Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells

Stem Cell Research (2012) 8, 416–426

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REVIEW

Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells☆ Adam L. Goulburn a , Edouard G. Stanley b , Andrew G. Elefanty b , Stewart A. Anderson a,⁎ a

Department of Psychiatry, Weill Cornell Medical College, Box 244, 1300 York Avenue, New York, NY 10065, USA Monash Immunology and Stem Cell Laboratories, Building 75, STRIP1, West Ring Road, Monash University, Clayton, Victoria, 3800, Australia b

Received 2 July 2011; received in revised form 29 November 2011; accepted 3 December 2011 Available online 13 December 2011 Contents Introduction . . . . . . . . . . . . . . . . . . . . . Emergence and regionalization of the mammalian Neural induction of ESCs . . . . . . . . . . . . . . Generation of cortical interneurons from ESCs . . Therapeutic applications of cortical interneurons Conclusion . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

. . . . . . forebrain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Efforts to differentiate embryonic stem cells (ESCs) toward neurons and glia has primarily focused on generating motor neurons, midbrain dopaminergic neurons or oligodendrocytes ☆ Funding support. This work was supported by grants from the Australian Stem Cell Centre, the National Health and Medical Research Council (NHMRC) of Australia and the Juvenile Diabetes Research Foundation. EGS and AGE are Senior Research Fellows of the NHMRC. SAA acknowledges the support from the National Institutes of Health (NIH), The Starr Foundation and NYSTEM. ⁎ Corresponding author. E-mail addresses: [email protected] (A.L. Goulburn), [email protected] (E.G. Stanley), [email protected] (A.G. Elefanty), [email protected] (S.A. Anderson).

1873-5061/$ - see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.scr.2011.12.002

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to target neural afflictions such as amyotrophic lateral sclerosis (ALS), spinal cord injury, and Parkinson's disease. Over the last few years, studies have emerged exploring the generation of forebrain interneurons from mouse (m) and human (h) ESCs. Multiple lines of evidence implicate dysfunction of forebrain interneurons in the symptomatology of major neuropsychiatric illnesses, including schizophrenia, autism, Tourette disorder, bipolar disorder, and epilepsy. This dysfunction involves distinct subtypes of interneurons, which differ in their neurochemistry, connectivity, and physiological characteristics. There has been a proliferation of differentiation methods directed at producing putative cortical interneurons from ESCs. The purpose of this review is to present the progress made in generating GABAergic cortical interneurons from ESCs and to outline the implications of this research for treating neuropsychiatric disease.

Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells

Emergence and regionalization of the mammalian forebrain After gastrulation, the region of ectoderm induced to acquire neural character gives rise to the neural plate, an epithelium that subsequently forms the central and most of the peripheral nervous systems. Following neural plate formation, cellular movements result in the formation of bilateral neural folds (O'Rahilly et al., 2006), which flank an interceding neural groove, the first identifiable morphological structure in the developing nervous system (~ E7–8 in mouse, ~E16–18 in humans) (O'Rahilly et al., 2006). The lengthening neural plate is broader rostrally, where the three major brain divisions of the brain – prosencephalon, mesencephalon and rhombencephalon – are identifiable. The prosencephalon or forebrain is further subdivided into the telencephalon and diencephalon (Fig. 1A). Studies in mice demonstrate that the most rostral end of the neural plate undergoes extensive proliferation and morphogenetic movements, transforming the telencephalon into a set of paired vesicles with regionally restricted

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dorsoventral markers including Dlx1/2, Nkx2.1, Gsx2, Lhx6 and Pax6 (~E12–13 in mouse) (Fig. 1B) (Rallu et al., 2002a). In a combinatorial arrangement, these markers herald the subsequent emergence of discrete proliferative zones along the dorsoventral axis of the telencephalon (~ E12–14 in mouse) (Fig. 1B). The pallial telencephalon can be divided into the anterior/lateral neocortex and the posterior/medial hippocampus, cortical hem and choroid plexus. Similarly, the pallidal or subpallial telencephalon gives rise to the preoptic area, medial ganglionic eminence (MGE), the lateral ganglionic eminence (LGE) and the caudal ganglionic eminence (CGE) (~ E12–14 in mouse) (Fig. 1) (Hebert and Fishell, 2008; Marin and Rubenstein, 2001; Nat and Dechant, 2011; Wonders and Anderson, 2006). Pivotal to initial patterning of the telencephalon is the transcription factor Foxg1, with specification of the telencephalic primordium dictated by the regionalized expression of this transcription factor (Hebert and Fishell, 2008). Subsequent ventral patterning of the telencephalon primarily relies on Shh. Shh is first expressed in the underlying axial mesoderm (prechordal plate and notochord) before being

Figure 1 Regionalization of the key proliferative zones within the embryonic mouse forebrain. (A) Sagittal section through the brain of an ~ E12.5 mouse showing the main subdivisions of the forebrain, the telencephalon and the diencephalon. Anterior is to the left. (B) Coronal hemisection through the telencephalon highlighting the approximate expression patterns of selected transcription factors that are implicated in regulating telencephalic patterning and differentiation. *Gli3 expression extends into the subpallium, however, the highest expression is found within the pallium. **At this developmental age Pax6 expression continues into the dorsal-most region of the LGE (hatched lines) (Corbin et al., 2003). (C) Telencephalon of an ~ E12.5 mouse indicating some of the main domains. The interaction between Shh (ventral) and Gli3 (dorsal) establishes the dorsoventral (D–V) axis in the telencephalon. (D) Schematic showing the primary location for precursor cells that generate neurons expressing the neurotransmitters glutamate, GABA and acetylcholine. Cholinergic neurons of the striatum (Str) and the globus pallidus (GP) are also generated from within the MGE (patches of dots) with GP cholinergic neurons emerging from the ventral-most domain of the MGE. Also shown are the primary migration pathways of precursors that populate different telencephalic domains. The tangential migration of progenitor cells from both the LGE and MGE to the cortex, which occurs perpendicular to the radial migration observed within cortical domains. Anterior–posterior (A–P) and dorsal–ventral (D–V) axis shown. Abbreviations: POa, anterior preoptic area; Cx, cortex; H, hippocampus.

418 restricted to the ventral territories of the prosencephalon itself, specifically the MGE, and the hypothalamus (Liu and Niswander, 2005; Rallu et al., 2002a). Shh is essential for normal ventral telencephalic development with the morphogen promoting the emergence of ventral domains by antagonizing the dorsalizing effects of the zinc finger protein Gli3 (Fig. 1B) (Hebert and Fishell, 2008). In this signaling pathway, Shh represses the conversion of Gli3 from the activator (Gli3) to the repressor isoform (Gli3R) (Hoch et al., 2009; Sousa and Fishell, 2010). With the Gli3 expression domain extending into the subpallium, it has been suggested that it is the ratio between the two Gli3 isoforms that is important to dorsoventral telencephalic patterning (Fig. 1B) (Fotaki et al., 2006). Partitioning of the dorsal telencephalon centers on the paired-box transcription factor Pax6. At the neural plate stage in the mouse, Pax6 expression is observed throughout the telencephalic anlagen, with later expression restricted primarily to the dorsal pallium neocortex domain (Pax6 expression has been shown to extend into the dorsal-most region of the LGE) (Fig. 1B) (Manuel and Price, 2005; Rossant and Tam, 2002). Therefore, regionalized expression of Pax6 is important in defining the pallial–subpallial boundary that divides the ventral cortical primordium from the dorsal domain of the subpallium (Hebert and Fishell, 2008). (See (Hebert and Fishell, 2008) for a more detailed review on the early genetics of telencephalic development). In mice, the ganglionic eminences (GEs) of the subpallium are the primary sites from which cortical inhibitory γ aminobutyric acid (GABA)-ergic interneurons are generated (Figs. 1C, D). These interneurons undertake a tangential migration from the ventricular zones of the subpallium before integrating in the laminar layers of the cortex (Figs. 1C, D) (Corbin and Butt, 2011; Marin and Rubenstein, 2001). Likewise, there is strong evidence to suggest that human interneurons also originate in the ventral telencephalic MGE (Batista-Brito and Fishell, 2009; Fertuzinhos et al., 2009; Letinic et al., 2002). However, unlike rodents, primates may have an additional location of interneuron genesis in the subventricular zone of the cerebral cortex (Fertuzinhos et al., 2009; Letinic et al., 2002; Petanjek et al., 2009). Depending on the scheme of classification, cortical interneurons comprise at least 15 different subtypes (BatistaBrito and Fishell, 2009; Moore et al., 2010 and see Ascoli et al., 2008 for a categorization of cortical interneuron subtypes). GABAergic cortical interneurons critically balance activity of functioning excitatory glutamatergic projection neurons in the neocortex; a balance that is necessary for the proper functioning of neuronal networks (reviewed in Batista-Brito and Fishell, 2009 and Corbin and Butt, 2011). Defects in the development and migration of GABAergic interneurons from the GE domains have been implicated in neuropsychiatric illnesses such as epilepsy, autism and schizophrenia (Batista-Brito and Fishell, 2009; Levitt et al., 2004).

Neural induction of ESCs There are two commonly used methods for directed ectoderm differentiation of ESCs. The first employs adherent ‘flat-cultures’ in which ESCs are differentiated either on

A.L. Goulburn et al. extra cellular matrix-coated surfaces or on neural inducing cell layers, while in the second protocol, ESCs are differentiated as three-dimensional aggregates, termed embryoid bodies (EBs) (Fig. 2) (Erceg et al., 2009; Keller, 2005). Ectodermal flat-culture differentiation systems frequently use inductive bone marrow-derived stromal cell layers, such as MS5 (Aubry et al., 2008; Barberi et al., 2003; Ideguchi et al., 2010) or the calvarial marrow-derived line PA6 (Fig. 2) (Irioka et al., 2005; Kawasaki et al., 2000; Watanabe et al., 2005). The neural inducing activity associated with PA6 cells has been termed ‘stromal cell-derived inducing activity’ (SDIA) (Kawasaki et al., 2000). Consistent with established embryological theories concerning neural induction, initial experiments indicated that SDIA impinged on BMP signaling, although the exact mechanisms of action and the molecular identity of SDIA are still unknown (Kawasaki et al., 2000). Nevertheless, ESC co-culture with stromal cell lines has derived various neural cell types including GABAergic neurons, dopaminergic neurons, spinal motoneurons and peripheral sensory neurons (reviewed in Erceg et al., 2009). The generation of a variety of neural sub-lineages also reflects a key limitation of stromal feeder co-culture differentiations. Despite the use of exogenous growth factors to direct differentiation, control over the inducing activities of the stromal layer is limited, predisposing the system to heterogenous differentiation outcomes. In addition, stromal layers introduce the risk of pathogen transfer between cell lines, an issue that would need to be addressed prior to the use of neural derivatives for clinical applications. Strategies have been developed for directing neural ESC differentiation in stromal-free media supplemented with specific growth factors in a time and concentration dependent manner to influence the course of the neural differentiation (reviewed in Erceg et al., 2009; Denham and Dottori, 2009 and Petros et al., 2011). These procedures have allowed the generation of a variety of neural lineages and specific neuronal subtypes, including midbrain dopamine neurons, spinal motor neurons and cortical projection neurons (Gaspard et al., 2008; Lee et al., 2000; Wichterle et al., 2002). Many protocols include the inhibition of BMP, Nodal and Wnt signaling pathways to achieve neural induction (Fig. 2) (for review see Gaspard and Vanderhaeghen, 2010). To this end, signaling inhibitors such as Noggin, Lefty (Lefty1, Lefty2, or LeftyA) and Dickkopf-1 (Dkk-1) have all been used to promote ESC differentiation to early neurectodermal derivatives (Eiraku et al., 2008; Pera et al., 2004; Perrier et al., 2004; Smith et al., 2008; Verani et al., 2007; Watanabe et al., 2005; Watanabe et al., 2007). These inhibitors block mesendoderm formation and permit the adoption of a default neurectodermal differentiation program. In mESCs, Kamiya and colleagues have shown that the zinc-finger nuclear protein, Zfp521, is an important BMP molecular regulator during early neurectodermal specification (Kamiya et al., 2011). In addition, recent work suggests that fibroblast growth factor-2 (FGF2) can also enhance neural induction, acting at a stage of differentiation akin to primitive ectoderm (epiblast) (Cohen et al., 2010). However, it is also important to bear in mind the species differences that may exist in the emergence of neuroepithelial/neuroectoderm (NE) precursors. For example, recent studies by Zhang and

Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells

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Figure 2 Differentiation of ESCs towards neural lineages. Undifferentiated ESCs can be differentiated with or without the use of a co-culture stromal layer. Stromal layer free differentiation platforms employ either a flat culture system using various tissue culture surface coatings or three-dimensional embryoid body (EB) aggregates. Generally, neural induction involves the use of mesendoderm signaling antagonists/inhibitors in order to transition undifferentiated ESCs towards an anterior neural precursor fate marked by the expression of genes including Otx2, Nestin and Pax6. Recent studies have indicated that FGF2 may also contribute to neural induction. Subsequently, these neural precursors can be directed towards regionalized neural cell types using morphogens and growth factors. For example retinoic acid (RA) has been shown to generate caudal cell types, Wnt genes have been implicated in producing dorsalized cells, and Shh application has been demonstrated to induce ventral cell phenotypes. For effects of specific growth factors refer also to recent reviews (Erceg et al., 2009; Gaspard and Vanderhaeghen, 2010).

colleagues revealed a novel role for PAX6 in early human, but not mouse NE specification (Zhang et al., 2010). Their study demonstrated that PAX6 was a transcriptional determinant of human NE and that distinct isoforms coordinated the transition from pluripotency to the NE fate by inhibiting pluripotent and activating NE expressed genes respectively (Zhang et al., 2010). After neural induction, a second series of growth factors has been used to direct differentiation towards neural subtypes (Fig. 2). For example, early studies suggested that continued exposure to FGF favored neuronal differentiation, while formation of glial cells was promoted by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) (Barberi et al., 2003; Reubinoff et al., 2001). Reubinoff and colleagues also reported that the extra-cellular matrix components, laminin and fibronectin, influenced the balance between neuronal or glial differentiation (Reubinoff et al., 2001). Furthermore, several factors have been shown to direct ESC-derived neural precursors towards region specific cell types, including retinoic acid (RA) (caudal, posterior), sonic hedgehog (ventral) and Wnts (dorsal) (reviewed in Erceg et al., 2009).

Despite the success of protocols in initiating neural induction from ESCs, the large-scale application of these methodologies is constrained by the high cost of recombinant growth factors. Because of this, recent studies have explored the use of small molecules to induce step-wise transitions from undifferentiated ESCs toward neural cell types (Ding and Schultz, 2004; Ding et al., 2003). In this context, Chambers and colleagues demonstrated that hESCs could be efficiently converted into neural derivatives by employing a combination of the BMP inhibitor Noggin and the small molecule Activin signaling antagonist, SB431542 (Chambers et al., 2009). In this system, Noggin and SB431542 collaborate to block the SMAD signaling required for mesendoderm formation, thus promoting neural differentiation (Chambers et al., 2009).

Generation of cortical interneurons from ESCs Within the developing forebrain, cells are identified based on their regional, morphological, and positional characteristics in combination with their gene expression patterns.

420 These attributes vary during the course of development, indicating that components contributing to the identification of a specific cell are also time dependent. However, the critical inputs of embryonic positional information are not available in an in vitro culture. Therefore, assignment of cellular identity relies more heavily on the gene expression profile of the cell in question, as well as aspects of its developmental history. In the case of hESC-derived cell types, detailed knowledge of the changes in gene expression during the genesis of human neural lineages is extremely limited. As a consequence, classification of such cell types is based upon the gene expression profiles of ‘corresponding’ cells within the developing mouse brain. This approach, however, does not account for potential species differences in the biology of the human and mouse forebrain development. With these caveats in mind, Fig. 3 summarizes key genes whose expression may be utilized to classify forebrain cells generated from differentiating ESCs. Differentiating ESCs adopt an anterior (rostral) neural character both in the absence of exogenous growth factor stimulation and also upon the blockade of mesendoderm signaling by exogenously added inhibitors (Elkabetz et al., 2008; LaVaute et al., 2009; Pankratz et al., 2007; Smukler et al., 2006; Wataya et al., 2008; Zhang et al., 2010) (for

A.L. Goulburn et al. reviews see Gaspard and Vanderhaeghen, 2010 and Nat and Dechant, 2011). This ESC model of neural differentiation is consistent with the classical embryological view of neural induction, whereby the first neural fate adopted is of anterior character. This is subsequently respecified to a posterior fate by signals emanating from more caudal domains of the developing embryo (Gaspard and Vanderhaeghen, 2010). Interestingly, and in contrast to these previous ESC reports, a study by Peljto and colleagues reported that in the absence of any added inductive factors it was possible to generate a subset of Hb9-expressing caudal neurons from mESCs (Peljto et al., 2010). However, as previously mentioned, it is important to note that differences exist between the neural commitment and differentiation of mESCs and hESCs and it is not clear whether this result will also hold true for hESC neural differentiation. Studies to derive cortical interneurons have centered on guiding ESCs toward ventral telencephalic precursors, primarily defined by the co-expression of Foxg1 and Nkx2.1 (Fig. 3). These progenitors could be further differentiated to neurons that expressed the neurotransmitter GABA (Barberi et al., 2003; Watanabe et al., 2005; Watanabe et al., 2007). Initial neural induction was achieved by blocking mesendoderm formation with inhibitors of Wnt, BMP or Nodal signaling. Once

Figure 3 Key genes whose expression distinguishes forebrain lineages derived from differentiating ESCs. ESCs are differentiated towards an anterior neuroepithelial (NE) precursor marked by Otx2, Six3 and Lhx2. Alternatively the upregulation of Hox genes (Hoxb4, Hoxb9) is diagnostic of more posterior cell types (gray). Foxg1 is employed as a marker of telencephalic identity (red), with the cell surface marker Forse1 also predominantly marking emerging telencephalic precursors. Nkx2.1 expression in conjunction with Foxg1 is thought to mark ventral telencephalic cells that can generate GABA + neurons. Foxg1 + cells lacking Nkx2.1 expression resemble dorsal telencephalic (cortical) cells. GABA + cells generated from ventral telencephalic Nkx2.1 + precursors contribute to cortical tissues, with Nkx2.1 expression downregulated (dotted line). Nkx2.1 +Foxg1 − cells are likely to represent diencephalic hypothalamus progenitors (green) capable of generating TH + and GABA + neurons. Nkx2.1 cells can also give rise to telencephalic-derived oligodendrocytes (orange). In this schematic, these cells are assumed to derive from Foxg1 + cells, although formal proof is lacking because Foxg1 expression was not used to identify ESCs differentiating towards forebrain-derived oligodendrocytes.

Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells established, the resultant anterior neural cell population was ventralized using Shh (Li et al., 2009; Watanabe et al., 2005; Watanabe et al., 2007). Examination of the differentiated cultures revealed a range of neural cell types expressing GABA, GAD and DARPP32 (Fig. 3) (Aubry et al., 2008; Li et al., 2009; Watanabe et al., 2005). A number of stromal-free differentiation systems have been described for the generation of cortical interneurons from ESCs (Li et al., 2009; Watanabe et al., 2005; Watanabe et al., 2007). Watanabe and colleagues described a method to direct mESC differentiation towards telencephalic precursors using suspension EB cultures in serumfree media containing specific inhibitors and growth factors (Watanabe et al., 2005). In order to track the conversion of mESCs to a neural fate, the group employed mESCs in which green fluorescent protein (GFP) was inserted by homologous recombination to the locus of the early neurectodermal marker, Sox1. Their study indicated that the first 5 days of differentiation were critical for achieving efficient telencephalic differentiation (Watanabe et al., 2005). During this phase, mesendoderm signaling inhibitors (LeftyA and Dkk-1) promoted the formation of telencephalic progenitors, as measured by the emergence of cells expressing Foxg1 (Watanabe et al., 2005). Subsequent use of dorsalizing (Wnt3a) and ventralizing (Shh) factors induced expression of dorsal (Pax6) and ventral (Nkx2.1) markers respectively within this Foxg1 + population, although the lack of a specific tag to isolate viable Foxg1 + cells meant that it was not possible to unequivocally determine the precursor–progeny relationships between cell types (Watanabe et al., 2005). In this study, ~5–15% of the total cell population was Foxg1 +Nkx2.1 +, and could therefore be nominally classified as ventral telencephalic-like and ~5–8% of the cells were Foxg1 −Nkx2.1 +, probably representing a ventral diencephalic hypothalamic precursor (Watanabe et al., 2005; Wataya et al., 2008). Although the study by Watanabe also reported the emergence of GABA + neurons, it was not possible to discern whether these arose from Foxg1 +Nkx2.1 + (telencephalic) or Foxg1 −Nkx2.1 + (diencephalic) progenitors (Yee et al., 2009). Moreover, since there are large populations of telencephalic GABAergic neurons that are not of an Nkx2.1-lineage, such as medium spiny projection neurons of the striatum, or nearly all GABAergic interneurons of the olfactory bulb, these populations may be present as well. In order to more clearly define the emergence of ventral telencephalic derivatives from mESCs, Maroof and colleagues used a transgenic Lhx6 reporter cell line to visualize and isolate cortical interneuron precursors (Maroof et al., 2010). A direct target of Nkx2.1 (Du et al., 2008), Lhx6 is expressed in most MGE-derived interneurons from the time of the cell cycle exit through postnatal maturation (Lavdas et al., 1999; Liodis et al., 2007). Employing a GFP reporter regulated by the Lhx6 promoter (Maroof et al., 2010), the authors identified early subpallial derivatives in vitro (Fig. 4A) and investigated their in vivo potential. Their differentiation protocol used Noggin to initiate neural induction, and regionalized cell types were generated through the application of specific factors including insulin growth factor-1 (Igf1), Fgf2 and Shh. Transplantation experiments showed that the Lhx6::GFP + cells could migrate extensively in the tangential plane, a feature of MGE-derived precursors when compared to transplants of other forebrain regions

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(Wichterle et al., 2001; Wichterle et al., 2003). In addition, they differentiate into GABA + cortical interneurons expressing subgroup markers somatostatin (SST) or parvalbumin (PV), and displayed the corresponding SST or PV interneuron-like electrophysiological characteristics. However, with only 2% of total cells expressing Lhx6::GFP, further protocol optimization will be necessary in order to generate higher yields of interneuron precursors. In 2007, Watanabe and colleagues transferred their mESC differentiation regimen to hESCs, using the Rho-associated kinase (ROCK) inhibitor to reduce the apoptosis associated with single cell dissociation of hESCs (Watanabe et al., 2007). Neurectoderm induction was achieved by blocking mesendoderm formation with inhibitors for BMP (BMPRIAFc), WNT (DKK-1) and NODAL (LEFTYA) signaling during the first 24 days of the differentiation (Watanabe et al., 2007). This resulted in ~ 30% of total cells expressing FOXG1 at day 35, of which ~96% were PAX6 + (dorsal) and less than ~1% were NKX2.1 + (ventral) (Watanabe et al., 2007). However, treatment with SHH from days 24–35, increased the proportion of NKX2.1 + cells to ~40% of the FOXG1 + cells (Watanabe et al., 2007). Li and colleagues reported that the coordination of SHH and WNT signaling determined ventral and dorsal telencephalic fates in differentiating hESCs cultures (Li et al., 2009). The protocol reported in this study included a physical separation step in which ‘definitive neuroepithelial’ cells (Li et al., 2009; Pankratz et al., 2007; Zhang et al., 2001), marked by the appearance of neural rosette structures, were purified from surrounding non-neural cells (Zhang et al., 2001). Similar to other studies, telencephalic commitment was assigned on the basis of FOXG1 expression relatively late in the differentiation program (day 31) (Watanabe et al., 2007; Li et al., 2009). Li and colleagues concluded that, in the absence of exogenous morphogens, telencephalic cells from differentiating hESCs adopted a dorsal fate primarily through activation of the WNT signaling pathway and its impact on the ratio of the GLI3 activator/repressor isoforms (Li et al., 2009). This conclusion was supported by the finding that inhibiting WNT signaling, or addition of SHH, could result in ~85% of the total population expressing NKX2.1 (Li et al., 2009). End-stage GABA + cells were generated in these differentiating cultures, however as with the study performed by Watanabe, whether these GABA + neurons were derived from FOXG1 +NKX2.1 + precursors could not be determined. To further highlight this point, Maroof et al., noted widespread expression of GABA throughout their differentiation cultures, demonstrating that while all Lhx6::GFP + cells were GABA +, not all GABA + cells were LHX6::GFP +. They concluded that GABA expression alone could not be used as a definitive marker of ventral telencephalic-derived cortical interneurons (Maroof et al., 2010). In the ESC studies described above, two general principles are apparent. First, neural differentiation was achieved through inhibition of mesendoderm induction pathways, implying that, within each culture system, endogenous mesendoderm promoting signals were present that required inhibition. Second, after neural induction, the ‘naïve’ neural progenitors were patterned using a second round of treatment with dorsalizing factors such as Wnt3a or ventralizing factors including Shh.

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Figure 4 Genetically engineered reporter lines assist in the identification of telencephalic cells differentiated from ESCs. (A) Maroof et al., demonstrated the ability to track in vitro the onset of Lhx6 expression within a mixed neural culture using a transgenic Lhx6::GFP mESC line. (B) Goulburn et al., reported the generation of a human ESC line in which GFP was inserted into the NKX2.1 locus using homologous recombination. This line was used to isolate and further differentiate NKX2.1 expressing cells, and subsequently demonstrate a precursor-progeny relationship between hESC derived NKX2.1+ progenitors and GAD (GABA) expressing neurons in vitro.

We have recently reported the generation of an NKX2.1 GFP/w targeted hESC line using homologous recombination (Goulburn et al., 2011). Using the reporter line, we showed the emergence of NKX2.1-GFP + ventral forebrain progenitors within differentiating ‘spin EB’ cultures using a serum-free defined medium (Fig. 4B) (Ng et al., 2008a; Ng et al., 2008b). Neural induction was achieved without the use of exogenous mesendoderm signaling inhibitors but rather by the combinatorial application of FGF2 and retinoic acid (RA) (Goulburn et al., 2011). Cultures consistently generated 5–15% NKX2.1-GFP + cells, with GFP + precursors emerging by day 14 of the differentiation. While the use of RA in the derivation of anterior neural cell types is contentious, there is evidence to support its role in ventral telencephalic patterning (Bissonnette et al., 2011; Schneider et al., 2001), and in forebrain GABAergic differentiation (Chatzi et al., 2011). The results in our study demonstrated both a time and concentration dependent requirement for RA in generating NKX2.1-GFP + cells, consistent with previous telencephalic ESC studies (Li et al., 2009; Watanabe et al., 2005). For example, Watanabe demonstrated that early treatment of mESCs (day 3–10) with RA had a caudalizing effect on cultures, whereas later treatment (day 6–10) did not (Watanabe et al., 2005). At these later treatment times, expression of anterior markers, Otx2 and Foxg1 persisted even with RA application (Watanabe et al., 2005). Likewise, Li and colleagues showed that RA treatment of neurally committed differentiating hESCs at day 17 had little effect and did not appear to induce expression of caudal genes nor result in the inhibition of FOXG1 expression (Li et al., 2005; Li et al., 2009). Nevertheless, it would be interesting to compare the transcriptional profiles and developmental

potentials of human NKX2.1-GFP + precursors generated using the multiple published protocols, especially given that there may be additional domains of NKX2.1 expression within the human forebrain. For example, although pallidal expression of NKX2.1 appears to be similar during mouse and human development, NKX2.1 is also strongly expressed in the human fetal cortex, in the proliferative zone, cortical plate and layer I (Fertuzinhos et al., 2009; Rakic and Zecevic, 2003; Zecevic et al., 2005). The NKX2.1 GFP/w hESC line enabled us to isolate viable NKX2.1-expressing cells, allowing the unequivocal identification of their progeny. NKX2.1-GFP + cells generated GABA + cells both in vitro and in vivo (Fig. 4B), and a small proportion of cells expressed somatostatin (SST), a marker of more mature cortical interneurons. Interestingly, NKX2.1-GFP + progenitors also generated tyrosine hydroxylase (TH +) cells indicating a potentially mixed telencephalic/diencephalic nature of the precursor population, although TH + GABAergic interneurons are also present in primate neocortex. While Watanabe and colleagues reported in their mESC study that approximately half of the counted Nkx2.1 population was Foxg1 −, our results showed that the RA-induced NKX2.1-GFP + cells emerged as FOXG1 −, before upregulating the transcription factor during extended culture of the harvested cells (Goulburn et al., 2011; Watanabe et al., 2005). Recently, Danjo and colleagues have demonstrated ventral telencephalic subregional specification of mESCs by employing patterning factors in a temporal and combinatorial manner (Danjo et al., 2011). Using a Foxg1::Venus knockin mESCs (Watanabe et al., 2005), their study demonstrated that low concentrations of the morphogen Shh at early

Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells time-points of addition generated telencephalic LGE cell derivatives. These LGE cell types could differentiate into mature medium-sized spiny striatal neurons both in vivo and in vitro (Danjo et al., 2011). In contrast, high concentrations of Shh or the Hedgehog agonist SAG at later time-points generated MGE and CGE cell subtypes (Danjo et al., 2011). Use of different FGF growth factors post Shh application was shown to alter the balance between MGE- and CGE-derived cortical interneuron subgroups (Danjo et al., 2011). The study also highlighted the importance of transplanting pure populations of differentiated cells in order to prevent tumor formation. The results of this study mirror the effects of Shh in mouse forebrain development (Rallu et al., 2002b), highlighting the profound effects that small increments in growth factor concentration may have on the generation of distinct telencephalic cell populations from which cortical interneurons are derived in a mESC setting. It will also be interesting to examine whether similar results hold true when applied to hESC differentiation regimes.

Therapeutic applications of cortical interneurons Cortical GABAergic interneuron precursors have the remarkable ability to migrate extensively and survive after transplantation into the postnatal cortex, making these cells an attractive candidate for use in cell-based therapy for seizures or other neuropsychiatric illnesses (Maroof et al., 2010). Several recent studies have highlighted the potential therapeutic benefits of embryonic MGE-derived precursors and the inhibitory interneurons that they generate. Martinez-Cerdeno and colleagues reported on the potential benefits of using embryonic MGE precursors for Parkinson's disease (Martinez-Cerdeno et al., 2010). In their study, E14.5 MGE precursors were transplanted into the striatum in a rat Parkinson's disease model (6-hydroxydopamine (6-OHDA)) with results demonstrating that the transplanted MGE cells integrated into the host circuitry and improved motor symptoms (Martinez-Cerdeno et al., 2010). Similarly, Baraban and colleagues utilized embryonic MGE precursors as potential treatments for epilepsy (Baraban et al., 2009). By grafting MGE precursor cells into the postnatal neocortex in a mouse model of epilepsy, Baraban and colleagues demonstrated an increase in GABA-mediated synaptic and extrasynaptic inhibition and a significant reduction in the frequency and duration of seizures (Baraban et al., 2009). Furthermore, other laboratories have demonstrated reduced seizure frequency following transplantation of MGE-derived cells into the cortex in various rodent models of epilepsy (Calcagnotto et al., 2010a,b; De la Cruz et al., 2011; Waldau et al., 2010; Zipancic et al., 2010). While these studies are preliminary, it is tantalizing to think that MGE precursors may be used as a cell therapy for a variety neurological and neurodevelopmental disorders. Therefore, generating sufficient MGE-like precursors from ESCs for transplantation, and thereby overcoming the limitations associated with tissue donation, could be of enormous benefit. However, with ESC-derived cortical interneuron research in its infancy, a more careful analysis is needed to better define and characterize the MGE-like progenitors. For example improvements

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in protocols to yield greater frequencies of precursor cells and long-term transplantation experiments to examine neuronal survival, migration and maturation need to be performed. That Maroof et al., could detect mES derived interneurons in mouse neocortex transplants 6 months following neonatal transplantation, bodes well for the potential therapeutic value of this system. The generation of ESC-derived MGE precursors also provides an opportunity to analyze homogenous populations of human inhibitory interneurons. Growth factors and compounds can be assayed to determine their capacity to preferentially direct interneuron subtypes, offering important insights into early human forebrain interneuron fate determination (McKernan et al., 2010). Compound libraries can be used to screen for agents that influence neuronal firing or inhibition, enhance synaptic functioning, or impact upon survival and proliferation of cortical interneurons. Such screens could lead to the identification of new cortical interneuron-associated therapeutics. Although not discussed in detail in this review, the emergence of iPSC technology will now permit the modeling of neuropsychiatric diseases in vitro (Cundiff and Anderson, 2011). For instance, mutations to the X-linked Aristaless related homeobox (Arx) gene have been implicated in improper migration of GABAergic interneurons from the subpallium proliferative zones to the cortex, potentially resulting in autism and epilepsy (Friocourt and Parnavelas, 2010). Similarly, a translocation involving the gene Disrupted in Schizophrenia-1 (DISC1), which appears to be crucial for intracellular mechanisms associated with neurite outgrowth, identifies this locus as a candidate involved in the genesis of schizophrenia (Kamiya et al., 2005; Pletnikov et al., 2008). Derivation of disease cell lines (for example see Brennand et al., 2011) from patients carrying such candidate gene mutations has the potential to result in greater understanding of disease etiology as well as enabling exploration of downstream signaling targets, resulting in the possible development of new therapeutics.

Conclusion Dysfunction of GABAergic interneurons of the cerebral cortex has been implicated in a variety of major neuropsychiatric illnesses, including schizophrenia, autism, anxiety, and epilepsy. However, both our imperfect knowledge of the development and function of human cortical interneurons, and our lack of understanding of the manner in which disease related genes may influence these processes, hinder our ability to understand, prevent or treat interneuron-related mental illness. HESCs represent an important source of human interneurons that could be used to map normal development, to explore genetic influences on interneuron development and function, and to study genetic–environmental interactions in forebrain development. ESCs, therefore, have great potential to enhance our understanding the molecular basis of neuropsychiatric illnesses and for the development of novel preventions and treatments, including cell therapies. Despite the tremendous potential of ESCs for understanding interneuron-related disease, major challenges remain to be solved before this promise can be realized. For example,

424 since distinct interneuron subtypes are differentially affected in various disorders (Lewis et al., 2011; Sibille et al., 2011), how will we generate these subtypes from human stem cells? Since key aspects of interneuron function depend not only on the presence of class-defining markers, but also on specific patterns of inputs, intrinsic activity, and axonal targeting onto other neurons, what assays will allow us to study these features? Second, how do we differentiate ESCs into distinct interneuron types, and what system can we use to define these subtypes outside of the human brain? The studies reviewed in this manuscript relating to human GABAergic interneurons differentiated from ESCs complement limited human embryological data. With most prior models for human forebrain specification based on the mouse, more recent investigations now serve to highlight both the similarities and disparities between mammalian species and remind us of the complexity of human prosencephalic development. We are confident that the ongoing studies of human ESC neural differentiation will result in continuing progress in unraveling the complexities of human cortical interneuron development.

Acknowledgments We would like to thank all the members of the Elefanty and Stanley Lab and the Anderson lab for their discussions regarding this review. In particular, we acknowledge Tim Petros for help with both forebrain migration schematics and Asif Maroof for the Lhx6 epifluorescence images.

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