The generation of neurons or glia that can be used to replace lost or damaged tissue is a key step in stem cell-based therapies designed to treat neurodegenerative disease and neurological disorders. Several different candidate sources have been proposed for producing these cells. Based upon their proliferative and pluripotential properties, embryonic stem cells (ESCs) are an attractive alternative to fetal or adult-derived central nervous system (CNS) tissue. The derivation of specific neuronal or glial cell types from ESCs invariably includes the production of neural stem cells (NSCs), the self-renewing, pluripotential stem cell of the nervous system, capable of differentiating into neurons, astrocytes, and oligodenrocytes (Temple,2001; Doetsch,2003). The desired fully differentiated cell can then either be produced in vitro before transplant by using signals that mimic the appropriate in vivo environment, or by transplanting the ESC-derived NSCs and relying upon the host environment to provide the appropriate cues.
Neurons and glia were among the first differentiated derivatives generated from mouse ESCs after their isolation over 25 years ago (Evans and Kaufman,1981; Martin,1981) and have successfully been derived from human ESCs as well (Thomson et al.,1998). Although both mouse and human ESCs are pluripotent early embryo-like cells, able to differentiate into derivatives of all three primary germ layers, they have several differences, including distinct growth factor requirements: leukemia inhibitory factor (LIF) and bone morphogenetic proteins (BMPs) for mouse ESCs and activin and fibroblast growth factor (FGF) for human ESCs (Brons et al.,2007; Tesar et al.,2007). This discrepancy is explained by recent reports documenting the isolation of pluripotential cells from the epiblast stage mouse embryo (Brons et al.,2007; Tesar et al.,2007). These mouse EpiSCs are more similar than mouse ESCs to human ESCs, based upon colony morphology, cloning efficiency, in vivo potential in chimeras, growth factor requirements, and transcriptional profile. Protocols for producing neural derivatives from ESCs were initially designed for mouse ESCs and modified for application to human ESCs. The recent realization that human ESCs reflect a later stage pluripotential cell type than mouse ESCs, epiblast vs. inner cell mass, may help in the future refinement of these protocols using human ESCs.
Several recent reviews have examined the conditions required to produce specific neuronal and glial subtypes, including neurons with specific transmitter profiles, from mouse and human ESCs (Dang and Tropepe,2006; Wilson and Stice,2006; Zhang,2006). We will focus here on the early steps of ESC neurogenesis, the transition from ESC to NSC. Multiple protocols have been established that produce ESC-derived NSCs. We will examine these protocols and compare their strengths and weaknesses as approaches for generating material for transplant. Several criteria will be considered, including the efficiency and yield of NSCs, the purity of the final population, the ability of the NSCs to produce all subtypes of neurons and glia upon subsequent induction, the ease and reproducibility of the approach, and the time required to go from ESC to NSC. We will also note whether the approach has been applied to human ESCs.
These protocols are based upon the mechanisms governing both neural induction during embryogenesis and neurogenesis in the adult CNS. Radial glia cells are the NSCs of the embryonic CNS, and astrocyte-like stem cells the NSCs of the adult brain (Doetsch,2003). Recent studies suggest that ESC-derived NSCs can have the properties of embryonic radial glia (Liour and Yu,2003; Plachta et al.,2004; Liour et al.,2006; Nat et al.,2007) and are able to proliferate and generate all types of neural derivatives, neurons, astrocytes, and oligodendrocytes. What are the similarities and differences between ESC-derived NSCs and those derived from the embryonic or adult CNS? In a direct comparison, mouse ESC-derived NSCs proliferate more readily than mouse somatic NSCs and prefer adherent conditions over suspension culture conditions (Colombo et al.,2006). The ESC-derived NSCs also differentiated into neural cell types with a higher efficiency than somatic NSCs and were able to produce more cell types, demonstrating a broader potential (Colombo et al.,2006). Transcriptional profiling demonstrated a close similarity between NSCs of both origins, but also showed that ES-derived NSCs displayed positional molecular information that indicated a broader potential than somatic NSCs, specifically including rostral spinal cord-specific markers (Colombo et al.,2006). In contrast, a similar gene profiling analysis comparing human ESC-derived NSCs with fetally derived NSCs led to the conclusion that there was limited similarity between the two cell types (Shin et al.,2007).
NSCs and progenitors derived from the embryonic or adult CNS have restricted potential that reflects their site of origin along the rostral/caudal and dorsal/ventral axes and are characterized by the expression of region-specific transcription factors (Temple,2001; Zhang,2006). On the other hand, ESC neurogenesis mimics embryonic neurogenesis by first producing multipotent NSCs that express Sox2, Sox3, Otx2, and Pax6, which characterize anterior neurectoderm identity, that can subsequently differentiate into neural progenitors with more restricted identity based upon the expression of region-specific transcription factors (Wilson and Edlund,2001; Barberi et al.,2003; Xia et al.,2007). Highly enriched populations of motor neurons can be induced by treatment of early stage anterior neurectoderm-like progenitors, but not later stage progenitors with regional identity, with appropriate signaling molecules (Zhang et al.,2001). Addition of caudalizing signals, such as retinoic acid (RA) or FGF2 can promote a spinal cord identity and subsequent production of motor neurons (Wichterle et al.,2002), whereas addition of Wnt and Nodal antagonists promotes production of telencephalic progenitors (Watanabe et al.,2005). Treatment with FGF8 and Sonic Hedgehog promote production of ventral midbrain precursors and subsequent differentiation of dopaminergic neurons (Lee et al.,2000; Barberi et al.,2003).
In addition to comparing available protocols for NSC production, we will also identify the molecular signals required for differentiation, survival, and proliferation ESC-derived NSCs. First, we will describe the basic mechanisms of neural induction and how these principals are applied in the various protocols used to generate neurons and glia from ESCs. We will then summarize more recent studies that have used ESC neurogenesis to identify the conditions and signals required to promote the production of NSCs from ESCs. Finally, we will integrate the conclusions from these studies to synthesize a unified model for NSC production.
HOW TO MAKE NEURAL DERIVATIVES FROM ESCS
Two basic approaches have been identified: (1) mimicking the environment that produces neurectoderm in the embryo by providing appropriate cell–cell interactions and signals through embryoid body formation, or (2) depriving the ESCs of both cell–cell interactions and signals by low density culture in serum-free medium, evoking a default mechanism for NSC differentiation. Some protocols combine aspects of both of these two approaches, promoting cell–cell interactions to facilitate formation of all three primary germ layers followed by neural lineage-specific selection under defined conditions. For both mouse and human ESCs, the quality of the starting ESC population is critical for the successful production of neural derivatives. As is the case for many in vitro protocols, cell populations at any stage may be heterogeneous and additional purification steps, such as fluorescence-activated cell sorting (FACS), may be required to obtain the desired cell type. Table 1 documents the properties of the six protocols described below.
|Protocols||1. RA induction (4−/4+)||2. MEDII CM induction||3. Serum-free selection||4. Stromal co-culture||5. Low density clonal neurosphere||6. Monolayer Serum-free|
|Mouse ESC lines used||D3, CCE||E14, D3||J1, CJ7, D3, R1||ESC: CJ7, AB2.2,E14,ESB5 ntES:C4, C15,C16,CN1,2, CT2||R1||46C (E14 derived) 15 clones of ESC|
|Coculture, CM, or other factors||None||HepG2 conditioned medium||None||Stromal cell (MS5,S17,PA6 etc)||LIF||none|
|Initial plating density||Not quantified >1×105 cells/ml||1×105 cells/ml||2-2.5×104cells/cm2||50 cells/cm2||1-20 cells/microwell||0.5-1.5x104 cells/cm2|
|Culture type||Suspension||Suspension||Suspension + adherent||Adherent||Suspension||Adherent|
|Serum or serum replacement||10% FBS+ 10% newborn calf serum||10% FBS||10% FBS for EB then no serum||15% serum replacement||No serum||No serum|
|Days to reach NSC peak||8||7||10-12||6||3 (4 hrs in PBS)||5|
|% NSC at peak||39% neuron-like Cellsb||Nearly 100% 95.7% NCAM+||>80%||High, not quantified||100%||75%|
|NSC marker||βIII tubulin||Sox1, Sox2, nestin||nestin||nestin, NCAM, Musashi||nestin||Sox1, nestin|
|NSC Regional identity||NA||Otx1 (fore- and midbrain) En1, En2 (midbrain)||Otx1 (fore- and midbrain) En1 (midbrain)||No specific regional identity||Emx2 (forebrain) HoxB1 (hindbrain)||NA|
|Differentiation potential of derived NSC||NA||Neurons, glia (>95%), neural crest||Neurons (Map2) Astrocytes (GFAP) Oligodentrocytes (O4)||Neurons (dopaminergic, serotonin, GABAergic and motor neurons with high efficiency), glia||Neurons (Map2) Astrocytes (GFAP) Oligodendrocytes (O4)||Neurons (GABA, TH) Astrocytes (GFAP) Oligodendrocytes (CNPase)|
|Other lineages||Many other lineages present||None||Non-neural lineages selected against in serum-free medium||None||primitive endoderm present (GATA4), no mesoderm or definitive endoderm||Non-neural cell types and Oct4+ cells present|
|Key references:||Bain et al.,1995 Bibel et al.,2004||Rathjen et al.,2002||Okabe et al.1996||Barberi et al.,2003 Kawasaki et al.,2000||Tropepe et al.,2001 Smukler et al.,2006||Ying et al.,2003|
Mimic the Embryo Environment: Embryoid Body Intermediates Make Neurectoderm
Initial studies demonstrating the generation of neural cell types from mouse ESCs were based upon first forming embryoid body intermediates. ESC-derived embryoid bodies are similar to embryoid bodies derived from teratocarcinoma stem cells, first described by Stevens and Pierce (Pierce and Dixon,1959; Stevens,1959) and subsequently demonstrated to undergo a similar pattern of differentiation to isolated inner cell masses cultured in vitro (Martin et al.,1977).
When mouse ESCs are removed from their feeder layer, and placed in suspension culture in the absence of the growth factor LIF, which promotes maintenance of the pluripotential state (Smith et al.,1988), they form aggregates, which within 2–4 days consist of an outer layer of hypoblast-like cells (extraembryonic visceral endoderm) surrounding an epiblast-like core (primitive ectoderm; Martin et al.,1977; Rathjen et al.,2002; Maye et al.,2004). At this stage, the embryoid body resembles the anterior prestreak stage embryo, with the epiblast-like core able to generate derivatives of all three primary germ layers; definitive endoderm, mesoderm, and ectoderm (Keller,2005). The embryoid body core continues to express the ESC marker Oct4 and begins expressing the primitive ectoderm marker FGF5 (Hebert et al.,1991; Rathjen et al.,2002; Maye et al.,2004). Between days 6 and 8, the core undergoes cavitation and forms an inner epithelial layer (Coucouvanis and Martin,1995). Cells within this layer can be committed to definitive ectoderm, characterized by Sox2 and Otx2 expression (Rathjen et al.,2002; Maye et al.,2004). Subsequently, a morphological transformation to columnar epithelium, resembling the neural tube, is accompanied by the expression of neurectoderm-specific markers such as Sox1 and Six3 (Rathjen et al.,2002; Maye et al.,2004).
Conditions that direct the differentiation of the embryoid core toward neurectoderm, at the expense of mesoderm or definitive endoderm derivatives, increase the pool of cells that can subsequently differentiate into neurons or glia. In the embryo, signals from a region of the anterior extraembryonic visceral endoderm (AVE) are known to promote anterior neurectoderm differentiation and the visceral endoderm may serve a similar function in the embryoid body (Thomas and Beddington,1996). More posterior in the embryo, the notochord supplies signals that pattern the developing neural tube, but no comparable structure is present in embryoid bodies (Stern,2005). Work from our laboratory suggests that the signaling molecule Indian Hedgehog, secreted by the visceral endoderm, promotes neurectoderm differentiation in vitro. Embryoid bodies derived from ESCs carrying loss of function mutations for components of the Hedgehog signaling pathway are able to generate primitive ectoderm, but not neurectoderm or neurons (Maye et al.,2004). The Hedgehog signaling cascade is best known for its role in ventral patterning (Briscoe et al.,2000; Wichterle et al.,2002); however, several recent studies support an additional role in NSC survival (Britto et al.,2000; Machold et al.,2003; Cayuso et al.,2006) and proliferation (Machold et al.,2003; Ahn and Joyner,2005; Palma et al.,2005) in the embryonic and adult mouse brain.
Retinoic acid protocol.
The yield of neural lineage cells generated by embryoid bodies can be dramatically increased by the addition of RA (Guan et al.,2001; Gottlieb,2002; Table 1; Fig. 1, protocol 1). RA has a well-established patterning role during development, for example in posteriorizing CNS tissue (Durston et al.,1989; Li et al.,2005) and ESC derivatives (Zhang,2006). Numerous studies also support a role for RA in embryonic neurogenesis in particular regions at specific times, and a recent study examining the effect of RA deficiency on granule cell differentiation suggests that RA is required for adult hippocampal neurogenesis (Jacobs et al.,2006). RA promotes the differentiation of pluripotential teratocarcinoma cells into neural progenitors and neurons (Jones-Villeneuve et al.,1983; Bain and Gottlieb,1994), and this treatment was applied to ESCs to promote neural induction. ESC embryoid bodies cultured for 4 days were treated for an additional 4 days with RA (4−/4+) and then attached to adhesive substrates for 7 days, and the extent of neuronal differentiation was determined based upon cell morphology, expression of cell type specific markers, and electrophysiological measurements (Bain et al.,1995). Although no markers for NSCs were assayed, there was a yield of up to 40% neuron-like cells, vs. just a few percent observed in the cultures not treated with RA. Related protocols increased the yield of neural derivatives, including glutamatergic neurons and motor neurons, with RA treatment (Wichterle et al.,2002; Bibel et al.,2004; Li et al.,2005). Subsequent studies from other groups have established that the NSCs generated using the RA protocol may have restricted differentiation potential. For example, when neural progenitors generated by RA induction were transplanted to the embryonic chick neural tube, they showed limited capacity to differentiate, compared with ESC-derived neural progenitors generated in the absence of RA (Plachta et al,2004). Additional drawbacks of this approach are the presence of numerous cell types in the final product, including mesoderm derivatives, and the length of culture time required, up to 2 weeks. This approach has been applied to hESCs (Schuldiner et al.,2001).
MEDII conditioned medium induction.
Studies from the Rathjen laboratory have demonstrated that conditioned medium from a human hepataocellular carcinoma cell line Hep-G2 can promote the homogeneous differentiation of primitive ectoderm-like (EPL) cells from mouse ESCs (Rathjen et al.,1999). Embryoid bodies formed from these cells do not have an outer visceral endoderm layer, but the remaining cells are able to form an essentially pure neurectoderm-like epithelial layer. Most cells at this stage expressed the NSC markers Sox1 (Pevny and Lovell-Badge,1997) and nestin (Hockfield and McKay,1985; Table 1; Fig. 1, protocol 2). The conditioned medium appears to contain signals that are capable of inducing neurectoderm differentiation at the expense of mesodermal and endodermal derivatives. These NSCs do not appear to have a restricted regional identity and, when subjected to different signaling factors, can be further induced to generate neurons or glia, as well as neural crest (Rathjen et al.,2002). However, the molecules in the Hep-G2–conditioned medium responsible for this activity have not been identified. This protocol has not yet been applied to human ESCs.
Embryoid body selection in defined medium.
This protocol has been used extensively by many groups and actually combines use of an embryoid body intermediate, in which cells of all three primary germ layers arise, followed by a neural lineage-specific selection step (Okabe et al.,1996; Guan et al.,2001; Wobus et al.,2001). This approach begins with early stage embryoid bodies. These are then plated on adhesive substrates in a minimal serum-free medium, ITSFn, which contains insulin, transferrin, selenium, and fibronectin (Table 1; Fig. 1, protocol 3). During several days of culture, non-neurectoderm cells, including undifferentiated ESCs, extraembryonic endoderm and mesoderm derivatives, die, leaving a neurectoderm-derived neural progenitor population, based upon expression of nestin, Sox1, Sox2, and other NSC markers (Okabe et al.,1996; Carpentino and Grabel, unpublished observations). Subsequent plating on laminin substrates in the presence of FGF2 promotes NSC proliferation, and at this stage cultures may be 80–95% NSCs (Okabe et al.,1996). NSCs generated using this approach have been used to produce a variety of specific neural and glial derivatives, including oligodendrocytes (Brustle et al.,1999), dopaminergic neurons, and glutamatergic neurons (Lee et al.,2000; Bibel et al.,2004). The strength of this method is the relative purity of the final product and the ability to generate large numbers of NSCs upon FGF2 addition, whereas weaknesses include the extended time it takes to generate NSCs, 10 days to two weeks, and the variability in the quality of the final product, which can be contaminated with mesodermal derivatives, likely due to the multiple steps. An accelerated version of this approach, which combines the selection and proliferation steps, produces a similar end product (Colombo et al.,2006). The embryoid body selection protocol has also been applied to the derivation of neurons from human as well as mouse ESCs (Carpenter et al.,2001; Zhang et al.,2001).
The Default Pathway
Based on work in nonmammalian species, particularly Xenopus and chick, a default hypothesis emerged for neural specification, proposing that the absence of signals mediates the decision to become neural vs. non-neural. Several studies support this hypothesis for ESC neural differentiation as well, but as observed for Xenopus and chick, the story appears to be more complicated than a simple default mechanism (Stern,2005). Positive factors are also required, and once the neural transition is achieved, signals are necessary to promote the survival and proliferation of these committed cells. In addition, there appear to be multiple intermediate stages between ESC and NSC, based upon both the expression of stage-specific markers and the distinct response of cells at different stages to extracellular signals.
The experiments of Hilde Mangold and Hans Spemann identified a region of the gastrula stage amphibian embryo, the dorsal lip of the blastopore, with the ability to induce ectopic neural tissue in cells otherwise fated to become epidermis (Spemann and Mangold,1924). Similar organizer-like regions were subsequently identified in many other species: Hensen's node in chick (Waddington,1936), the shield in teleosts (Luther,1935; Oppenheimer,1936), and the node and AVE in the mouse (Waddington,1936; Beddington and Robertson,1998). The identification of the dorsal lip of the blastopore led to several decades of unsuccessful attempts to establish the molecular nature of the induction signal. Finally, in the 1990s, a story began to emerge identifying several inhibitors of transforming growth factor-beta (TGF-β) family members, particularly BMP4, as neural inducers. The spatial and temporal pattern of expression of these inhibitors, including chordin, noggin, and follistatin, was also consistent with their role in neural induction (Smith and Harland,1992; Sasai et al.,1994,1995). These data suggested that simply antagonizing BMP signaling could promote neural specification, and several lines of experimentation supported this notion, including the observation that isolated animal caps dissociated to single cells to minimize signaling events, could form neural tissue after re-aggregation (Born et al.,1989; Godsave and Slack,1989; Sato and Sargent,1989). The story is not that simple, however, with additional factors now determined to play positive, as well as negative, roles in promoting neural fate. Several lines of investigation have established a positive role for FGFs in neural induction, both by down-regulating BMP targets, and by a BMP-independent mechanism (De Robertis and Kuroda,2004; Linker and Stern,2004). Also, several studies demonstrated an inhibitory role for Wnt signaling and a positive inductive role for antagonists of this pathway (Wilson et al.,2001). In contrast, some studies suggest Wnt signaling promotes neural induction (Baker et al.,1999). Whether this pathway acts to inhibit or promote neural differentiation depends upon the time of exposure to the signal and concentration of the signal present.
Given the evidence for a default pathway of neural induction, in which the absence of signals promotes this lineage specification, several approaches for promoting neural differentiation of ESCs include a selection step of culture in a serum-free, nutrient-poor neurobasal medium. These media were initially developed for culturing pure populations of NSCs derived from embryonic or adult neurogenic brain regions (Reynolds and Weiss,1996). These conditions are successful at supporting enrichment for NSCs by promoting the selective apoptosis of non-neural derivatives and excluding inhibitory factors, such as BMPs, present in serum. In particular, culture at low density under LIF-free, serum-free conditions promotes apoptosis of Oct4-positive ESCs (Tropepe et al.,2001; Barberi et al.,2003; Yamane et al.,2005). NSCs are more resistant to these conditions, perhaps due to autocrine signals.
Direct differentiation: culture at very low density in defined medium.
A series of studies from the van der Kooy group have demonstrated the transition of ESCs to NSCs at very low clonal densities (1–20 cells/well) under feeder-free conditions in serum-free defined medium (Tropepe et al.,2001; Table 1; Fig. 1, protocol 5). This differentiation occurs directly without embryoid body formation and includes the differentiation of an intermediate cell type, the primitive NSC. These studies use a neurosphere-based suspension assay for production of NSCs. ESCs first generate LIF-dependent primitive neurospheres that contain nestin-positive primitive NSCs that continue to express Oct4. These mature into LIF-independent, FGF/endothelial growth factor (EGF) -dependent neurospheres, that contain nestin-positive, Oct4-negative definitive NSCs. LIF is required at early stages of primitive NSC differentiation, most likely for survival of differentiating ESCs and to prevent neural differentiation. In addition, primitive neural stem cell differentiation is inhibited by BMP4 and promoted by the BMP antagonist noggin (Smukler et al.,2006).
The best evidence for a default mechanism of NSC differentiation comes from the observation that 80% of ESCs plated at low cell densities in phosphate buffered saline (PBS) begin to express nestin and Sox-1 within 4 hours (Smukler et al.,2006). Most of these cells, however, die due to the absence of conditions to support their survival and proliferation. This protocol, therefore, provides an excellent system to assay putative survival and proliferation factors. Forebrain and hindbrain markers have been observed in neuronal derivatives of these NSCs and neurons, astrocytes, and oligodendrocytes have been generated from these neurospheres (Tropepe et al.,2001). The differentiation of primitive NSCs from ESCs, however, is a rare event under these conditions, with only 0.2% of ESCs plated going on to form sphere-forming colonies in defined medium. This result suggests that this method is not an efficient means of producing NSCs, or their derivatives.
Direct differentiation: feeder-independent ESCs cultured at moderate density.
Direct differentiation of ESCs to NSCs can also be obtained under serum-free conditions in monolayer culture at moderate cell densities, but is most effective using ESCs adapted to growth under feeder-free conditions (Table 1; Fig. 1, protocol 6, Fig. 2B). This differentiation protocol was established by the Smith laboratory using the mouse ESC line 46C, which expresses green fluorescent protein (GFP) under the control of the promoter for the neurectoderm and NSC marker gene Sox1, providing an easy readout for the NSC transition in live cultures. Cells are plated at around 1 × 104/cm2 on gelatin-coated dishes in the absence of LIF in serum-free defined medium (N2B27). By 5 days, up to 75% of the cells express GFP as well as nestin. The transition to NSCs is not homogeneous and is accompanied by the death of significant numbers of GFP-negative cells in the cultures, suggesting that these conditions are effective at NSC production, at least in part due to selective survival of these cells. Surviving GFP-positive cells form rosettes in which cells elongate and align radially, in a manner reminiscent of neural tube formation, as well as ectoderm and neurectoderm differentiation in ESC embryoid bodies.
BMP signaling appears to inhibit, while FGFs support NSC production in this system. Addition of BMP4 inhibits the transition to Sox1-GFP-positive NSCs, whereas treatment with the BMP antagonist noggin promotes it. Several different neural derivatives have been generated using this method. The advantages of this protocol include the relative rapid direct emergence of NSCs (4–6 days), and the ability to isolate a pure NSC population using FACS (Chung et al.,2006; Fukuda et al.,2006). In addition, in 5 days, 10,000 ESCs can produce 1–5 million cells, 70% of which are NSCs, making this an efficient means of NSC production. Disadvantages include the presence of other cell types and reliance upon a feeder-independent ESC line for maximal efficiency of NSC production, but the purity of the NSC population can be improved through trypsin-based passage (Conti et al.,2005). The NSC lines derived in this manner retain pluripotency and are able to differentiate into neurons, astocytes, and oligodendrocytes (Glaser et al.,2007). This direct differentiation protocol has been adapted for use with human NSCs (Zhang et al.,2001).
Stromal cell co-culture.
The concept of the stem cell niche is based largely on studies of hematopoietic stem cells indicating that their survival, proliferation, and differentiation rely upon interaction with bone marrow stromal cells (Kaushansky,2006). Surprisingly, these cells also have the ability to direct the differentiation of NSCs from both mouse and human ESCs. ESCs are plated at clonal densities on stromal cell lines (Table 1; Fig. 1, protocol 4). A variety of different stromal lines have proven effective. By day 6, NSC differentiation is apparent, based on expression of a variety of markers, including neural cell adhesion (NCAM) molecule and nestin (Kawasaki et al.,2000; Barberi et al.,2003). It is key to the success of this co-culture approach that the cells be plated at low density to minimize cell–cell interactions and that serum be removed from the medium and serum replacement used in its place, suggesting that serum contains an inhibitor of the NSC transition, a conclusion supported by the observation that BMP4 suppresses neural differentiation under these conditions. The identity of the factor, or factors, provided by the stromal cells, be it permissive or instructive, has not yet been elucidated, but studies suggest that both a soluble factor and direct contact between ESCs and stromal cells may be required, because fixed feeder cells retain neural inducing activity (Kawasaki et al.,2000). Advantages of this protocol include the relatively pure population of multipotent neural derivatives that can be induced to produce neural subtypes with high efficiency, and the absence of an embryoid body intermediate, allowing generation of NSCs in a few days. NSCs generated using this approach have been used to produce many neural derivatives, including dopaminergic and γ-aminobutyric (GABA) acid-ergic neurons (Kawasaki et al.,2000; Barberi et al.,2003) by treatment with region-specific signals.