassemble into neural rosettes as columnar epithelia.When isolatedfrom neural rosettes in culture, NPCs can be amplified due to theirself-renewing capacity and further differentiated into a wide range

of neural cell types (13, 14). Neural crest cells are typically foundinterspersed with neural rosettes in such cultures and can beobtained only as a highly enriched cell population by cell-sortingtechniques (15, 16). Alternative methods for generating neuralcrest cells from pluripotent cells have been described, but these usecoculture on feeder layers (16, 17), are relatively inefficient, andalso require cell sorting to generate highly enriched populations.These methods all involve complex, multistep procedures thatproduce relatively low yields of p75+ Hnk1+ neural crest cells.These issues highlight the limitations of current approaches and,consequently, severely limit their utility in scale-up applications fortissue engineering, regenerative medicine, and drug-screeningapplications. An efficient, single-step method for generation ofneural crest cells from pluripotent cells would therefore representa significant advancement in understanding neural crest cell bi-ology and its biomedical application.The heterogeneity of neuroectoderm cultures from which neu-

ral crest cells are currently isolated led us to reevaluate the generalapproach from a cell-signaling perspective. Although low levels ofBMP and Activin A signaling are considered prerequisites for thegeneration of NPCs from human embryonic stem cells (hESCs)and human induced pluripotent stem cells (hiPSCs), the potentialrole of Wnt in specifying neural crest cells has not been evaluated.This is somewhat surprising considering the well-established rolefor canonicalWnt signaling in neural crest development in vivo (3–5). In this report, we describe a highly efficient, one-step methodfor efficient generation of neural crest-like cells (NCSCs) fromhESCs and hiPSCs that involves concurrent activation of canonicalWnt signaling under conditions of low global Smad signaling.Cultures arising under these conditions are composed of highlyenriched NCSCs that are devoid of other contaminating neuro-ectoderm cell types. NCSCs can be maintained over extendedperiods in culture while retaining developmental potential forperipheral neurons and mesenchymal cell-derived osteocytes,chondrocytes, and adipocytes. These findings significantly increasethe opportunities for the use of neural crest cells and their deriv-atives in tissue engineering, regenerative medicine, and drug-screening applications.

ResultsActivation of the Wnt Pathway Redirects Neural Progenitors Towarda Neural Crest Fate. Human pluripotent cells can be efficientlydifferentiated into Pax6+ NPCs by simultaneous inhibition ofActivin 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: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113746108/-/DCSupplemental.

19240–19245 | PNAS | November 29, 2011 | vol. 108 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1113746108

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generated under these conditions (Fig. 1B and Fig. S1), which isconsistent 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 highlyenriched cultures of NCSCs, FACS isolation of Hnk1+ p75+ cellsfromNPC cultures has been themethod of choice to obtain hESC-derived neural crest cells (18). Because canonical Wnt signalingperforms known roles in promoting neural crest formation invertebrate development (3–5), we asked whether concomitant

activation of Wnt signaling combined with global Smad inhibitionwould more efficiently divert early neuroectoderm away from aNPC fate toward a neural crest-like identity. This was initiallytested 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 canonicalWnt pathway, as indicated by the activation of a β-catenin–de-pendent luciferase reporter (Fig. S2). Concurrent Smad inhibitioncombined with activation of Wnt signaling (+BIO) after 12 d

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Fig. 1. hESC (WA09) differentiation to neuroprogenitor cells is inhibited by Wnt signaling. (A) Schematic summarizing differentiation of pluripotent cellsinto 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 p75bright population in NPC cultures generated by treatment with SB431542 and Noggin. (D) Real-time PCR data for Ap2 and Pax6 from p75dim or p75bright sorted 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 p75bright Hnk1bright cells in a dose-dependentmanner. 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.

Menendez et al. PNAS | November 29, 2011 | vol. 108 | no. 48 | 19241

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severely inhibited the formation of Pax6+ cells while markedlyincreasing the percentage of p75+ cells (Fig. 1B). These cultureslost 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 p75bright cells present in NPC culturesobtained by treatment with Noggin and SB 431542 (Fig. 1C).p75bright cells expressed high levels of the neural crest marker Ap2but low levels of the NPC marker Pax6 (Fig. 1D). p75dim cells, onthe other hand, expressed high levels of Pax6 transcript but lowlevels of Ap2, indicating that p75dim and p75bright cells representNPC and neural crest-like populations, respectively (Fig. 2).These data indicate that the minor population of p75bright neuralcrest-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, we performed dose–responseexperiments where the GSK3 inhibitor BIO or Wnt3a were addedto cultures treated with SB 431542 and Noggin. Increasing theamount of BIO (0–2 μM) or recombinant Wnt3a (0–50 ng/mL)increased the proportion of Hnk1bright p75bright cells in a dose-

dependent manner (Fig. 1 E and F). As the concentration of BIOwas reduced, the proportion of p75dim cells increased, indicatingthat cells were being directed to a Pax6+ p75dim fate (Fig. 1C). Thiswas confirmed by comparing p75dim and p75bright cells that weregenerated following treatment with an intermediate concentrationof BIO (0.5 μM). Quantitative RT-PCR analysis shows that p75dim

cells expressed elevated levels of Pax6 and Sox2 but low levels ofAp2, p75, and Slug transcripts (Fig. S4A). Conversely, p75bright

cells show elevated levels of Ap2, p75, and Slug transcripts but lowlevels for Sox2 and Pax6. Immunostaining also confirmed thatSox2 expression showed a dose-dependent response to BIO whenSmad signaling was suppressed, consistent with cells being directedaway from a neural progenitor fate toward a neural crest-likeidentity at higher concentrations (Fig. S4B). As expected, thepluripotent cell markers Nanog and Oct4 were lost over a broadrange of BIO concentrations in the presence of SB 431542 (FigS4B). Elevating Wnt signaling in the context of low Smad activitytherefore directs cells away from a Pax6+ p75dim population to aneural crest-like fate.

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Fig. 2. hESC differentiation to neural crest cells requires Wnt signaling and is antagonized by Activin A and BMP pathways. (A) Flow cytometry analysis ofWA09 hESCs treated as indicated for 15 d. Cells were analyzed by probing with antibodies for p75 and Hnk1. The percentage of double negative and positivecells is indicated in the bottom left and top right, respectively, of each graph. (B) Immunocytochemistry and bright field (Lower Right) of WA09 hESCs treatedwith 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-PCRtranscript 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.

19242 | www.pnas.org/cgi/doi/10.1073/pnas.1113746108 Menendez et al.

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Next, we performed further analysis to define the signalingrequirements required to efficiently specify hESC-derived Hnk1+

p75+ neural crest-like cells. Addition of SB 431542, BIO, andNoggin (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 thelow level of basal BMP-dependent Smad1,5,8 activity in these cells(Fig. S5). Addition of BMP4 to SBio-treated cultures, however,suppressed the transition to an Hnk1bright p75bright state showingthat BMP signaling antagonizes this pathway (Fig. 2A). Recom-binant Wnt3a can substitute for BIO when combined with SB431542, but BIO and SB 431542 by themselves are ineffective.To characterize the Hnk1bright p75bright cell population in fur-

ther detail, immunostaining was performed using antibodies forneural crest markers p75, Ap2, Hnk1, and the NPC marker Pax6.This showed that>90%of SB 431542 and BIO (SBio)-treated cellswere 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,self-renewing population over extended periods of culture in SBio-containing media (>25 consecutive passages). Similar results wereobtained when different hESC lines and hiPSCs were treated withSBio-containing media (Figs. S6 and S7). We conclude that acti-vation of canonical Wnt signaling combined with low Smad2,3 andSmad1,5,8 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. Theneural crest is a multipotent population of cells arising from neuralectoderm in vertebrate embryos, capable of forming a diverse ar-ray of cell lineages. To characterize the developmental potential ofhESC-derived NCSCs described in this report, we asked if thesecells could differentiate into lineages previously shown to begenerated by hESC-derived neural crest-like cells (15). The ex-periments designed to answer this question were performed onWA09-derived NCSCs that had been self-renewed for >10 pas-sages to establish that the developmental potential of these cellswas retained over time. First, we confirmed that NCSCs have thecapacity for neural differentiation as previously described (12).

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Fig. 3. Peripheral neurons derived from hESC-derived (A, C, andD) and hiPSC-derived (B) neural crest-like stem cells. BIO and SB 431542-treated NCSCs weredifferentiated 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.)

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D Fig. 4. Differentiation of WA09-de-rived NCSCs into mesenchymal progen-itors. (A) Schematic illustrating possibledifferentiation pathways for NCSCs. (B)Bright-field view of mesenchymal cellsgenerated from NCSCs after treatmentfor 4 d with 10% FBS-containing media.(Scale bar, 100 μm.) (C) Loss of p75 ex-pression detected by flow cytometry asNCSCs are converted to mesenchymalcells. (D) Flow cytometry analysis show-ing marker expression (blue) in NCSCsand mesenchymal cells. Red, isotypecontrol. The percentage of positive cellsfor each antigen is shown in eachquadrant of each graph.

Menendez et al. PNAS | November 29, 2011 | vol. 108 | no. 48 | 19243

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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 asimilar number expressed peripherin/neurofilament 4 (Fig. 3A),indicative of peripheral neurons. Similar results were obtainedwith 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-ciently converted to a cell type withmesenchymal properties over a4-d period (Fig. 4 A and B). Mesenchymal cells produced werehighly enriched for mesenchymal stem cell markers such as CD73,CD44, CD105, and CD13 but lost expression of p75 (Fig. 4 C andD). We also showed that hESC-derived mesenchymal cells couldbe converted into smooth muscle cells, chondrocytes, osteocytes,and adipocytes (Fig. 5). Many of these observations were repeatedusing NCSCs derived from other hESC lines (RUES1, RUES2)and from hiPSCs (Figs. S8 and S9). In summary, neural crest-likecells generated by our highly efficient one-step method usingsmall-molecule compounds is capable of multilineage differenti-ation. This is comparable to the developmental potential of neuralcrest cells isolated from NPC cultures by FACS sorting reportedpreviously (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 theDiO cell tracer were implanted into Hamburger-Hamilton (HH)stage 8–10 chicken embryos (n= 26) along the boundary betweenthe neural and nonneural ectoderm at the level of the formingforebrain and midbrain (Fig. 6A). Of the 19 embryos in whichaggregates remained in place, migrating cells were observed in 13.Seventy-two hours after injection, fluorescently labeled cells wereobserved in the head and pharyngeal regions (Fig. 6B), includingthe 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 ofinjected NCSCs and their ability to generate peripheral neurons

in vivo, we assessed hNA-positive cells for expression of the neuralmarkers Tuj1 or peripherin. Double hNA/Tuj1-positive cells wereobserved in small clusters throughout the head mesenchyme (Fig.6 G–J). hNA/peripherin-positive cells were also found in themesenchyme and incorporated into host cranial ganglia (Fig. 6 C–F). The injected cells therefore migrate and differentiate intoperipheral neurons in vivo, which is consistent with the expectedcharacteristics of neural crest cells.

DiscussionSeveral reports have described the generation of neural crestprogenitor cells from human pluripotent cells. These involve co-culture on PA6 or M5 feeder layers (15, 17), differentiationthrough an embryoid body stage (23), and differentiation along aneuroectoderm pathway using inhibitors of the Smad pathway(18). The latter represents a culture system primarily designed togenerate Pax6+ NPCs. Minor amounts of p75+ neural crest cellsproduced in this system are likely to be a consequence of signalingheterogeneities in the culture dish. None of these approachesrepresents a guided approach to specifically generate neural crestcells and, as a major downside, require a cell-sorting step to isolatehighly enriched neural crest cell populations. This is obviously asignificant obstacle that must be circumvented for the utility ofneural crest cells to be fully realized in an experimental and celltherapy setting. This report describes a guided differentiationstrategy specifically for the purpose of generating neural crest cellswithout significant amounts of other ectoderm-derived lineages.At the molecular level, neural crest cells generated by our method

peripherin/hNA Tuj1/DiO/hNA

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Fig. 6. In vivo migration and differentiation of WA09 hESC-derived NCSCs.(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 acranial ganglion area and differentiated to peripheral neurons. Cells wereprobed with antibodies for peripherin and hNA and counterstained with DAPI(DNA). (Scale bar, 50 μm.) (G–J) Images of the samemicroscopic field showing acluster of human NCSCs (hNA-positive) in the head mesenchyme adjacent tothe neural tube. Many of the cells are also Tuj1-positive. (Scale bar, 20 μm.)

chondrocytes

osteocytes

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Fig. 5. Differentiation of NCSC-derived mesenchymal cells. (A) Schematicshowing the lineages capable of being formed from neural crest-derivedmesenchymal cells in culture. (B) Bright-field picture (Left) after differentiationinto calponin+ smooth muscle actin+ (SMA) smooth muscle cells. (C) Oil red O-stained adipocytes and (Right) a bright-field image of adipocytes showing oildroplets. (D) Osteocytes produced by differentiation of neural crest-derivedmesenchymal 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.)

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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 crestformation during vertebrate development (3–5). The signalingconditions for neural crest progenitor specification from hESCsand hiPSCs involve inhibition of GSK3, an antagonist of Wntsignaling, and inhibition of Activin A/Smad signaling with SB431542. BMP4 antagonized neural crest specification, but inhib-itors such as Noggin were not required due to the low basal level ofSmad1,5,8 signaling in our system. The activation of Wnt signalingwas sufficient to divert cells from a Pax6+NPC fate to a p75+ neuralcrest cell fate, both of which require low levels of global Smad sig-naling. Wnt therefore controls a molecular switch that determinesdifferential ectoderm fates arising from human pluripotent cellsin culture. These results indicate that suppression of Wnt signalingwith Dkk, for example, may be a useful approach to reduce thenumber 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 methodis highly efficient and cost-effective and precludes formation ofcontaminating Pax6+ NPCs. Removing the need for FACS-assis-ted purification provides a platform from which the basic biologyof neural crest cells can be better understood and better applied todisease modeling. This approach also establishes a starting pointfor the generation of neural crest cells at a scale that can be used inapplications in tissue engineering and regenerative medicine.

Materials and MethodsStem Cell Culture. WA09 (WiCell), RUES1, RUES2 (A. Brivanlou, The RockefellerUniversity, New York) hESCs, and the hiPSC lines Fib2-iPS4 and Fib2-iPS5(George Daley, Children’s Hospital, Boston) were cultured on Geltrex-coatedplates (Invitrogen) in chemically definedmedia containingHeregulin β (10ng/mL),Activin A (10 ng/mL), LR-Igf (200 ng/mL), and Fgf2 (8 ng/mL) as describedpreviously (24).

Neuroprogenitor Cell, Neural Crest, and Mesenchymal Cell Differentiation. NPCdifferentiation was performed as described (18). Briefly, cells were plated onGeltrex-coated plates in defined media without Activin A, supplemented with20 μMSB 431542 (Tocris) and 500 ng/mL Noggin (R&D Systems) for 11 d with orwithout 2 μM of BIO (GSK3 Inhibitor IX; Calbiochem) or 15 ng/mL Dkk (R&DSystems). For direct neural crest differentiation, cells were plated at a densityof 1 × 105 cells/cm2 in defined media lacking Activin A and supplemented with2 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 peripheralneuron differentiation, neural crest cells were plated in poly-L-ornithine/

laminin or Geltrex-coated four-well chamber slides. The following day, SBmedia 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), ascorbicacid (200 μM), and dbcAMP (0.5 mM). Cells were grown for 10–14 d andassayed by immunocytochemisty. For mesenchymal differentiation, neuralcrest cells were cultured in media containing 10% FBS and passed every 4–5 d.Osteocyte, adipocyte, and chondrocyte differentiation was performedaccording 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 TableS1. Secondary antibodies were Alexa Fluor 488- and Alexa Fluor 555-conju-gated (Invitrogen). DNA was visualized by staining with DAPI. For flowcytometry, 1 million live cells dissociated with Accutase were resuspended inPBS and incubated for 30 min on ice with primary conjugated antibodies(Table S1). Unconjugated p75 (neurotrophin receptor) and Hnk1 antibodieswere 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 RNeasyMini Kit (Qiagen). One microgram of total RNA was used for cDNA synthesisusing the iScript cDNA kit (BioRad). Ten nanograms of cDNA were used forreal-time PCR with Taqman assays (Applied Biosystems) on a MyiQ real-timePCR iCycler (BioRad). Each sample was analyzed in duplicate, and gene ex-pression was normalized to GAPDH. For Western blot analysis, hESCs wereplated 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 extractwere loaded in 12% SDS gels, transferred to nitrocellulose membranes, andprobed with antibodies for pSmad1,5,8, Smad1 (Cell Signaling Technolo-gies), and Cdk2 (Santa Cruz Biotechnology).

In Ovo Transplantation.Neural crest cellswerepassagedwithAccutaseandthenseeded on agarose-coated plates to form small-sized aggregates. Three dayslater, cell aggregates were labeled with the fluorescent dye DiO (3,3'-dio-ladecyloxacarbocyanine perchlorate) (Invitrogen) as described (24). A slit wascut intoHH stage 8–10 chicken embryos in ovo at the junction of the nonneuralectoderm and the forming neural tube, and a small DiO-labeled cell aggregatewas 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 theNational Institute of Child Health and Human Development (to S.D.), by GrantGM75334 from the National Institute for General Medical Sciences (to S.D.), andby Grant P41RR018502 from the National Center for Research Resources (to S.D.).

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Correction

CELL BIOLOGYCorrection for “Wnt signaling and a Smad pathway blockadedirect the differentiation of human pluripotent stem cells tomultipotent 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/pnas.1113746108).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-coatedplates (Invitrogen) in chemically defined media containingHeregulin β (10 ng/mL), Activin A (10 ng/mL), LR-Igf (200 ng/mL), and Fgf2 (8 ng/mL) as described previously (24)” shouldinstead appear as “WA09 (WiCell), RUES1, RUES2 (A. Brivanlou,The Rockefeller University, New York) hESCs, and the hiPSClines 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-likegrowth factor-1 receptor and ERBB2 receptor signaling. Blood 110:4111–4119.

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