Neural Crest Cells: From Developmental Biology to Clinical Interventions Parinya Noisa1,2 and Taneli Raivio*1,3

Neural crest cells are multipotent cells, which are specified in embryonic ectoderm in the border of neural plate and epiderm during early development by interconnection of extrinsic stimuli and intrinsic factors. Neural crest cells are capable of differentiating into various somatic cell types, including melanocytes, craniofacial cartilage and bone, smooth muscle, and peripheral nervous cells, which supports their promise for cell therapy. In this work, we provide a comprehensive review of wide aspects of neural crest cells from their developmental biology to applicability in medical research. We provide a simplified model of neural crest cell development and highlight the key external stimuli and intrinsic regulators that determine the neural crest cell

Definition and Migration of Neural Crest Cells Neural crest cells confine a multipotent and migratory cell population, uniquely found in vertebrate embryos. During gastrulation, as neural plate patterning is initiated in the ectoderm, the progenitors for neural crest, neuroectoderm, and placodes are organized at the edge of the prospective neural plate (Fernandez-Garre et al., 2002). The presumptive neural crest cells are initially specified at the edge of neural plate, bordering between neuronal and nonneuronal ectoderm, and requiring contact-mediated tissue interactions between the neural plate and surface ectoderm (Moury and Jacobson, 1990). Neural crest cells locate within the dorsal part of the neural tube prior to their migration to various embryonic tissues (Fig. 1). Neural crest cells actively migrate throughout the body and give rise to multiple cell types, such as neurons and glial cells of the peripheral nervous system, melanocytes of the skin, craniofacial cartilage, the dentin, dental pulp, and alveolar bone of the head (Fig. 2). Rather than migrating randomly, neural crest cells appear to follow precise, region-specific pathways, and, consequently, they are usu-

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Institute of Biomedicine/Physiology, University of Helsinki, Finland School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand 3 Children’s Hospital, Helsinki University Central Hospital, Finland 2

Conflict of Interest: Authors confirm that there is no competing financial interest. Supported by a grant from the Academy of Finland, the Sigrid Jus elius Foundation, and the Research Funds of the Helsinki University Central Hospital. *Correspondence to: Prof. Taneli Raivio, Institute of Biomedicine/Physiology, Biomedicum Helsinki. Haartmaninkatu 8, P.O. Box 63, 00014 University of Helsinki, Helsinki, Finland. E-mail: [email protected] Published online 16 September 2014 in Wiley Online Library (wileyonlinelibrary. com). Doi: 10.1002/bdrc.21074

C 2014 Wiley Periodicals, Inc. V

fate. Defects of neural crest cell development leading to several human disorders are also mentioned, with the emphasis of using human induced pluripotent stem cells to model neurocristopathic syndromes. Birth Defects Research (Part C) 102:263–274, 2014. C 2014 Wiley Periodicals, Inc. V

Key words: neural crest cells; neurocristopathies; human pluripotent stem cells

ally classified into five distinct populations, namely cranial, cardiac, vagal, trunk, and sacral neural crest cells (Theveneau et al., 2012). Cranial neural crest cells exhibit a unique ability to differentiate into cartilage, bone, connective tissues, and sensory and parasympathetic ganglia. Cardiac neural crest cells give rise to the aorticopulmonary septum, conotruncal cushions, aortic arch smooth muscle, and parasympathetic cardiac ganglia. Vagal neural crest cells generate the majority of neurons and glia of the enteric nervous system. Trunk neural crest cells contribute to neurons and glia of the peripheral nervous system and pigment cells of the skin. Finally, sacral neural crest cells cooperate with the vagal neural crest to form a small portion of the enteric nervous system within the caudal hindgut. It is noted that, although neural crest cell fate acquisition is influenced by intrinsic positional information (Le Douarin et al., 2008), distinct region-specific neural crest cells share developmental potentials, as shown by an in vivo transplantation study and ex vivo culture experiment (Le Douarin et al., 2003). For example, trunk neural crest cells can differentiate to mesenchymal lineages after being challenged with appropriate extracellular cues (Shah et al., 1996). Importantly, neural crest cells are a self-renewing population, and can be clonally propagated and serially subcloned in vitro (Trentin et al., 2004). While neural crest cells initially are multipotent, their fates are progressively restricted soon after the commencement of cell migration. Prior to this, neural crest cells undergo an epithelial-mesenchymal transition (EMT), which involves tight-to-gap junction transition (Aaku-Saraste et al., 1996), modification of the apical-basal cell polarity (Newgreen and Gibbins, 1982), alteration of cell adhesion molecules (Nakagawa and Takeichi, 1995), and they acquire mesenchymal migratory characteristics (Sauka-Spengler

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FIGURE 1. Emergence of neural crest cells. Picture presents the emergence of neural crest during embryogenesis. Neural crest cells are induced at the border of the neural plate during neurulation. After neural tube closure, neural crest cells delaminate from the dorsal neural tube along the body axis.

FIGURE 2. Developmental potential of neural crest cells. Neural crest cells are multipotent and able to migrate throughout the body, where they give rise to specific cells, for example melanocytes, Schwann cells, and enteric neurons.

and Bronner-Fraser, 2008). EMT endows neural crest cells the ability to depart the dorsal neural tube to their target organs, where they stop and differentiate. The cranial neural crest cells are the first to delaminate before neural tube closure in the mouse, Xenopus, and zebrafish, while cranial neural crest cells in chick begin the migration soon after apposition of the neural folds (Mayor et al., 1995; Selleck and Bronner-Fraser, 1995). At more caudal level, trunk neural crest cells migrate from the dorsal aspect of the forming neural tube. Neural crest cells display a wide range of migration strategies, either solitary or collective cell migration, and some of the cells migrate as a stream of individual cells, chains, or cell sheets along the body axis (Kulesa et al., 2010). Midbrain emerging neural crest cells migrate as uniform sheets of cells, while hindbrain and trunk neural crest cells follow a segmental migratory pattern (Sechrist et al., 1993; Bronner-Fraser, 1995). The paths of migration are directed by molecular guidance cues, and mediated by the binding of external ligands to receptors on the surface of neural crest cells. For example, Cxcl12/Sdf1 chemokine of the lateral ectoderm interacts with the neural crest cell receptor Cxcr4 in Xenopus embryos (Theveneau et al., 2010). After reaching specific territories, neural crest cells restrict and commit their fate in response to instructive signals of surrounding tissues. Aberrations of neural crest cell migration during embryogenesis contribute to clinical presentations of various neurocristopathies, for instance neuroblastoma, craniofacial anomalies, and pigmentation defects.

Neural Crest Cell Specifying Signaling Pathways Neural crest cell development is recognized as a step-wise process, which involves both external stimuli and intrinsic factors to modulate induction, migration, and differentiation. The signals implicated in neural crest cell fate have been extensively elucidated. Bone morphogenetic protein (BMP), fibroblast growth factor (FGF), Wnt, and Notch signaling appear to play essential roles in the development of neural crest cells. Initially, BMP signaling, together with other molecules, primes neural crest fate of the neural plate border. The level of BMP activity required to induce neural crest cells is intermediate between those required for the formation of dermal ectoderm and neural plate. BMP signaling is modulated by the activity of BMP antagonists, such as chordin, noggin, and follistatin, which are produced by the underlying paraxial mesoderm (Marchant et al., 1998). Inhibition of BMP signaling by injection of a dominant negative BMP receptor or Noggin into a zygote of Xenopus embryo leads to the expression of neural crest markers. In addition to neural crest cell induction, BMP signaling is also found to pattern embryonic ectoderm from anterior to posterior progressively during gastrulation (Tucker et al., 2008). FGFs are also important for neural crest induction. Several FGFs are expressed in a spatiotemporal manner and associated with the induction of neural crest cells. FGF signaling can efficiently transform non-neural ectoderm into neural crest cell fate, without inducing ectopic mesodermal

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tissue (Yardley et al., 2012). In Xenopus embryos, Fgf ligands, in particular Fgf8, can trigger neural crest induction through the activation of STAT pathway (Nichane et al., 2010). FGF/MAPK signaling cascade is required in the gastrula epiblast for avian neural crest induction and prevents the ectopic expression of lateral ectoderm markers (Stuhlmiller et al., 2012). In addition, FGF signaling prevents premature neural crest cell migration and specification by opposing the activity of retinoic acid (Martinez-Morales et al., 2011). Several experiments suggest that FGF signaling induces neural crest cell fate by activating Wnt signaling, which in turn promotes neural crest formation in the ectoderm (Hong et al., 2008). In contrast, blocking of Wnt signaling alters neural crest-inducing activity of FGFs (de Croze et al., 2011). Canonical WNT signaling has long been proposed to be involved in neural crest lineage specification via its intracellular component, b-catenin. In chicken embryos, addition of Wnt3a to prospective neural epiblast explants can eliminate the expression of neural markers and induce expression of Msx1, Hnk1, and Slug. Conversely, inhibition of Wnt signaling restricts the expression of those neural crest genes, indicating an instructive role of Wnt signaling for neural crest induction (Patthey et al., 2009). In mouse, Wnt1 and Wnt3a are expressed shortly before neural crest cell expansion, migration, and differentiation, but do not play roles at the initial induction (Ikeya et al., 1997). Double homozygous null mutant mice for Wnt1/Wnt3a initially express Ap-2a normally, but its expression is lost from the migrating neural crest cells, which have severe abnormalities in generating neural crest cell derivatives (Ikeya et al., 1997). In humans, activation of canonical Wnt signaling leads to efficient generation of neural crest-like cells from human pluripotent stem cells at the expense of neuroepithelial cells (Menendez et al., 2011). Notch proteins are transmembrane receptors, activated by Ligands from neighboring cells. Notch-mediated lateral inhibition is employed to inhibit neurogenesis, and in vertebrates it promotes neural crest formation and development (Cornell et al., 2002; Mead and Yutzey, 2012). In chicken embryos, Notch signaling defines the neural crest domain on neural plate by regulating the levels of Bmp4 (Endo et al., 2002). Homozygous null mutant mice for Delta1, a Notch ligand, display proper neural crest generation, but show abnormal neural crest cell migration and differentiation (De Bellard et al., 2002). An appropriate level of Notch signaling, revealed by conditional gain and loss of Notch signaling activity, is needed for correct neural crest cell proliferation, migration, and differentiation in mouse embryos (Mead and Yutzey, 2012). Specific inhibition of Notch signaling in neural crest cells has demonstrated the requirement of Notch for vascular smooth muscle differentiation, and enteric nerve cell maintenance (High et al., 2007; Okamura and Saga, 2008). Importance of Notch signaling in neural crest cell differentiation was

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also recently revealed in humans by studying pluripotent stem cells. Activation of Notch signaling is required for the generation and maintenance of premigratory neural crestlike cells, while neural crest-derived neurons are produced when Notch signaling is inhibited (Noisa et al., 2014). Finally, a recent study suggests that extracellular matrix (ECM) is also involved in neural crest cell formation. In chicken embryos, the ECM protein, anosmin, provides autocrine regulation of cranial neural crest cell formation by enhancing Fgf8 activity and inhibiting Bmp5 and Wnt3a signaling (Endo et al., 2012). Depletion or locally enhancing the level of anosmin revealed its crucial role in neural crest cell formation. Crosstalk among signaling pathways and ECM thus appears an important determinant to trigger the activity of neural crest transcriptional circuitry.

Transcription Factors and Epigenetic Regulators of Neural Crest Cell Development Neural crest cell fate commitment is ensured by the coordinated actions of several neural crest specifier genes, including the zinc finger transcription factors Slug and Snail, HMG box transcription factors SoxE, paired-box transcription factors Pax3/7, and forkhead transcription factors FoxD3. In Xenopus, Slug, and Snail can be detected at neural crest-forming regions of the neural plate border by late gastrula stages (Mayor et al., 1995). Expression of Slug is regulated by Wnt signaling, as the activity of Slug promoter depends on its Lef1-binding site (Vallin et al., 2001). Overexpression of Slug and Snail leads to expanded expression of neural crest markers and excess production of migratory neural crest cells (Aybar et al., 2003). Slug is known to regulate the signals involved in mesodermal induction of neural crest. Morpholino-based inhibition of Slug disrupts mesoderm formation and decreases in Snail RNA levels in Xenopus embryos (Zhang et al., 2009). It is suggested that the loss of neural crest cells in Slug mutant embryos might involve mesoderm-derived secreted factors, such as bmp4 and wnt8 (Shi et al., 2011). Another family of genes implicated in neural crest formation are the SoxE genes (Sox8, Sox9, and Sox10), which encode HMG-box containing transcription factors. Sox10 is expressed in neural crest cells and later in glial lineage (Britsch et al., 2001). Inhibition of Sox10 in Xenopus embryos with morpholino antisense oligonucleotides prevents the formation of neural crest cells and reduces the expression of Slug and FoxD3 (Honore et al., 2003). Mutations of Sox10 in mice and zebrafish result in a defective generation of neural crest derivatives, including peripheral neurons, glia, and melanocytes (Dutton et al., 2001; Pingault et al., 2002). Another SoxE gene, Sox9, is expressed in neural crest cells and appears to be involved in neural crest formation. Forced expression of Sox9 in chicken embryos not only leads to overproduction of neural crest

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cells, but also biases these cells toward melanocytes and glia (Cheung and Briscoe, 2003). A mammalian neural crest-specific enhancer of Sox9 has been identified and required the activity of b-catenin, supporting a direct link between neural crest-inducing Wnt signal and Sox9 function (Bagheri-Fam et al., 2006). PAX proteins contain distinguishing paired box-DNA binding domains. Pax3 and the closely related Pax7 have been implicated in diverse developmental events, and are expressed in the developing neural crest cells. Pax3 mutations in mice lead to various neural crest defects and, in some cases, myogenic deficiencies (Kassar-Duchossoy et al., 2005; Sato et al., 2010; Olaopa et al., 2011). In Xenopus embryos, Pax3, together with Zic1, triggers the early neural crest gene regulatory network by direct activation of multiple key neural crest specifies, for instance Slug/ Snail, FoxD3, and Twist1 (Plouhinec et al., 2014). Pax3 may modulate neural crest-specifying signals, for example Wnt, through the induction of key pathway regulators, such as Axin2 and Cyp26c1 (Plouhinec et al., 2014). Pax3 and Zic1 also cooperatively drive the migration of neural crest cells and differentiation of several neural crest derivatives, that is, melanocytes and craniofacial cartilage (Milet et al., 2013). In chicken embryos, Pax7 is required for neural crest formation in vivo, since blocking its translation inhibits expression of the neural crest markers, Slug, Sox9, and Sox10 (Basch et al., 2006). Homozygous Pax72/2 mice die shortly after birth, due to defective development of cranial neural crest cells (Mansouri et al., 1996). FoxD3, a member of forkhead transcription factors, is expressed by presumptive neural crest cells. FoxD3 is an early marker of neural crest progenitors in several species, including Xenopus, chick, zebrafish, and mouse (Dottori et al., 2001; Kos et al., 2001; Pohl and Knochel, 2001). In Xenopus, overexpression of FoxD3 promotes neural crest cell induction, and its dominant negative form leads to a loss of neural crest marker expression (Sasai et al., 2001). Ectopic expression of FoxD3 in avian embryos causes increased delamination of neural crest cells from the neural tube (Dottori et al., 2001), while in FoxD3 mutant zebrafish neural crest cells fail to migrate (Stewart et al., 2006). Morpholino-mediated FoxD3 depletion results in precocious migration and restricted cell fate of neural crest cells (Kos et al., 2001), and the formation of melanocytes could be suppressed by exogenous FoxD3, suggesting that FoxD3 may function in neural crest cell lineage specification (Dottori et al., 2001). Chromatin structure is known to regulate stem cell maintenance, proliferation, and differentiation. Recently, several epigenetic regulators have been demonstrated to play essential roles for neural crest development. Chd7, an ATP-dependent chromatin remodeler, is required for the formation of multipotent migratory neural crest cells, both in Xenopus and human. Chd7, together with Brg1 (a SWI/ SNF family member), occupies distal regulatory elements and activates the expression of neural crest cell transcrip-

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tional circuitry, including Sox9, Slug, and Twist1 (Bajpai et al., 2010). In mice, Chd7/Brg1 complex binds on the PlexinA2 promoter to activate PlexinA2 expression, which is essential for navigation of neural crest cell migration into proximal cardiac outflow tract (Li et al., 2013). Another epigenetic factor is DNA methyltransferases (DNMTs), which recognize CpG islands and newly methylated DNA by catalyzing the transfer of a methyl group to cytosine residue, and thereby repressing expression of target genes (Cheng and Blumenthal, 2008). In chicken embryos, Dnmt3a promotes neural crest specification by directly mediating repression of neural genes, Sox2 and Sox3, and the knockdown of Dnmt3a expression causes ectopic Sox2 and Sox3 expression at the expense of neural crest markers (Hu et al., 2012). Histone deacetylase (HDAC) is an enzyme that removes acetyl moieties and results in chromosome condensation and gene repression. Recently, it was shown that HDACs play a crucial role in timing of trunk neural crest cell specification in chick (Murko et al., 2013). Inhibition of HDACs by Trichostatin A induces neural tube defects and, concomitantly, upregulation of several neural crest markers, including Pax3, Sox9, and Sox10 (Murko et al., 2013). In mice, HDACs control lineage specification of neural crest cells. HDAC1/2 is essential for the specification of neural crest cells, Schwann cells, and satellite glia by inducing Pax3 expression (Jacob et al., 2014). Taken together, functional elucidations of transcription factors and epigenetic regulators that control neural crest cell fate have provided an insight into molecular mechanisms of neural crest cell development, which might explain the pathology of several neurocristopathic syndromes. The complete picture of both external inducing signals and internal programming factors will lead to a better route of treatment for such diseases.

Neural Crest-Related Human Diseases Clinical presentations of neurocristopathies are variable and cover a wide range of phenotypic features. Forty years ago, Bolande (1974) divided neurocristopathies to simple and complex ones including those with syndromic features. A modification presented by Martucciello et al. (2012) adds the possible neoplastic component to both categories. Examples of simple neurocristopathies include albinism and Hirschsprung’s disease, whereas neuroblastoma and pheochromocytoma represent examples of simple neurocristopathies with a neoplastic component. Complex neurocristopathies include entities such as neurofibromatosis and multiple endocrine neoplasias 2 and 3 (Martucciello et al. 2012). However, the above classifications do not include craniofacial syndromes of neural crest origin; neural crest-derived tissues and cells are also frequently affected in Down syndrome, and in animal studies ethanol has been shown to affect cranial neural crest cells

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FIGURE 3. Strategy to derive neural crest cells from human pluripotent stem cells. Directed differentiation of human pluripotent stem cells (hPSCs) toward neural crest cells in chemically defined systems is implemented by using several strategies. A: The double SMAD inhibition neuralizes hPSCs to adopt a neuroepithelial (NEP) fate. Neural crest cells can be obtained at the periphery of the colonies and purified by antibody-based flow cytometry (Lee et al., 2007). B: Modulation of WNT and SMAD signaling allows efficient derivation of migratory neural crest cells from hPSCs at day 10 of the induction (Menendez et al., 2011). C: Modification of the previous protocol to modulate WNT and BMP signaling in N2B27 medium permits hPSCs to convert into premigratory neural crest-like cells at day 10 of the induction (Noisa et al., 2014). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

with a possible link to human fetal alcohol syndrome (Cartwright and Smith, 1995). Our research group has a long-standing interest in elucidating the genetic and developmental basis of Kallmann syndrome (KS), and the role of neural crest in the pathogenesis of KS is rapidly emerging. KS consists of decreased/absent sense of smell (that typically is associated to hypoplasia or aplasia of olfactory bulbs) and congenital hypogonadotropic hypogonadism. The degree of gonadotropin deficiency is variable, ranging from severe forms (microphallus, cryptorchidism, absent puberty, and infertility) to reversal of hypogonadotropism later in life (Sidhoum et al., 2014). Associated phenotypic features (i.e., not related to the reproductive system) include cleft lip and/or palate, synkinesia, hearing loss, and renal and limb anomalies (Costa-Barbosa et al., 2013). During the last few years, several of the many genes underlying KS have been shown to be key players in the formation and development of neural crest. First, mutations in KAL1, a gene encoding a secreted ECM protein anosmin-1, have long known to underlie X chromosomal recessive KS (Franco et al., 1991; Legouis et al., 1991). For long, anosmin-1 has been known to bear axon guidance and branch-promoting activity (Soussi-Yanicostas et al., 2002), and only recently Endo et al. (2012) demonstrated that

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anosmin-1 is essential for cranial neural crest formation and induction of Fgf8 (another gene implicated in the etiology of KS) in the chick, as discussed above. Second, as much as 75% of patients with CHARGE syndrome (coloboma, heart defects, retardation of growth and development, genital anomalies, and ear anomalies) carry mutations in CHD7. Interestingly, there is a clear genetic and phenotypic overlap between CHARGE syndrome and KS, as exemplified by the finding that 3–5% of patients with KS do have mutations in CHD7 without the full-blown CHARGE syndrome (Kim et al., 2008; Jongmans et al., 2009). Third, another genetic overlap between KS and a known neurocristopathy was recently described when Pingault et al. (2013) showed that mutations in SOX10 underlie KS with deafness. Before these findings, mutations in SOX10 were known to underlie Waardenburg syndrome (WS) types 2E and 4C and its neurological variant Peripheral demyelinating neuropathy, Central dysmyelination, WS and Hirschsprung’s disease (Inoue et al., 2002; Bondurand et al., 2007). SOX10 is important for the development of many cell types, including melanocytes, enteric ganglia cells, structures of the inner ear, and glial cells—especially olfactory ensheathing cells (OECs) (Barraud et al., 2013). Interestingly, Sox10 KO mouse recapitulates KS phenotype in that these mice do not have OECs (Barraud et al., 2013). The clinical impetus of these findings is clear as, according to current recommendations, all patients with KS and hearing loss should be first screened for mutations in CHD7 and SOX10 (Costa-Barbosa et al., 2013; Pingault et al., 2013); these patients also frequently display semicircular canal hypoplasia/aplasia. Taken together, these examples share light on the molecular genetic and phenotypic overlap between rare syndromes that were initially categorized purely on clinical basis.

The Derivation of Neural Crest Cells from Human Pluripotent Stem Cells Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs), are capable of expanding indefinitely and differentiating into all human germ layers both in vitro and in vivo (Itskovitz-Eldor et al., 2000; Lensch et al., 2007). hESCs are derived from the inner cell mass of blastocyst embryos, while human iPSCs are generated from somatic cell reprogramming by the overexpression of essential transcription factors, Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006). This feature makes hPSCs an attractive cell source for the derivation of desired cell types, in particular here neural crest cells. To date, several groups have reported the success of neural crest cell generation from hPSCs, employing different strategies, either coculture (Pomp et al., 2005), cell aggregate (Curchoe et al., 2010), or chemically defined system (Lee et al., 2007; Menendez et al., 2011). However, it appears

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fied induction medium contains Wnt activator, 1Azakenpaulone, together with a BMP inhibitor, Dorsomorphin, in N2B27 medium (Fig. 3C). After 10 days of induction, these cells robustly expressed neural crest specifier genes, including PAX3, SLUG, and TWIST1 (Noisa et al., 2014). The cells were premigratory neural crest cells, but phenotypically shifted to p75/HNK1-positive migratory cells upon application of FGF8 and Notch inhibitor. The premigratory neural crest-like cells can be clonally expanded and differentiated to neural crest derivatives, such as peripheral sensory neurons, adipocytes, chondrocytes, and osteocytes (Noisa et al., 2014). These premigratory neural crest-like cells allow studying the early events of human neural crest lineage commitment and also neural crest fate specification. FIGURE 4. Schematic of tissue-specific progenitor cells derived from multipotent neural crest cells. Neural crest cells can serve as precursors for tissuespecific progenitor cells, which should be safe for cell replacement purposes.

that a chemically defined and monolayer differentiation protocol for hPSCs is more preferable than the other systems, because it enables investigators to directly visualize the differentiation process and thereby neural crest cell fates (Fig. 3). The pioneering report for neural crest cell generation in a chemically defined culture is achieved by using SMAD inhibitors, Noggin, and TGFb inhibitor SB431542 (Lee et al., 2007). Neural rosette phenotypes were obtained after 7 days of the differentiation, which presented as a mixed population of central nervous system neural progenitors, marked by PAX6, and neural crest cells, marked by p75/HNK1 (Fig. 3A). Expression of p75 and HNK1 was observed primarily in the cells located at the periphery of the rosettes. The neural crest cell population can be isolated by antibody-based flow cytometry against p75/HNK1 for further culture and analysis, and the purified cells retain the ability of in vitro propagation and multipotentcy. Soon thereafter, a single step protocol for neural crest derivation from hPSCs was invented (Menendez et al., 2011). The protocol is based on the activation of canonical Wnt signaling under conditions of low global SMAD signaling (Fig. 3B). Wnt agonist, BIO, was supplemented along with the dual SMAD inhibitors to bias hPSCs toward neural crest fate and avoid other neuroectoderm cell types. After day 15 of the differentiation, the resulting neural crest cells, marked by p75/HNK1 expression, can be maintained over extended periods in culture, while retaining developmental potential for peripheral neurons and mesenchymal cell-derived progeny. Nevertheless, it is noted that most currently available neural crest differentiation protocols present the derivation of migratory neural crest-like cells, marked by the expression of p75 and HNK1. To generate pre-migratory neural crest-like cells, this differentiation protocol was modified (Menendez et al., 2011). The modi-

Modeling of Neural Crest-Related Diseases by Induced Pluripotent Stem Cells Mice, chick, and zebrafish have long been used to study vertebrate development. However, in the ultimate quest to understand the mechanisms of human development, with the goal of preventing and treating developmental defects, animal modeling falls short. In contrast, previous reports indicate that the differentiation of hPSCs in culture follows hierarchical sets of signals that regulate generation of germ layers and specific cell types in humans (Hay et al., 2008; Wu et al., 2010). Importantly, iPSCs derived from somatic cells of patients with monogenic disease-causing mutations, now provide a suitable model for studying disease mechanisms. The first example of successful modeling of a neural crest-related disease with iPSCs (Familial Dysautonomia, FD) was published in 2009 (Lee et al., 2009). FD is a disease characterized by the degeneration of sensory and autonomic neurons. Most FD patients carry mutations in the IKBKAP gene, resulting in reduced levels of normal IKBKAP protein, and potentially affecting cell motility (Close et al., 2006). To address the impact of IKBKAP gene on FD pathology, fibroblasts from FD patients were induced to pluripotent state (called FD-iPSCs), and differentiated toward neural crest cells. As compared with the control cells, the mutant transcript of IKBKAP predominated in neural crest cells derived from FD-iPSCs, suggesting a mechanism of disease specificity (Lee et al., 2009). Significantly, FD-iPSCs-derived neural crest cells showed reduced cell migration and neuronal differentiation. Aberrant phenotypes of FD-iPSCs derived neural crest cells could be rescued by Kinetin, a candidate drug for reversing abnormal IKBKAP splicing. Continuous Kinetin treatment increased expression of key peripheral neuronal markers, and enhanced neuronal differentiation from neural crest cells derived from FD-iPSCs. In addition, largescale compound screening identified SKF-86466 to be able to rescue IKAP protein level and to prevent the loss of

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FIGURE 5. Schematic illustration of human induced pluripotent stem cells for disease modeling and experimental cell replacement. Patient-specific iPSCs hold a premise for disease-modeling and cell therapy. Patient’s somatic cells are obtained (A) and induced to pluripotency (B). iPSCs can be corrected for the diseasecausing mutations (C) prior to further differentiation into specific cell types (D). Finally, these cells could be used for cell replacement (E).

autonomic neuronal marker expression in FD-iPSCsderived neural crest cells, demonstrating the feasibility of iPSCs in identifying novel candidate drugs for the treatment of neurocristopathies (Lee et al., 2012). Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder with an intronic GAA repeat expansion in the frataxin (FXN) gene, leading to partial reduction of FXN transcript (Campuzano et al., 1996) and causing mitochondrial dysfunction and increased sensitivity to oxidative stress of the affected cells (Schmucker and Puccio, 2010). FRDA-iPSCs showed normal pluripotent stem cell characteristics, but reduced expression of FXN mRNA and protein (Liu et al., 2011). All FRDA-iPSCs retained pathological GAA repeat expansion. As peripheral sensory neurons (PSNs), which derive from the neural crest, are the most affected population in FRDA (Morral et al., 2010), FRDA-iPSCs were differentiated toward PSN via neural crest cells. No significant differences were found in the expression of neural crest and PSN markers between FRDA- and control iPSCs; however, PSNs derived from FRDA-iPSCs failed to upregulate the expression of FXN, both at the mRNA and protein level (Eigentler et al., 2013). This FXN expression discrepancy suggests its developmental effect in PSNs that may accumulate over time and consequently manifest in neurodegeneration. More recently, pathologic mechanisms of hypopigmentation defects Hermansky-Pudlak (HP) and Chediak-Higashi (CH) syndromes were recapitulated by using iPSCs (Mica et al., 2013). HP syndrome has been recognized as a genetically heterogeneous group of autosomal recessive disorders that share in common aberrant formation, transport, and

fusion of intracellular vesicles of lysosomal lineage (Wei, 2006). CH is caused by mutations in the lysosomaltrafficking regulator gene and the main clinical manifestations include partial albinism, bleeding diathesis immunodeficiency, and progressive neurological deterioration. The mechanisms of CH have remained enigmatic, but are related to the persistence of abnormally large lysosome-related organelles, including the melanosomes (Tchernev et al., 2002; Durchfort et al., 2012). Due to their enlarged size, melanosomes in CH patients cannot be efficiently transferred to neighboring keratinocytes, which leads to hypopigmentation (Introne et al., 1999). As these diseases are involved in defects in melanosome vesicle formation and trafficking, the disease modeling paradigm by using patientspecific iPSCs was to first generate neural crest cells and then further differentiate them to melanocytes. All control and HP/CH-iPSCs were first efficiently differentiated into neural crest cells with a brief activation of WNT signaling, and subsequently, mature melanocytes expressing PMEL, MITF, and SOX10, were derived by the treatment of BMP4 and EDN3 at comparable efficiencies from each of the patient and control iPSCs. HP-iPSC derived melanocytes faithfully reproduced the ultrastructural features of diseaseassociated pigmentation defects, a near-complete loss of pigmentation at the macroscopic level and when quantified after cell lysis (Mica et al., 2013). CH melanocytes exhibited pigmentation levels lower than those observed with an African-American control line, but comparable to those observed with a Caucasian control. These phenotypes are compatible with the hypothesis that CH affects melanosome transfer rather than the production. All disease-related

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phenotypes were consistent among melanocytes derived from HP/CH-iPSCs, indicating that disease behavior reflects genome-specific differences. Altogether, these reports have reinforced the feasibility of using patient-specific iPSCs to model various neural crest-related diseases. It is noted that most modeled diseases are early onset disorders, suggesting that iPSCs may be particularly suitable to model developmental disorders. iPSCs can also provide dynamic systems to investigate molecular mechanisms underlying disease pathology, which will potentially lead to the development of novel therapeutic strategies.

ative disorders, the recent advancement of genome editing technology has extended the promise of iPSCs to also cure congenital defects (Fig. 5). The rapid development of genome editing technology is a mainstay for the generation of healthy iPSC lines for patients with genetic diseases (Corti et al., 2012; Fong et al., 2013). Several neurocristopathy syndromes, such as CHARGE, Waadenburg-Shah, Hirschsprung, and Kallmann, are affected by genetic mutations of genes that alter division, survival, migration, or differentiation of neural crest cells. Therefore, genetic correction of neurocristopathy syndromes-derived iPSCs will provide healthy neural crest cells that might be applicable for cell-based therapy in the future.

Future Perspectives

Acknowledgment

The availability of neural crest cell differentiation protocols from hPSCs serves as a starting platform for making human neural crest cells and their derivatives applicable for various purposes, including developmental biology study, toxicant screening, and cell replacement. Together with high throughput screening technology, hPSC-derived neural crest cells have already led to the finding of novel candidate compounds for treating FD (Lee et al., 2012). Importantly, the currently available protocols provide valuable tools for redefining unanswered questions of the basic biology of human neural crest cells, including discrepancy of anterior/posterior identity of human neural crest cells, characterization of the lineage potential of premigratory/postmigratory human neural crest cells, and molecular control of specific cell fate decision of certain human neural crest cell derivatives. Another key advantage of using iPSCs is the personalized cell-based therapy for tissue degeneration and damage. Spinal cord injury and age-related macular degeneration are the two diseases that have now undergone clinical trials for iPSC-based therapy (Cyranoski, 2013; Nakamura and Okano, 2013). It has been shown that, after xenografting, iPSC-derived neural crest cells can survive, integrate, and differentiate within the host animals (Saadai et al., 2013), and promote the repairing process in diseased organisms (Xu et al., 2013). However, neural crest cells are multipotent, and could give rise to undesired cell types in recipient tissues. It is challenging to develop specific priming protocols to drive multipotent neural crest cells toward tissue specific progenitor cells that should be safe for cell replacement (Fig. 4). In addition, retroviral transduction of reprograming factors results in random exogenous DNA integration that can trigger tumor formation (Li et al., 2002). Proteinand chemical-based iPSC generation methods are possible options and show the promising success in mouse fibroblasts (Zhou et al., 2009; Hou et al., 2013). The same technology has been applied to human cells as well (Kim et al., 2009). Furthermore, a clinical iPSC cultivation system needs to be safe and devoid of xenogenic contamination (Durruthy-Durruthy et al., 2014). Besides treating degener-

The authors thank Dr. Timo Tuuri for critical reading.

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