Isolation and directed differentiation of neural crest stem cells ... - Nature

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Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells Gabsang Lee1, Hyesoo Kim1,4, Yechiel Elkabetz1,4, George Al Shamy2, Georgia Panagiotakos2, Tiziano Barberi3, Viviane Tabar2 & Lorenz Studer1,2 Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest–related disorders.

Human embryonic stem (hES) cells provide an in vitro assay to study human development and a potential source of specialized cells for use in regenerative medicine1. Significant efforts have been devoted to characterizing the neural potential of hES cells, and protocols have been developed to achieve neural induction2 and differentiation toward specialized neuron subtypes, including midbrain dopamine3 neurons and spinal motoneurons4,5. Although progress in the specification of central nervous system (CNS) cell types from hES cells has evolved rapidly, the ability to control peripheral nervous system (PNS) specification has remained limited. A recent study characterized neural crest differentiation from cloned bovine blastocysts via a neural rosette intermediate6. Other studies report the presence of neural crest derivatives among hES cell progeny, including peripheral neurons and melanocytes7,8. However, neural crest development is thought to emerge from pluripotent precursors, and a systematic understanding of neural crest differentiation from hES cells requires the isolation of NCS cells. NCS cells have been well characterized in chick and murine systems9–11. Defects in the complex processes that choreograph neural crest development are involved in a wide range of human diseases12–16. Despite the importance of neural crest cell biology in development and disease, the isolation of an NCS cell of human origin has remained elusive. Efficient strategies for the isolation of NCS cells will be essential for studies of human neural crest development and disease and will provide opportunities in regenerative medicine. Here we report the isolation, propagation and directed differentiation of NCS cells from hES cells. Gene expression analysis confirmed a molecular profile compatible with NCS cell identity.

Clonal analysis showed that hES cell–derived NCS cells are capable of multilineage differentiation toward neural crest lineages in vitro. After transplantation into the developing chick embryo, the cells migrated extensively and contributed to neural crest structures, including peripheral ganglia. Cells subcutaneously injected into adult murine hosts survived, did not form teratomas and contributed to mesenchymal tissues in dermis and muscle. RESULTS Derivation of neural crest precursors Previous studies have shown that hES cell–derived neural rosettes can be directed toward various region-specific CNS fates3–5. Here we tested whether neural rosette cultures also have the potential to differentiate toward PNS fates. Neural induction and rosette formation were performed as described previously3 (using hES cell lines WA-09, I-8 and RUES1-eGFP). Rosettes were mechanically isolated and replated on polyornithine-laminin–precoated culture dishes (passage 1, P1) and characterized for the expression of neural crest precursor markers. Expression of p75 and HNK1 was observed primarily in cells located at the periphery of rosettes (Fig. 1a,b). Cells in the center of neural rosettes expressed the neuroepithelial marker Pax6 (Fig. 1c). Expression of the neural crest marker AP2 was observed predominantly in cell clusters surrounding rosettes (Fig. 1d). Neural crest development in vivo is modulated by extrinsic patterning factors that determine dorso-ventral identity within the developing neuroepithelium. We observed a significant increase in p75+ putative neural crest precursors upon exposure of hES cell–derived neural rosettes to fibroblast growth factor (FGF)2 (2.5-fold ± 0.8, P o 0.05)

1Developmental Biology Program, and 2Department of Neurosurgery, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10021, USA. 3Division of Neuroscience, Beckman Research Institute of The City of Hope, 1500 E. Duarte Road, Duarte, California 91010, USA. 4These authors contributed equally to this work. Correspondence should be addressed to L.S. ([email protected]).

Received 15 May; accepted 8 November; published online 25 November 2007; corrected after print 8 July 2008; doi:10.1038/nbt1365

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or bone morphogenic protein (BMP)2 (2.8-fold ± 1.2, P o 0.01). Treatment with FGF and BMP antagonists inhibited induction of p75+ cells (0.4-fold ± 0.3 fewer cells with SU5402 treatment and 0.7-fold ± 0.2 fewer cells with Noggin treatment) (Fig. 1e). These findings indicate that neural crest precursors spontaneously emerge in cultures of hES cell–derived neural rosettes, and that their number can be regulated by extrinsic signals.

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or forebrain identity. Gene expression data were confirmed by RT-PCR analysis (Supplementary Fig. 1 online) and by quantitative immunocytochemistry (Fig. 2d–e). Brn3A is a known marker of murine postmitotic sensory neurons but is also expressed in immature migrating neural crest precursors18. We observed strong Brn3A expression in Tuj1+ neurons 2 d after plating of p75+ cells, but we also observed weak expression in Tuj1– precursors (Fig. 2f). One of

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Molecular characterization and clonal analysis Fluorescence-activated cell sorting (FACS) analysis showed that most hES cell–derived p75+ neural crest precursors coexpressed HNK1 (Fig. 2a) and CD49d (integrin-a4 subunit; Fig. 2b), another NCS cell marker17. Analysis of additional neural crest markers and markers associated with mesenchymal, endothelial, glial or hematopoietic fate yielded a surface marker profile compatible with neural crest precursor identity (Supplementary Table 1 online). We next performed gene expression analysis (Affymetrix U133A) to identity global mRNA profiles in double-positive (p75+/HNK1+) versus double-negative (p75–/HNK1–) P1 rosette progeny (Fig. 2c and Supplementary Table 2 online). Transcripts most highly enriched in the p75+/HNK1+ cells included key markers of neural crest development (Brn3a, 32-fold; AP2, 19-fold; Pax3, eightfold; Snail, fivefold) and known NCS cell markers (Sox10, 17-fold; ErbB3, 12-fold; p75, sevenfold; integrin-a4, sevenfold). The p75–/HNK1– cells were highly enriched in transcripts associated with CNS precursor and/

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Figure 1 Cells expressing neural crest markers are present in cultures of hES cell–derived neural rosettes and can be induced through extrinsic cues. (a) Coexpression of p75 and HNK1 in passage 1 (P1) neural rosette cultures. (b) The expression of p75 in the periphery of rosettes. (c,d) Neural rosette (Pax6+) was surrounded by neural crest cells (AP2+). (e) Fold increase in the percentage of p75+ cells, measured by FACS analysis, upon exposure to various cytokines and signaling antagonists in P1 rosette cultures (*, P o 0.05; **, or , P o 0.01; , P o 0.001). Scale bars, 50 mm.

Figure 2 Molecular characterization of neural crest precursor cells in hES cell–derived neural rosettes. (a) FACS analysis for p75 and HNK1 in P1 neural rosette cultures. (b) Representative FACS analysis for coexpression of CD49d in p75+ gated population. (c) Graph for the top increased (blue) and top decreased (red) genes comparing p75+/HNK1+ versus p75–/HNK1– populations as assessed by microarray analysis (Affymetrix U133A). (d) Immunocytochemical analysis of FACS-purified p75+ cells for markers of cell proliferation (Ki67) and neural crest identity (AP2, p75). (e) Quantification of the percentage of p75+ cells expressing a given marker immediately after isolation by FACS. (f) Strong expression of Brn3a in Tuj1+ neurons (arrow) and weak expression in Tuj1– precursors (arrowhead) derived from p75+ cells 2 d after plating. (g) Quantification of cell migration: p75+ cells showed significantly increased migration behavior compared with p75– cells as assayed in 10 min intervals over 6-h period starting 6 h after plating of FACS-purified cells (mean ± s.e.m.; n ¼ 149 and n ¼ 125 for p75+ and p75– population; P o 0.001). Scale bar in (d) corresponds to 50 mm in (d) and 25 mm in (f).

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the functional features that distinguish early PNS and CNS precursors is the migration rate on fibronectin. Time-lapse microscopy at 6 and 24 h after FACS purification revealed that the migration rates of p75+ cells were more than threefold higher than those of p75– cells, (Fig. 2g) with peak migration rates of 100 mm/h (Supplementary Fig. 2 and Supplementary Movies 1 and 2 online). Clonal analysis showed that the average plating efficiency of p75+/ HNK1+ cells was 5–20%. After in vitro expansion of FACS-purified p75+/HNK1+ cells for 2 months, single-cell cultures were established and cultured for an additional 3 weeks before analysis. We observed three major differentiated cell types: Peripherin+ and Tuj1+ neurons, glial fibrillary acidic protein (GFAP+) Schwann cells and smooth muscle actin (SMA+) myofibroblasts. Clonal analysis showed the multipotentiality of p75+/HNK1+ cells (Fig. 3a). An average of 65% of all clones differentiated toward neurons, glia and myofibroblasts. A total of 25% of the clones differentiated to myofibroblasts and neurons, and 10% of the clones gave rise to myofibroblasts only. All clones that yielded neuronal progeny contained a substantial fraction of Peripherin+ cells (12–89% of Tuj1+ cells).

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Figure 3 Identification of neural crest stem cell potential by clonal assay and sphere-forming assay. (a) Three representative examples of clonal cultures derived from hES cell–derived NCS cells and stained with markers of myofibroblast (SMA), neurons (Tuj1) and Schwann cells (GFAP). (b–d) FACS-purified neural crest precursors gave rise to spheres positive for neural stem cell markers (Nestin, Musashi1 and Vimentin). (d) Blue color shows nucleus (DAPI). (e,f) Whereas spheres derived from both p75+ and p75– cells were highly proliferative (Ki67), those derived from p75– cells were largely negative for neural crest markers (AP2/p75). (g,h) Quantification of the efficiency of sphere formation and the capacity to form secondary and tertiary spheres in p75+ and p75– populations. (i) Effect of FGF2 and EGF on sphere formation in p75+ neural crest cells (*, P o 0.05; **, P o 0.01). Scale bars, 50 mm.

Freshly isolated p75+/HNK1+ cells cultured in the presence of FGF2 and epidermal growth factor (EGF) on ultra-low-attachment plates formed spheres. Cells within spheres expressed neural stem cell markers, including Nestin, Musashi1 and Vimentin (Fig. 3b–d) while retaining expression of the neural crest markers (Fig. 3e). The establishment of PNS sphere cultures was previously reported for primary mouse NCS cells19. Spheres derived from p75–/HNK1– hES cell progeny were also positive for Nestin, Mushashi-1 and Vimentin (data not shown) but were mostly negative for neural crest markers (Fig. 3f). Sphere formation by p75+/HNK1+ and p75–/HNK1– cells occurred at similar efficiencies (Fig. 3g), and sphere formation potential was maintained for at least three passages in vitro (Fig. 3h). Sphere formation efficiency was dependent on extrinsic addition of FGF2 and EGF (Fig. 3i). Whereas differentiation of p75+/HNK1+ spheres resulted in a bias toward neural crest derivatives, cultures derived from p75–/HNK1– populations were enriched in GABA+ neurons and GFAP+ astrocytes negative for O4 and MBP (Supplementary Fig. 3 online).

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Directed differentiation of hES cell–derived NCS cells We next assessed the differentiation potential of hES cell–derived NCS cells after various periods of in vitro expansion in medium containing FGF2 and EGF (Fig. 4a). Neuronal differentiation (Fig. 4b,c) was induced by withdrawal of FGF2/EGF and exposure to BDNF, GDNF, NGF and dibutyryl cyclic AMP (dbcAMP), yielding peripheral sympathetic neurons (Tyrosine hydroxylase (TH)+/Peripherin+) and sensory neurons (Brn3a+/Peripherin+). An average of 25% of all colonies contained Brn3A+ putative sensory neurons whereas 2% of all colonies contained TH+ neurons. Most TH+ cells coexpressed peripherin and the noradrenergic marker dopamine b-hydroxylase. In contrast, TH+ neurons derived from p75– cells were negative for peripherin (Supplementary Fig. 4 online). Schwann cell differentiation, as assessed by the expression of S100b, GFAP and MBP+, was induced in the presence of CNTF, neuregulin 1b and dbcAMP (Fig. 4d,e). Notably, quantification of neuronal versus glial differentiation showed that hES cell– derived NCS cells do not yield Schwann cells immediately after isolation but only after additional in vitro culture (Fig. 4f). These data were confirmed in clonal cultures maintained for 4 weeks after p75 isolation. In such short-term-expanded clones, no GFAP+ cells were detected, whereas identical clones maintained for 2 months readily yielded Schwann cell progeny (Fig. 3a). In shortterm-expanded clones, 55% of colonies contained peripheral neurons and myofibroblasts. A total of 45% of the clones yielded myofibroblasts only, and no pure neuronal clones were observed. These data suggest that a cell-intrinsic temporal switch in competency occurs during NCS cell progression, similar to that observed for astrocytic differentiation during CNS stem cell progression20–23.

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f Figure 4 Differentiation of hES cell–derived NCS cells toward peripheral nervous system lineages. (a) Schematic illustration of experimental design. hES cell–derived NCS cells were induced to differentiate following 30, 60 and 120-d periods of in vitro expansion. (b–e) Representative images of differentiated hES cell-NCS cells stained with the markers for sympathetic neurons, sensory neurons and Schwann cells. Blue color marks nucleus (DAPI). (f) Quantification of neuronal and glial differentiation after various periods of in vitro expansion. Scale bars, 50 mm.

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Vertebrate neural crest cells are known to contribute to various structures outside the PNS including the head mesenchyme24,25. Clonal analyses during avian cranial neural crest development identified cells with mesodermal and/or ectodermal potential26,27. To examine differentiation potential toward mesenchymal lineages,

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Figure 5 Characterization and differentiation of mesenchymal precursor cells derived from hES cell–derived NCS cells. (a) Morphology and CD73 expression by FACS in hES cell–derived NCS cells and hES cell–derived NCMP cells upon exposure to serum-containing medium. (b) FACS analysis of the surface marker profile of CD73+ FACS-purified NCS cell–derived mesenchymal precursors. (c–f) Adipogenic (c), chondrogenic (d), osteogenic (e) and smooth muscle cells (f) were selectively induced from hES cell–derived NCMP cells. Blue color shows nucleus (DAPI). Scale bars, 50 mm.


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coexpressed a set of surface markers characteristic of mesenchymal stem cell fate, including Stro-1, CD29, CD73, CD146 and CD44 (Fig. 5b). We used established mesenchymal stem cell differentiation protocols30,31 and showed that mesenchymal precursors generated from hES cell–derived NCS cells were capable of adipocytic (Fig. 5c), chondrogenic (Fig. 5d) and osteogenic (Fig. 5e) differentiation. We recently reported the isolation of skeletal muscle cells from hES cell–derived mesenchymal precursors using a method that involved isolation by FACS of neural cell adhesion molecule (NCAM)+ progeny29. Mesenchymal precursors in the previous study were thought to arise from paraxial mesodermal precursors29. Mesenchymal precursors derived in the present study from NCS cell cultures contained 4–9% NCAM+ cells. However, when NCAM+ progeny were isolated by FACS, only few MyoD+ cells were detected, and none of these cells expressed committed myocyte lineage markers such as myogenin. Most NCAM+ cells expressed markers typical of smooth muscle fate (Fig. 5f). In vivo analysis of hES cell–derived NCS cell progeny We assessed the in vivo potential of hES cell–derived NCS cells after transplantation into the developing chick embryo and after subcutaneous injection into adult nonobese diabetic/severe combined immunodeficiencient (NOD/SCID) mice (Fig. 6a). FACS-purified hES cell–derived NCS cells, cultured for o5 d before transplantation, were grafted into the intersomite space of H&H stage 10–12 chick embryos. Human cells were identified by the expression of humanspecific markers and labeling with the cell tracer DiI. To confirm faithfulness of DiI staining, we used DiI-labeled dead cells (repeated

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Figure 6 In vivo transplantation of hES cell– derived NCS cell progeny. (a) Schematic illustration of the in vivo transplantation studies into the developing chick embryo and into adult NOD/SCID mice. (b–e) Analysis of cell migration (b,c) and images for differentiation into Tuj1+/ Islet+ neurons in host sympathetic ganglion (d) and in locations close to endogenous dorsal root ganglia (e). Red arrow in b shows location of the injection site. The outline of neural tube is marked with white line. The yellow box in left panel marks the area enlarged in right panel. (f–j) hES cell–derived NCS cells injected subcutaneously into adult NOD/SCID mice. Human cells (hNCAM+) were often associated with skeletal muscle cells (f) and expressed precursor markers such as human specific nestin (hNestin) and Vimentin (g). Hematoxylin-Eosin and Lyve1 staining in h and i revealed the presence of lymphatic vessels in areas where human cells were located. In j human cells in these areas expressed NG2 and SMA compatible with a pericyte- or smooth muscle phenotypes. Scale bar, 100 mm.

snap freezing/thawing cycles) as controls (Supplementary Fig. 5 online). Three days after in ovo transplantation, we observed extensive migration of hES cell–derived NCS cell progeny throughout the embryo from the hNCAM/SMA site of transplantation following ventral, anterior and posterior migration routes (Fig. 6b–c). Although many human cells continued to express neural crest precursor cell markers, we found evidence for in vivo differentiation toward Tuj1+/Islet+ neurons located within host peripheral ganglia (Fig. 6d–e). Histological analysis 6–8 weeks after transplantation into adult NOD/SCID mice showed NCAM+ human cells widely dispersed and often associated with skeletal muscle or lymphatic vessels. Human cells expressed multiple precursor cell markers and markers characteristic of pericyte-like populations (Fig. 6f–j). We did not observe signs of teratoma formation in any of the animals tested, as demonstrated by the absence of a-fetoprotein, Oct-4 and SSEA4 in immunohistochemistry analysis and by histological assessment (H&E) (data not shown). These data show in vivo survival and differentiation compatible with neural crest precursor identity in both embryonic and adult hosts.

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DISCUSSION We have demonstrated the isolation of NCS cells from hES cell– derived neural rosette cultures. Several observations suggest that NCS cells may arise directly from neural rosettes, including the high efficiency of deriving p75+/HNK1+ cells from P1 rosette cultures, the location of p75+ cells at the periphery of rosettes and the responsiveness of rosette-stage cultures to extrinsic cues known to specify neural crest development in vivo. However, proof of a direct lineage relationship will require clonal analysis of rosette-stage cells. Numerous studies have described molecular signals related to the induction of neural crest, including secreted growth factors of the BMP, FGF and Wnt families32,33. Effects of FGF2 and BMP2 on neural crest differentiation in vitro have been reported34. In the case of hES cell–derived neural rosette cultures, we postulate that BMP2 induces

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ARTICLES dorsal cell fate–determination genes35, whereas FGF2 promotes the observed here given the lag of 42 months from onset of FGF2/EGF loss of epithelial organization and the exit of neural crest precursors treatment to acquisition of Schwann cell competency. We previously reported the isolation of hES cell–derived mesenchfrom rosette structures25. The large number of cells with a neural crest profile in P1 rosette cultures was surprising given that similar condi- ymal precursors using a different protocol that involves a mesotions are routinely used for the generation of hES cell–derived CNS endodermal rather than a neural crest cell intermediate28,29. In progeny. This suggests that studies aimed at generating pure popula- mouse and avian development, progeny from neural crest and paraxial tions of CNS precursors should consider the possible presence of mesoderm closely interact in the formation of connective tissue neural crest derivatives capable of forming proliferating neurosphere- elements24. A recent study reported molecular markers that distinlike structures in response to FGF2/EGF exposure. guish mesenchymal precursors of mesodermal and neural crest origin Gene expression data and clonal analysis showed that p75+/HNK1+ during craniofacial development24,43. As shown here, the main differcells in P1 rosette cultures are highly enriched in neural crest ence between mesenchymal precursors derived from NCS cells and precursors and NCS cells. However, p75+ cells also expressed markers our previously published mesenchymal precursor populations is the of more differentiated neural crest derivatives. Previous studies have lack of skeletal muscle progeny and a decreased efficiency in adipocytic used negative selection for the early Schwann cell marker P0 for differentiation. Smooth and skeletal muscle differentiation from further NCS cell purification. However, our cultures were devoid of cranial neural crest precursors in vivo has been described44,45. Exprescells expressing Schwann cell markers at the time of isolation. Alter- sion of HoxA2 (ref. 46) and differentiation potential toward chonnative markers that could be adopted for purification of NCS cells drogenic tissues47 suggest the presence of cranial neural crest include CD29 (ref. 13) and CD49d17 or CD133 (ref. 36). The plating precursors in our hES cell–NCS cell cultures. However, our study efficiency in our clonal assays was 5–20% which is 5–10 times lower has not shown that a single NCS cell can give rise to both mesenchthan the cloning efficiency for primary NCS cells, whereas the ymal and neural progeny. Although we did observe Tuj1 expression in percentage of clones capable of multilineage differentiation (450%) single cell–derived clones containing CD73+ progeny, these cells were was comparable to that of primary NCS cells9. negative for more mature markers such as Peripherin. Furthermore, We also showed that hES cell–derived NCS cells can generate serum exposure both at clonal density and in bulk culture transiently primary, secondary and tertiary neurosphere structures in vitro similar induced increased cell death within the p75+ population, further to those produced by primary NCS cells19. We did observe a decrease complicating lineage analyses. Therefore, we cannot formally rule in sphere formation potential in the presence of FGF2/EGF after more out that mesenchymal cells arise from a distinct lineage of p75+ cells than four passages, suggesting that FGF2/EGF may not be sufficient that is selectively capable of survival, clonal expansion and mesenchfor long-term self-renewal of hES cell–derived NCS cells in vitro. ymal differentiation in the presence of serum. Future studies are Although to our knowledge no previous study has described the required to conclusively address the stem cell nature of putative cranial prospective isolation of NCS cells from hES cell progeny, neural crest neural crest progeny derived from hES cells and to explore conditions precursors with differentiation potential toward melanocytes, neurons that direct anterior-posterior fate in hES cell-NCS cell cultures, and GFAP+ cells were isolated from mouse ES cells using cell sorting including the derivation of cultures enriched with cells of cardiac, for c-kit37. C-kit is a marker previously used in the identification of ES trunk or sacral neural crest identity. The transplantation data presented here demonstrate in vivo cell–derived melanocytes38 and is considered a marker of committed migratory melanocyte precursors in vivo39. It will be interesting in survival of hES cell–derived NCS cells in both developing and adult future studies to assess c-kit status in FGF2/EGF-expanded and vertebrate hosts. In vivo differentiation toward peripheral neuron, smooth muscle, but not Schwann cell fates is in agreement with differentiating hES cell–derived NCS cells. Temporal and regional changes in NCS cell competency have been our in vitro data showing that specification toward glial fates is a late reported17,40. Our data on Schwann cell differentiation indicate that event. This suggests that efforts aimed at restoring Schwann cell early NCS cells lack access to Schwann cell fate, although clonal function after peripheral nerve damage will require the use of cells derivatives of the same cells were capable of generating Schwann cells after in vitro culNeuron P ture. We propose that these changes in com+ + F, cAM (Peripherin , Tuj1 ) , GDN , NGF petency are due to cell-intrinsic temporal BDNF CNTF, Nrg, cAM Schwann cell P e changes in epigenetic state rather than to an + + fre (GFAP , MBP ) rum artifact of in vitro culture. The generation of Se neuronal and glial derivatives during PNS Se ru Adipogenic cell development follows a stereotypic sequence, m hES cells Neural crest IBXT + + sulin, (PPARγ , Oil red ) + + + + 41 xa, In e (p75 , HNK1 , AP2 ) D CD73 with neurogenesis preceding gliogenesis . AA + TGFβ3, FACS Chondrogenic cell p75 + + Similarly, glial fate specification in CNS Sorting (Collagen , Aggrecan ) FACS β-GP, Dexa, AA Sorting NCAM + development follows an early wave of excluFACS Osteogenic cell Mesenchymal sively neurogenic differentiation. Schwann + + (ALP , BSP ) cell + + cell competency and lack of GFAP expression (CD73 , Stro1 ) Smooth muscle cell + + may be related to epigenetic changes, includ(SM22α , Calponin ) ing direct methylation of the GFAP promoter, as shown during differentiation of fetal Figure 7 Schematic illustration for the isolation and differentiation of hES cell–derived NCS cell. Stepneural stem cells and ES cells21–23. Although wise isolation and differentiation of hES cell–derived neural crest cells using a combination of cell sorting, in vitro expansion and directed differentiation via extrinsic signals. Serum-free conditions allow FGF2-mediated expansion of cell fate poten- differentiation of NCS cells into both sensory and autonomic neurons as well as Schwann cells. Serum tial has been reported in O2A precursor cells exposure leads to rapid conversion of NCS cell cultures into CD73+ mesenchymal precursors suggesting in vitro42, we think that it is unlikely to that CD73+ cells may derive directly from NCS cells. However we cannot rule out alternative origin of account for the changes in competency mesenchymal cells (see Discussion for details).


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ARTICLES predifferentiated to the Schwann cell stage before transplantation. Association of human cells with skeletal muscle and lymphatic structures in the adult host suggests that hES cell–derived NCS cells can adopt a wide range of properties depending on environmental cues. However, transplantation into additional sites will be required to fully probe the in vivo potential of hES cell–derived NCS cells and to confirm in vivo safety in long-term studies. Our study reports conditions for the isolation of hES cell–derived NCS cells and the directed differentiation of a wide range of neural crest derivatives, including sensory and autonomic neurons, Schwann cells, myofibroblasts, adipocytes, cartilage and bone cells (Fig. 7). These data should facilitate efforts in tissue engineering, disease modeling and regenerative medicine. METHODS Cell culture. Undifferentiated hES cells, H9 (WA-09), RUES1-eGFP48, I-8 were cultured under growth conditions described previously3 on mitotically inactivated mouse embryonic fibroblasts (MEFs; Chemicon) For neural induction, hES cells were plated at 5-20 103 cells on a confluent layer of irradiated (50 Gy) stromal cells (MS-5) in 60-mm cell culture plates in serum replacement medium (Invitrogen) containing 2 mM L-glutamine and 10 mM b-mercaptoethanol. After 16 d in serum replacement medium, cultures were switched to N2 medium3. Medium was changed every 2–3 d, and growth factors were added as described previously3: 200 ng/ml sonic hedgehog, 100 ng/ml FGF8, 20 ng/ml brain-derived neurotrophic factor (BDNF) (all R&D Systems), and 0.2 mM ascorbic acid (AA) (Sigma–Aldrich). Rosettes structures were harvested mechanically at day 22–28 of differentiation (termed passage 0; P0) and gently replated on 15 mg/ml polyornithine/1 mg/ml laminin (PO/Lam)coated culture dishes in N2 medium (termed passage 1; P1). P1 cultures were supplemented with FGF2, AA, and BDNF or any alternative growth factor conditions as listed in main text. After 6–7 d of P1 culture, cells were mechanically triturated after exposure to Ca2/Mg2-free Hanks’ balanced salt solution (CMF-HBSS, 20 min at 25 1C) and labeled with antibodies for flow cytometry. FACS sorting (p75, Advanced targeting systems; HNK1, Sigma) was performed on a MoFlo (Dako). Sorted cells were plated on culture dishes precoated with PO/Lam and 10 ng/ml fibronectin (10–30 103 cells/cm2). hES cell–derived NCS cells were maintained in N2 medium supplemented with 20 ng/ml of FGF2 and 20 ng/ml of EGF changed every 2–3 d and passaged every 7–8 d. For sphere formation assay cells were dissociated and plated onto 6-well ultra-low-attachment plates (Costar, Corning). For serial sphere formation assays, cells were dissociated with Accutase (Innovative Cell Technologies). For clonal assays, hES cell–derived NCS cells were mechanically dissociated after exposure to CMF-HBSS for 20 min at 25 1C and spun at 200g for 5 min. The cell pellet was resuspended with 1 ml of N2 medium and filtered through 40 mm mesh. Filtered cells were counted and directly plated on 35-mm culture dishes coated with 15 mg/ml polyornithine/1 mg/ml laminin/10 ng/ml fibronectin at clonal densities (10–30 cells/cm2)9. They were grown in N2 medium supplemented with bFGF and EGF for 1 week followed by mitogen withdrawal and culture in N2 medium supplemented with BDNF, nerve growth factor (NGF, 10 ng/ml), glial cell line–derived neurotrophic factor (GDNF, 10 ng/ml), 1 mM dibutyryl cAMP, ciliary neurotropic factor (CNTF, 10 ng/ml) and neuregulin (20 ng/ml) for a period of at least 2 weeks. For directed differentiation of hES cell–derived NCS cells toward peripheral nerve or Schwann cells, FGF2/EGF-expanded hES cell–derived NCS cells were differentiated upon mitogen withdrawal in medium supplemented with BDNF, GDNF, NGF, dbcAMP (peripheral nerve) or supplemented with CNTF, neuregulin, bFGF (10 ng/ml), dbcAMP (Schwann cells). For mesenchymal differentiation (hES cell–derived neural crest-mesenchymal precursor cells (hES cell–derived NCMP cells)), hES cell–derived NCS cells were cultured in aMEM containing 10% FBS (FBS, Invitrogen) for 42 weeks in uncoated tissue-culture grade dishes. FACS sorting (CD73-PE; Pharmingen) was performed on a MoFlo as described previously28. For adipogenic differentiation, hES cell–derived NCMP cells were grown to confluence, followed by exposure to 1 mM dexamethasone, 10 mg/ml insulin, and

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0.5 mM isobutylxanthine (all Sigma) in aMEM medium containing 10% FBS for 43 weeks. For chondrogenic differentiation, hES cell–derived NCMP cells were induced in pellet culture by exposure to 10 ng/ml TGFb-3 (R&D Systems) and 200 mM AA in aMEM medium containing 10% FBS for 44 weeks. For osteogenic differentiation, hES cell–derived NCMP cells were plated at low density (1 103 cells/cm2) on tissue culture–treated dishes in the presence of 10 mM b-glycerol phosphate (Sigma), 0.1 mM dexamethasone, and 200 mM AA in aMEM medium containing 10% FBS for 3–4 weeks. For myogenic differentiation, hES cell–derived NCMP cells were passaged for 2–3 weeks in aMEM medium with 10% heat inactivated FBS (FBS). At that stage FACS sorting for NCAM (5.1H11, DSHB) was performed. NCAM+ cells were grown until confluent in aMEM medium with 10% FBS and then induced to terminally differentiate in N2 medium. Immunocytochemistry and flow cytometry. Cells were fixed in 4% paraformaldehyde/0.15% picric acid and stained with the primary antibodies (Supplementary Table 3 online). Appropriate Alexa 350, Alexa 488, Alexa 568, Cy5 labeled secondary antibodies (Molecular Probes) and/or DAPI counterstaining was used for visualization. For flow cytometry, cells were mechanically dissociated after exposure to CMF-HBSS for 20 min at 25 1C. To eliminate dead cell populations in FACS analysis, we used 7-AAD according to manufacturer’s recommendation. Cells were analyzed using FACScan (Becton Dickinson) and FlowJo software (Tree Star, Inc.). RT-PCR and Affymetrix analysis. Total RNA was extracted using the RNeasy kit and DNAse I treatment (Qiagen) to avoid genomic contamination. Undifferentiated hES cells, mouse fetal fibroblasts (feeder layer for hES cells), mouse stromal cells (MS-5), hES cell–derived p75+/HNK1+, p75–/HNK1– cells and chondrogenic, osteogenic, adipogenic and myogenic cells derived from hES cell–derived NCMP cells were collected and frozen for further RNA extraction. Total RNA (2 mg for each sample) was reverse transcribed (Superscript, Invitrogen). Primer sequences, cycle numbers and annealing temperatures are provided (Supplementary Table 4 online). For microarray, 5 mg of total RNA from p75+/HNK1+ and p75–/HNK1– cells were processed by the MSKCC Genomic core facility and hybridized on Affymetrix U133A human oligonucleotide arrays. Time-lapse analysis. Cells were monitored 6–24 h after FACS purification of p75+ and p75– cells and monitored for 1- to 24-h intervals on an Olympus IX81 microscope under Nomarski optics using a Hamamatsu ORCA CCD camera, in an enclosed chamber with temperature and CO2 control (Weather Station, Precision Control). Quantification was performed at 6 h after FACS and for a 6-h or 24-h observation interval. Cell migration was quantified by computer-assisted tracking of the center of the cell body in 10-min intervals (five 2-min frames) using a commercial imaging software package (Slidebook). Data were quantified for 6-h periods measured in 2-min intervals (180 frames at 1344 1024 resolution). In vivo transplantation. For in ovo transplantation, fertile eggs (CBT farms) were incubated at 37 1C in a humidified incubator. The hES cell–NCS cells in 3–4 d after cell sorting from P1 stage were stained with DiI solution (Invitrogen) according to manufacturer’s recommendation. After repeated washings, DiI-labeled hES cell–derived NCS cells were transplanted into the intersomite space of H&H Stage 10–12 chick embryos49. Eggs were incubated for 3 d post-transplantation. For subcutaneous transplantations, 200,000 hES cell–derived NCS cells were injected into the neck region of adult NOD/SCID mice. Animals were monitored for tumor formation 6–8 weeks after transplantation. Tissues from chick embryo and adult mouse were fixed in 4% paraformaldehyde and cryosectioned for immunohistochemical analysis. Statistical analysis. The data were processed using Prism 4.0c or Statistica software. Values are reported as means ± s.e.m. if not indicated otherwise. Comparisons among values for all groups were performed by one-way ANOVA. Bartlett’s test for equal variances and Newman-Keuls Multiple Comparison Test were used to determine the level of significance. Note: Supplementary information is available on the Nature Biotechnology website.

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ACKNOWLEDGMENTS We would like to thank Z. Dincer and M. Tomishima for technical advice and the Tri-institutional Stem Cell Research Facility at the Memorial Sloan-Kettering Cancer Center (MSKCC) for help in the time-lapse studies. We also would like to thank J. Itskovitz and M. Amit for providing the I-8 cell line, the MSKCC genomics core for performing microarray hybridizations, and members of the Studer, Tabar and Tomishima labs for helpful discussions. This work was supported through the Tri-Institutional Stem Cell Initiative funded by the Starr Foundation. AUTHOR CONTRIBUTIONS G.L., T.B., V.T. and L.S. designed the study. G.L., V.T. and L.S. analyzed the data and wrote the manuscript. G.L., H.K., Y.E., G.A.S., G.P. and L.S. performed the experiments. Published online at http://www.nature.com/naturebiotechnology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions

1. Joseph, N.M. & Morrison, S.J. Toward an understanding of the physiological function of Mammalian stem cells. Dev. Cell 9, 173–183 (2005). 2. Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001). 3. Perrier, A.L. et al. From the Cover: Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 101, 12543–12548 (2004). 4. Li, X.J. et al. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215–221 (2005). 5. Lee, H.J. et al. Directed differentiation and transplantation of human embryonic stem cell derived motoneurons. Stem Cells 25, 1931–1939 (2007). 6. Lazzari, G. et al. Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro. Stem Cells 24, 2514–2521 (2006). 7. Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B. & Goldstein, R.S. Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells. Stem Cells 23, 923–930 (2005). 8. Fang, D. et al. Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem Cells 24, 1668–1677 (2006). 9. Morrison, S.J., White, P.M., Zock, C. & Anderson, D.J. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell 96, 737–749 (1999). 10. Wong, C.E. et al. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J. Cell Biol. 175, 1005–1015 (2006). 11. Yoshida, S. et al. Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea. Stem Cells 24, 2714–2722 (2006). 12. Gitler, A.D., Brown, C.B., Kochilas, L., Li, J. & Epstein, J.A. Neural crest migration and mouse models of congenital heart disease. Cold Spring Harb. Symp. Quant. Biol. 67, 57–62 (2002). 13. Iwashita, T., Kruger, G.M., Pardal, R., Kiel, M.J. & Morrison, S.J. Hirschsprung disease is linked to defects in neural crest stem cell function. Science 301, 972–976 (2003). 14. Fuchs, S. & Sommer, L. The neural crest: understanding stem cell function in development and disease. Neurodegener. Dis. 4, 6–12 (2007). 15. Edery, P. et al. Mutations of the RET proto-oncogene in Hirschsprung’s disease. Nature 367, 378–380 (1994). 16. Pingault, V. et al. SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat. Genet. 18, 171–173 (1998). 17. Bixby, S., Kruger, G.M., Mosher, J.T., Joseph, N.M. & Morrison, S.J. Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 35, 643–656 (2002). 18. Fedtsova, N.G. & Turner, E.E. Brn-3.0 expression identifies early post-mitotic CNS neurons and sensory neural precursors. Mech. Dev. 53, 291–304 (1995). 19. Molofsky, A.V. et al. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425, 962–967 (2003). 20. Molne, M. et al. Early cortical precursors do not undergo LIF-mediated astrocytic differentiation. J. Neurosci. Res. 59, 301–311 (2000).


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21. Song, M.R. & Ghosh, A. FGF2-induced chromatin remodeling regulates CNTFmediated gene expression and astrocyte differentiation. Nat. Neurosci. 7, 229–235 (2004). 22. Shimozaki, K., Namihira, M., Nakashima, K. & Taga, T. Stage- and site-specific DNA demethylation during neural cell development from embryonic stem cells. J. Neurochem. 93, 432–439 (2005). 23. Fan, G. et al. DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling. Development 132, 3345–3356 (2005). 24. Noden, D.M. & Trainor, P.A. Relations and interactions between cranial mesoderm and neural crest populations. J. Anat. 207, 575–601 (2005). 25. Kang, P. & Svoboda, K.K. Epithelial-mesenchymal transformation during craniofacial development. J. Dent. Res. 84, 678–690 (2005). 26. Baroffio, A., Dupin, E. & Le Douarin, N.M. Clone-forming ability and differentiation potential of migratory neural crest cells. Proc. Natl. Acad. Sci. USA 85, 5325–5329 (1988). 27. Baroffio, A., Dupin, E. & Le Douarin, N.M. Common precursors for neural and mesectodermal derivatives in the cephalic neural crest. Development 112, 301–305 (1991). 28. Barberi, T., Willis, L., Socci, N.D. & Studer, L. Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med. 2, e161 (2005). 29. Barberi, T. et al. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat. Med. 13, 642–648 (2007). 30. Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999). 31. Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M. & Yoo, J.U. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238, 265–272 (1998). 32. Knecht, A.K. & Bronner-Fraser, M. Induction of the neural crest: a multigene process. Nat. Rev. Genet. 3, 453–461 (2002). 33. Le Douarin, N.M. & Dupin, E. Multipotentiality of the neural crest. Curr. Opin. Genet. Dev. 13, 529–536 (2003). 34. Sailer, M.H. et al. BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells. J. Cell Sci. 118, 5849–5860 (2005). 35. Mizuseki, K. et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc. Natl. Acad. Sci. USA 100, 5828–5833 (2003). 36. Marzi, I. et al. Purging of the neuroblastoma stem cell compartment and tumor regression on exposure to hypoxia or cytotoxic treatment. Cancer Res. 67, 2402–2407 (2007). 37. Motohashi, T., Aoki, H., Chiba, K., Yoshimura, N. & Kunisada, T. Multipotent cell fate of neural crest-like cells derived from embryonic stem cells. Stem Cells 25, 402–410 (2007). 38. Yamane, T., Hayashi, S., Mizoguchi, M., Yamazaki, H. & Kunisada, T. Derivation of melanocytes from embryonic stem cells in culture. Dev. Dyn. 216, 450–458 (1999). 39. Wilson, Y.M., Richards, K.L., Ford-Perriss, M.L., Panthier, J.J. & Murphy, M. Neural crest cell lineage segregation in the mouse neural tube. Development 131, 6153–6162 (2004). 40. White, P.M. et al. Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals. Neuron 29, 57–71 (2001). 41. Kalcheim, C. & Burstyn-Cohen, T. Early stages of neural crest ontogeny: formation and regulation of cell delamination. Int. J. Dev. Biol. 49, 105–116 (2005). 42. Kondo, T. & Raff, M. Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 289, 1754–1757 (2000). 43. Bhattacherjee, V. et al. Neural crest and mesoderm lineage-dependent gene expression in orofacial development. Differentiation 75, 463–477 (2007). 44. Etchevers, H.C., Vincent, C., Le Douarin, N.M. & Couly, G.F. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development 128, 1059–1068 (2001). 45. Korn, J., Christ, B. & Kurz, H. Neuroectodermal origin of brain pericytes and vascular smooth muscle cells. J. Comp. Neurol. 442, 78–88 (2002). 46. Trainor, P.A., Ariza-McNaughton, L. & Krumlauf, R. Role of the isthmus and FGFs in resolving the paradox of neural crest plasticity and prepatterning. Science 295, 1288–1291 (2002). 47. Abzhanov, A., Tzahor, E., Lassar, A.B. & Tabin, C.J. Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro. Development 130, 4567–4579 (2003). 48. James, D., Noggle, S.A., Swigut, T. & Brivanlou, A.H. Contribution of human embryonic stem cells to mouse blastocysts. Dev. Biol. 295, 90–102 (2006). 49. Goldstein, R.S. Transplantation of human embryonic stem cells to the chick embryo. Methods Mol. Biol. 331, 137–151 (2006).

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Corrigendum: Large-scale chemical dissection of mitochondrial function Bridget K Wagner, Toshimori Kitami, Tamara J Gilbert, David Peck, Arvind Ramanathan, Stuart L Schreiber, Todd R Golub & Vamsi K Mootha Nat. Biotechnol. 26, 343–351 (2008); published online 24 February 2008; corrected after print 8 July 2008


In the version of this article initially published, on p.348, column 2, paragraph 2, line 7, the following sentence was incorrect: “Statins block the synthesis of cholesterol—a precursor to ubiquinone….” It should have read “Statins block the synthesis of mevalonate, a precursor not only of cholesterol but also ubiquinone, ….” The error has been corrected in the HTML and PDF versions of the article.

Corrigendum: Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells Gabsang Lee, Hyesoo Kim, Yechiel Elkabetz, George Al Shamy, Georgia Panagiotakos, Tiziano Barberi, Viviane Tabar & Lorenz Studer Nat. Biotechnol. 25, 1468–1475 (2007); published online 25 November 2007; corrected after print 8 July 2008 In the version of this article initially published, a reference was missing from the first paragraph. A new sentence and the reference (no. 6) have been added: “A recent study characterized neural crest differentiation from cloned bovine blastocysts via a neural rosette intermediate6. Other….” Subsequent references have been renumbered. The corrections have been made in the HTML and PDF versions of the article.

Erratum: Looking forward, looking back Anonymous Nat. Biotechnol. 26, 475 (2008); published online May 2008; corrected after print 13 June 2008 In the version of this article initially published, in paragraph 4, the generic name and ligand given for Avastin are incorrect. The correct generic name is bevacizumab and its target is VEGF (vascular endothelial growth factor). The error has been corrected in the HTML and PDF versions of the article.

Erratum: Is personalized medicine finally arriving? Malorye Allison Nat. Biotechnol. 26, 509–517 (2008); published May, 2008; corrected after print 8 July 2008 In the version of this article initially published, Table 1 (pp. 510–511) contained two errors. In the entry for Agendia, the product Mammaprint was described as providing information on chemotherapy options for breast cancer patients. In fact Mammaprint is a prognostic test. In the entry for Genomic Health, the product Oncotype Dx was described as providing information on breast cancer recurrence. Oncotype DX also provides information on the response to chemotherapy. The error has been corrected in the HTML and PDF versions of the article.

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