Conversely, in the damaged brain, such as following ischemia, EGF promoted SVZ NPC proliferation with subsequent production of neuroblasts in SVZ and striatum (Ninomiya et al., 2006), also it induced migration of doublecortin positive precursors and their subsequent long term (13 weeks) survival as parvalbumin-expressing interneurons (Teramoto et al., 2003). A similar infusion following traumatic brain injury also induced SVZ proliferation, as well as SGZ proliferation, at early timepoints but did not promote longer term (4 weeks survival) of the newborn SGZ cells, many of which had differentiated into astrocytes rather than neurons (Sun et al., 2010). Similarly, in a rat model of ischemia EGF alone induced proliferation and some NSPC migration but did not promote regeneration unless combined with a later administration of erythropoietin (Epo) (Kolb et al., 2007), with similar results obtained for infusion of TGFα in a PD model (Cooper and Isacson, 2004). Part of the discrepancy between differentiation outcomes may lie in rat:mouse species differences. In general, mouse studies have indicated that EGF infusion can promote proliferation, migration and at least some neuronal differentiation while rat studies have shown proliferation, some migration and differentiation into glial cells, if they are found to differentiate at all. This may be due to EGF-induced dysplasia in rat but not mouse SVZ (Lindberg et al., 2012). In rats, the EGF-induced NSPC proliferative response generates SVZ/lateral ventricle polyps, which contain a higher percentage of cells expressing the transcription factors *Sox2*, *Olig2*, markers of proliferative NPCs, as well as Id1, an inhibitor of neuronal differentiation, concomitant with decreased expression of the neuroblast marker doublecortin. This suggests that in rat, EGF promotes proliferation of more glial fated NPCs that do not migrate to the same extent as mouse NPCs exposed to EGF. However, combination of EGF plus other growth factors appears to overcome some of the limitations of infusion of EGF alone to some extent.

## *EGF plus FGF2*

FGF2 is another potent NSPC mitogen, commonly used to induce proliferation of these cells in culture, usually in combination with EGF. Infusion of FGF2 by itself into the lateral ventricles of normal adult rats promotes NSPC proliferation and enhances neurogenesis in the OB (Kuhn et al., 1997). Following traumatic brain injury it has been shown to enhance SVZ and SGZ NSPC proliferation and increase the 4 weeks survival of newborn neurons (Sun et al., 2009), while FGF2 knockout mice show decreased hippocampal neurogenesis following seizure or ischemia (Yoshimura et al., 2001). In addition, conditional deletion of FGFR1, the major receptor for FGF2, decreases hippocampal neurogenesis and impairs memory consolidation (Zhao et al., 2007). Quite a few studies have used a combination of EGF and FGF2 infusion, which has generally promoted neural repair. In a rat global ischemia model, which led to loss of CA1 hippocampal neurons, EGF+FGF2 promoted proliferation of SVZderived cells which repopulated the damaged CA1 hippocampal neurons and promoted functional recovery (Nakatomi et al., 2002). Similar studies reported increased numbers of neural progenitors in hippocampus, as well as SVZ and hypothalamus (Oya et al., 2008). Comparable results were obtained following transient middle cerebral artery occlusion in rat, including increased neuronal numbers and survival (Tureyen et al., 2005). However, in an endothelin model of transient ischemia, while co-infusion of EGF+FGF2 promoted increases in SVZ neurogenesis, it decreased hippocampal neurogenesis and increased infarct volumes (Baldauf and Reymann, 2005) but the reasons for the different outcomes in these models is not clear. Increased proliferation and migration to dopamine deficient areas have also been observed following EGF+FGF2 infusion in a PD model (Winner et al., 2008).

## *Other factors that can promote NSPC proliferation*

Although EGF and FGF2 are possibly the most potent NSPC mitogens, particularly for SVZ-derived cells, other factors can also promote increases in NSPC numbers, although most have yet to be tested in brain injury/disease models. Infusion of ATP into the lateral ventricles of mice increased numbers of transit amplifying progenitor cells (Suyama et al., 2012). A mechanism by which this occurs may involve induction of FGF2 and TGF synthesis (Jia et al., 2011). VEGF can augment SVZ progenitor proliferation, as well as migration (Wittko et al., 2009; Calvo et al., 2011). Other factors have been shown to enhance proliferation induced by EGF and/or FGF2 without having a notable effect by themselves, such as growth hormone (GH) (McLenachan et al., 2009).

IGFs also play a role in several NSPC functions. IGF1 has different effects in the SVZ and dentate gyrus SGZ. Peripheral administration promotes hippocampal dentate gyrus NSPC proliferation and increases subsequent dentate gyrus neurogenesis (Aberg et al., 2000) and survival (Cheng et al., 2001). GH receptor knockout mice, which are serum-IGF1-deficient, do not show any alterations in hippocampal neurogenesis (Ransome and Turnley, 2008b), which may indicate that local hippocampal/dentate gyrus production of IGF-1 is important in supporting ongoing hippocampal neurogenesis (Sun et al., 2005a). However, peripheral administration of GH promoted hippocampal but not SVZ NSPC proliferation (Aberg et al., 2009). Indeed, in the SVZ IGF1 does not seem to regulate proliferation, rather being more important for NSPC migration; IGF1 null mice show an abundance of neuroblasts in the SVZ that have failed to migrate to the OB (Hurtado-Chong et al., 2009). IGF2 regulates proliferation in the dentate gyrus in an Akt-dependent manner (Bracko et al., 2012) and promotes survival of newborn hippocampal neurons as well as regulating hippocampal dependent fear extinction (Agis-Balboa et al., 2011).

## **NEUROTROPHINS**

Neurotrophin signaling mediated by BDNF interacting with TrkB receptors and to a much lesser extent NGF binding to TrkA, regulates several aspects of NSPC function. In the adult brain, TrkB expression and effects of BDNF are widespread but TrkA expression is very limited, primarily restricted to basal forebrain cholinergic neurons which project to the NGF-expressing hippocampus. Therefore, effects of BDNF in NSPC biology have been the most widely studied, although NGF infusion has been shown to promote the survival of normal newborn dentate granule cell neurons but not proliferation of their progenitors (Frielingsdorf et al., 2007). Intranasal delivery of NGF following focal ischemia in rats similarly did not promote proliferation of SVZ progenitors but enhanced subsequent newborn neuronal survival in the ipsilateral SVZ and injured striatum (Zhu et al., 2011).

*In vitro*, SVZ-derived neurospheres express TrkB and little/no TrkA or TrkC and addition of BDNF promoted a transient increase in newborn neuron numbers due to enhanced differentiation and neurite outgrowth, rather than a proliferative effect on the NSPCs (Ahmed et al., 1995). Further, BDNF is required for cultured hippocampal progenitor cells to adopt a neuronal fate (Bull and Bartlett, 2005).

In the dentate gyrus, TrkB is expressed predominantly by maturing but not proliferating neuroblasts (Donovan et al., 2008), which correlates with findings that BDNF is important for neuroblast migration, survival, and integration of new neurons. BDNF increases the number and survival of newborn neurons in the SVZ and OB (Kirschenbaum and Goldman, 1995; Zigova et al., 1998; Bath et al., 2008), in a p75 neurotrophin receptordependent manner (Young et al., 2007), and in striatum, caudate putamen and septum (Benraiss et al., 2001; Henry et al., 2007), dentate gyrus (Lee et al., 2002) as well as subcallosal neurons (Kim et al., 2011b). Knockdown of TrkB receptors and disruption of BDNF signaling resulted in decreased SGZ NSPC proliferation and neurogenesis, indicating that TrkB signaling may also have some proliferative effects in these cells (Li et al., 2008). Disruption of BDNF signaling also results in shorter dendrites and reduced spine formation, culminating in a lack of survival of newborn granule cells (Bergami et al., 2008; Gao et al., 2009a). In addition, conditional deletion of BDNF resulted in increased death of newborn neurons in mice following traumatic brain injury (Gao and Chen, 2009). However, conversely, enhanced long-term AAVmediated BDNF expression in the hippocampus was shown to inhibit neurogenesis following ischemia in rats (Larsson et al., 2002). The reason for this discrepancy is unclear and further work is required to delineate the mechanisms by which BDNF regulates adult neurogenesis following neural damage. Given that neural activity promotes BDNF-mediated TrkB activation (Aloyz et al., 1999) and TrkB signaling is required for activity-dependent differentiation of hippocampal NSPCs, BDNF is likely to be a critical factor to promote NSPC-derived newborn neuron survival and integration into neural circuitry. However, if too much BDNF-stimulation is provided, this may be at the expense of NSPC numbers in some situations but this needs further study for confirmation.

BDNF combined with a mitogen is more effective in enhancing NSPC function and may be a better therapeutic option. Neurogenic NSPCs can be cultured from SVZ of adult humans but requires a combination of FGF2 and BDNF for robust growth (Pincus et al., 1998). This combination also appears to be required for creating a neurogenic environment in normally non-neurogenic regions *in vivo* (Chen et al., 2007a). In addition, co-infusion of BDNF plus EGF was more effective than either factor alone at promoting long term striatal neuron survival following ischemic injury in mice (Im et al., 2010).

## **MORPHOGENS**

The morphogens Wnt, Shh, and Bone Morphogenic Proteins (BMPs) play a major role in neural development with some data to suggest they may also play a role in the NSPC response to neural damage.

## *Wnt signaling*

While Wnts are endogenously expressed in adult neurogenic niches (Wexler et al., 2009), their expression is not upregulated following neuronal damage, such as stroke (Morris et al., 2007). Wnt signaling via β-catenin, promotes NSC proliferation to regulate their maintenance (Lie et al., 2005; Adachi et al., 2007). This appears to be by regulating symmetric vs. asymmetric division, promoting symmetric division during neural regeneration (Piccin and Morshead, 2011). Wnt signaling is negatively regulated by glycogen synthase kinase 3 beta (GSK3β) and inhibitors of GSK3β have been proposed as candidate targets for neural repair (Mao et al., 2009). Inhibition of β-catenin signaling by the amyloid beta peptide resulted in reduced neurogenesis from NPCs grown from Alzheimer disease brains, which also have increased levels of GSK3β (He and Shen, 2009). Further, downregulation of β-catenin in a rat stroke model inhibited striatal neurogenesis (Lei et al., 2008), while overexpression of Wnt3a following focal ischemia promoted NSPC proliferation, neurogenesis and functional recovery (Shruster et al., 2012). However, following traumatic brain injury in mice, β-catenin activity was upregulated in cortical NG2+ oligodendrocyte/astrocyte progenitor cells, suggesting that it also plays a role in gliogenesis following injury (White et al., 2010).

## *Shh*

Intrathecal administration of Shh to rats following ischemia promoted SVZ NSPC proliferation and improved behavioral recovery (Bambakidis et al., 2012), while following spinal cord injury it promoted proliferation of nestin+ NSPCs surrounding the central canal leading to increased numbers of oligodendrocyte precursors and neurons (Bambakidis et al., 2003). Shh expression is upregulated in the SVZ and hippocampus following ischemia, potentially playing a role in increased proliferation in a Notch dependent manner (Sims et al., 2009; Wang et al., 2009); however in the hippocampus its expression was also upregulated in mature CA3 neurons, suggesting effects in mature neurons as well as progenitors (Sims et al., 2009). The source of Shh in the adult brain appears to be astrocytes. In the normal brain, astrocytes in neurogenic regions produce Shh and when transplanted they induce neurogenesis in non-neurogenic regions of the brain, such as cortex (Jiao and Chen, 2008). Following brain injury activated astrocytes upregulate Shh expression in response to pro-inflammatory stimuli, which subsequently promotes increased numbers of Olig2+ NSPCs (Amankulor et al., 2009); this may be via activation of quiescent endogenous cortical NSPCs derived from astrocytes (Ahmed et al., 2012). However, in different neuroinflammatory conditions, such as experimental allergic encephalomyelitis (EAE) or multiple sclerosis, the inflammatory response induces Shh in astrocytes as above but inhibits Shh-induced NSPC differentiation by subsequent downregulation of the Gli1 transcription factor (Wang et al., 2008).

## *Bone morphogenic proteins (BMPs)*

The role of BMP signaling in NSPCs and their role following CNS injury has recently been reviewed (Sabo et al., 2009) and so will only be covered briefly here. In general, BMP signaling inhibits neuronal and oligodendroglial differentiation of NPCs and promotes astrogliogenesis, during development (Gross et al., 1996) and in the adult (Lim et al., 2000), with somewhat different effects at different stages of development (Mehler et al., 2000). However, expression of the BMP inhibitor noggin in the SVZ (Lim et al., 2000) or SGZ (Bonaguidi et al., 2008) can obstruct this cascade and promote neurogenesis. Further, BMP signaling promotes non-neurogenic parenchymal astroglial cell fate while leukemia inhibitory factor (LIF), which also promotes astrogliogenesis, promotes cells of an astroglial progenitor cell phenotype (Bonaguidi et al., 2005). Demyelination induced expression of the BMP antagonist chordin in the SVZ, induced glial fate in neuroblasts to generate new oligodendrocytes in the corpus callosum (Jablonska et al., 2010). Conversely, BMP signaling in SVZ NSCs but not NPCs is required to promote a neurogenic rather than oligodendrogliogenic cell fate (Colak et al., 2008).

BMP signaling inhibits NPC proliferation (Gajera et al., 2010) and while inhibition of the BMP pathway increases neurogenesis initially, it eventually results in depletion of the NSC pool, leading to decreased neurogenesis in the dentate gyrus (Mira et al., 2010). This indicates that a fine balance of BMP signaling vs. inhibition is required to regulate appropriate NSC numbers and subsequent cell fate.

Given that BMP signaling promotes an astroglial fate and inhibits oligodendrogliogenesis and neurogenesis, the effect of inhibition of BMP signaling by infusion of noggin into the lateral ventricles has been examined in various models of neural injury. In cuprizone-induced demyelination models noggin infusion inhibited the cuprizone induced upregulation of BMP4 and its signaling pathways, decreased SVZ astrocyte numbers, increased oligodendrocyte numbers and promoted remyelination of the corpus callosum (Cate et al., 2010; Sabo et al., 2011), possibly by regulation of Olig2 function (Chen et al., 2012). Noggin infusion also produced similar results in a model of intraventricular hemorrhage (Dummula et al., 2011). In a brain injury model, noggin was found to be produced by reactive astrocytes and similarly promoted oligodendrocyte fate (Hampton et al., 2007). It was also recently shown that Bcl2 regulates neurogenesis in a striatal injury model, by increasing β-catenin expression and decreasing BMP4 expression, in a noggin independent fashion (Lei et al., 2012).

## **ERYTHROPOIETIN (Epo)**

Epo is a cytokine better known for its regulation of erythrocyte production. However it also has a number of functions within the CNS and on NSPC biology. Epo is expressed in the developing and adult SVZ (Shingo et al., 2001) and is required for endogenous embryonic and adult SVZ and SGZ neurogenesis (Tsai et al., 2006; Chen et al., 2007b). It promotes neurogenesis of SVZ NSPCs at the expense of multipotent progenitors in the normal rodent brain (Shingo et al., 2001) but in the SGZ this effect is transient, briefly increasing neuronal progenitor numbers but with no long term enhancement of survival (Ransome and Turnley, 2007). One of the mechanisms by which it may do this is by upregulation of Suppressor of Cytokine Signaling-2 (SOCS2) in NSPCs (Wang et al., 2004b). SOCS2, an intracellular regulator of cytokine signaling, promotes embryonic SVZ neurogenesis (Turnley et al., 2002; Scott et al., 2006), while in the hippocampus it promotes NSPC-derived newborn neuron survival (Ransome and Turnley, 2008b). It also promotes axonal growth of hippocampal neurons (Ransome and Turnley, 2008a), which may be a factor contributing to its enhanced neuroprotective effects following neural injury.

Following neural damage Epo has been found to be both neuroprotective and to promote neurogenesis (Wang et al., 2004a). Epo expression is induced/upregulated by hypoxia and thus many studies have examined its role post-ischemia or in ischemia/hypoxia models, although it has also been shown to be effective following traumatic brain injury. Conditional knockdown of the Epo receptor reduced post-stroke neurogenesis, with reduced proliferation and stroke-induced neuroblast migration to the cortex (Tsai et al., 2006). The Epo-induced NSPC migration appears to be indirect, rather inducing expression of matrix metalloproteases in endothelial cells, which in turn induced NSPC migration (Wang et al., 2006).  Inhibition of proliferation by infusion of AraC into the ventricles inhibited Epo-induced dentate gyrus neurogenesis and recovery of spatial learning (Zhang et al., 2012). Epo has also been shown to be neuroprotective in disease models, such as PD, in which it promoted SVZ NSPC proliferation and migration to the damaged striatum (Kadota et al., 2009).

One of the problems associated with use of Epo as a therapeutic for promotion of neural regeneration is that it generally increases the hematocrit at doses required for promotion of neurogenesis (e.g., 5000 U/kg). While this has not presented major issues for use in animal models, it raises concerns for potential clinical use. Therefore, several Epo derivatives have been developed to try to promote neuroprotective effects while avoiding effects on erythrocyte production. Carbamylated Epo does not bind to classical Epo receptor and does not stimulate erythropoiesis but does promote SVZ and dentate gyrus proliferation and neuronal differentiation of adult NSPCs (Wang et al., 2007; Leconte et al., 2011), while a different non-erythropoietic derivative asialo-Epo promoted SVZ-derived oligodendrogenesis (Kako et al., 2012). A peptide agonist of Epo, Epobis, has also recently been developed and shown to promote neuron survival and neurite outgrowth, but its effects on neurogenesis have yet to be determined (Pankratova et al., 2012).

## **CHEMOKINES AND CYTOKINES**

Chemokines form a family of small (8–14 kD), mainly basic, secreted molecules that are primarily known for regulating chemoattraction of immune cells to sites of tissue damage. They have been reported to have widespread non-immunological effects in the CNS, including regulation of neural cell proliferation, migration, survival, and synaptic transmission and can act in a paracrine or autocrine manner (Bajetto et al., 2002; Cartier et al., 2005).

Pro-inflammatory cytokines, such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) appear to be major regulators of chemokine and chemokine receptor expression in many tissues (Hiroi and Ohmori, 2003; Suyama et al., 2005). In the adult brain there is basal expression of chemokines, especially in neurogenic regions, while treatment with IFNγ and TNFα can significantly increase the expression of specific chemokines including CXCL1, CXCL9, and CCL2 (Turbic et al., 2011).

Adult NSPCs express a range of chemokine receptors and chemokines are expressed in different brain regions, with the highest levels in the OB, suggesting an as yet largely unexplored role for chemokines in regulating basal adult NPC migration (Turbic et al., 2011). Neurospheres derived from adult mouse SVZ-derived NPCs have been shown to express a range of chemokine receptors, including CCR1-8, 10 and CXCR1-6 (Tran et al., 2004). Functionally, specific chemokines, such as CXCL12/CXCR4 can promote NPC migration (Imitola et al., 2004; Tran et al., 2004; Dziembowska et al., 2005) and proliferation (Tran et al., 2004) or survival (Krathwohl and Kaiser, 2004; Dziembowska et al., 2005) *in vitro*. Following neural damage, NPC migration to site of injury is promoted by CXCL12 (Imitola et al., 2004; Itoh et al., 2009) and is mediated at least in part, by induction of metalloprotease expression in the NPCs (Barkho et al., 2008). CCL2, CCL3, and CXCL1 also promote NPC migration to striatum following quinolinic acid lesion (Gordon et al., 2009).

The gp130-associated cytokines, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), activate Janus kinase [JAK/signal transducer of transcription 3 (STAT3)], mitogen activated protein (MAP) kinase and PI-3K/Akt pathways following ligand binding. These cytokines have been shown to regulate NSC proliferation and differentiation (Turnley and Bartlett, 2000; Heinrich et al., 2003; Kamimura et al., 2003; Ernst and Jenkins, 2004). IFNγ, which signals via STAT1, and IFNβ which does not, both inhibit cultured adult NPC proliferation, but only IFNγ promotes neuronal differentiation (Wong et al., 2004; Lum et al., 2009). Specifically in the dentate gyrus, the activation of STAT3 from CNTF appears to be essential for the formation and maintenance of the NSCs (Muller et al., 2009).

## **OTHER FACTORS THAT PLAY A ROLE IN NSPC FUNCTION THAT MAY BE TARGETED FOR NEURAL REPAIR**

There are factors involved in neurogenesis other than extrinsic growth factors and cytokines, as discussed above. Some of these include membrane bound molecules, neurotransmitters and their receptors and blood vasculature.

## **EPH/EPHRIN SIGNALING AND NSPCs—A NEW TARGET TO PROMOTE NSPC PROLIFERATION, SURVIVAL, AND MIGRATION**

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are membrane bound molecules that signal bidirectionally, are generally involved in cell or axon guidance by repulsive mechanisms and which play a role following neural injury (reviewed in Goldshmit et al., 2006b). They have been receiving increasing interest of late as they have been shown to play major roles in inhibition of regeneration in the CNS following neural injury or disease, with deletion or blocking of various Ephs or ephrins promoting neural repair (Goldshmit et al., 2004, 2006b, 2011; Rodger et al., 2004; Liu et al., 2006; Fabes et al., 2007; Overman et al., 2012; Van Hoecke et al., 2012). EphA4 also plays a role in regulation of inflammation following neural injury (Munro et al., 2012), which could have secondary effects on NSPC responses.

Eph receptor signaling also regulates several aspects of NSPC biology but as yet has not been targeted to enhance NSPC responses following injury. The EphB2/ephrin-B2 pathway enables formation of the chain migration from the SVZ to the OB (Conover et al., 2000) and also controls the conversion of ependymal cells to astrocytic NSPCs (Nomura et al., 2010). EphB2 signaling via ephrin-B1 is also required for development of the dorsal half of the dentate gyrus, where it controls migration of the dentate progenitor cells (Catchpole and Henkemeyer, 2011). Ephrin-A2 signaling via EphA7 inhibits SVZ NSPC proliferation and subsequent neurogenesis (Holmberg et al., 2005), while conversely ephrin-A5 signaling via EphA7 induces their apoptosis (Depaepe et al., 2005). EphB2 and ephrin-B signaling however promotes SVZ NSPC proliferation, decreases migration, and promotes neuronal differentiation (Katakowski et al., 2005), with ephrin-B1 expression in SVZ NSPCs critical for maintenance of the proliferative NSPC state (Qiu et al., 2008), a role also played by EphA4 (Khodosevich et al., 2011). In addition, EphB3 is expressed by adult SVZ NSPCs and neuroblasts and EphB3 and ephrin-B3 knockout mice show increased neonatal and adult SVZ NSPC proliferation, indicating that this pathway normally inhibits their proliferation (Theus et al., 2010; Del Valle et al., 2011; Doeppner et al., 2011). Infusion of soluble ephrin-B3 into the lateral ventricles reversed the proliferation defect in ephrin-B3 knockout mice but not the EphB3 receptor knockout mice (Theus et al., 2010).

In the hippocampus, EphB1 and ephrin-B3 promote hippocampal NSPC proliferation and migration, as well as cell polarity (Chumley et al., 2007), while ephrin-A5 promotes hippocampal NSPC proliferation and neurogenesis, at least partly through regulation of the normal vascular system (Hara et al., 2010), which requires normal EphA4 (Goldshmit et al., 2006a) and EphB4 signaling (Colin-Castelan et al., 2011). Ephrin-B2, expressed by hippocampal astrocytes in the SGZ, induces neuronal differentiation of EphB4-expressing hippocampal NSPCs and this effect involves activation of β-catenin independent of Wnt signaling, with subsequent upregulation of proneural transcription factors (Ashton et al., 2012).

To date, little has been examined regarding the role of Eph/ephrin signaling following neural damage. Following traumatic brain injury, EphB3 expression was transiently downregulated, which may lift its inhibition of NSPC proliferation and this may be one of the mechanisms by which NSPCs are able to respond to injury, by increasing proliferation and survival (Theus et al., 2010). However, in ephrin-B3 knockout mice, while there was an increase in NSPC proliferation following ischemic injury, there was also enhanced cortical damage with an increased infarct volume (Doeppner et al., 2011) due to NSPCindependent effects of ephrin-B3, which will need to be taken into account when considering therapeutic use of Eph/ephrins. Conversely, in a model of PD infusion of soluble ephrin-A1 into the lateral ventricles, to activate EphA receptor/s, promoted SVZ NSPC proliferation and migration of these cells to striatum, where they subsequently differentiated in dopaminergic neurons (Jing et al., 2012).

## **OTHER FACTORS**

Following injury, one of the early mediators of the injury response is upregulation of nitric oxide (NO). NO has been reported to have proliferative (Zhang et al., 2001a; Reif et al., 2004) or anti-proliferative (Packer et al., 2003; Park et al., 2003; Moreno-Lopez et al., 2004) effects on basal SVZ and SGZ NSPCs, as well as following injury (Zhang et al., 2001a; Zhu et al., 2003; Sun et al., 2005b). This apparent discrepancy in effect appears to lie largely in the source of NO production and the subsequent signaling pathways induced. If the NO derives from neuronal nitric oxide synthase (nNOS) then the effect is largely anti-proliferative (Packer et al., 2003; Moreno-Lopez et al., 2004; Sun et al., 2005b), but if derived from inducible NOS (iNOS) (Zhu et al., 2003; Carreira et al., 2010) or endothelial derived NOS (eNOS) (Reif et al., 2004) then the effects are largely proliferative. This likely relates to the location of NOS as well as the concentration of NO produced. Further, infusion of NO donors that induce MAP kinase signaling (Carreira et al., 2010) or cyclic guanosine monophosphate (cGMP) signaling, promotes proliferation and neurogenesis in normal and ischemic rats (Zhang et al., 2001a). Indeed, both pathways appear to be involved in NSPC proliferation in response to NO, with activation of the MAPK pathway at shorter timepoints (6 h) and activation of cGMP at later timepoints (24 h) (Carreira et al., 2012). Recent work has indicated that the NO-cGMP pathway is also an important mediator of the neuro-proliferative effects of other factors, such as Neuropeptide Y in the hippocampus (Howell et al., 2005; Agasse et al., 2008; Cheung et al., 2012) and also explains the increased proliferation and neurogenesis observed with infusion of phosphodiesterase inhibitors, such as sildenafil and tadalafil, which lead to increased cGMP levels and improve outcome in stroke models (Zhang et al., 2003, 2006). cGMP levels also appear to regulate NSPC fate during embryonic development, with high levels promoting neuronal differentiation and low levels non-neuronal fate (Gomez-Pinedo et al., 2010), however, whether this is the case in adult remains to be determined.

Endocannabinoid signaling has been shown to regulate migration and neurogenesis in both the SVZ and dentate gyrus (Aguado et al., 2005, 2007; Jiang et al., 2005; Goncalves et al., 2008; Gao et al., 2010; Hill et al., 2010; Wolf et al., 2010; Oudin et al., 2011a,b). Other molecules involved in NPC migration include polysialated neural cell adhesion molecule (PSA-NCAM) (Cremer et al., 1994; Hu et al., 1996; Chazal et al., 2000), Slit-Robo (Wu et al., 1999), and integrins (Jacques et al., 1998; Murase and Horwitz, 2002). In basal conditions, reelin is a detachment signal for neuroblasts from the RMS at the OB (Hack et al., 2002), while overexpression of reelin in a demyelinating lesion led to an increase in ectopic migration of neuroblasts at the lesion site (Courtes et al., 2011). Many of these factors signal via the Rho kinase pathway, which is a downstream regulator of NPC migration (Leong et al., 2011) and targeting this pathway may circumvent the large number of external signals that converge on this pathway to allow more precise control of directed NPC migration and possibly neuronal differentiation and survival.  Following ischemia SVZ neuroblasts migrate to the infarct in close association with blood vessels (Ohab et al., 2006; Yamashita et al., 2006; Thored et al., 2007; Kojima et al., 2010).

NMDA receptors expressed in neuroblasts along the RMS are crucial to the integration of these neurons in existing OB circuitry (Platel et al., 2010), with glutamate released from astroglial cells in the glial tube that surrounds the migrating neuroblasts. NMDA receptor activation in newly born dentate gyrus granule cells also increases survival. Initial GABA depolarization promotes maturation of neurons in the dentate gyrus and OB (Saghatelyan et al., 2005; Ge et al., 2006) and this depolarization and subsequent Ca2<sup>+</sup> influx are required for dendrite initiation and elongation (Gascon et al., 2006). Agrin signaling is also necessary for integration and survival of newborn neurons in the OB: a loss of agrin leads to improper synapse formation while overexpression of agrin results in an increase in dendritic spines (Burk et al., 2012).

The migration distance for new neurons from the SGZ is relatively short as they travel into the granular layer above the SGZ, where guidance molecules may control this movement. NMDA receptor signaling is required for the proper migration of newborn granular cells in the dentate gyrus (Namba et al., 2011). This is achieved through the activation of Disrupted-in-schizophrenia (DISC1), as neurons without DISC1 migrate further into granular layer and into the molecular layer (Duan et al., 2007; Namba et al., 2011). DISC1 also controls the dendritic maturation of newborn granule cells through GABA depolarization of NKCC1 and activation of the Akt-mTOR pathway (Duan et al., 2007; Kim et al., 2012), while Reelin regulates migration and dendritic development of adult-generated hippocampal neurons (Teixeira et al., 2012). Whether targeting any of the above factors will enhance NSPC responses following neural injury largely remains to be determined.

## **CONCLUSIONS**

Many factors regulate the biology of NSPCs at different stages along their growth and maturation cycle. To date, the majority of studies which have administered extrinsic growth factors or cytokines have concentrated on the effect of a single factor at a time. However, some studies are now emerging where combinatorial effects of different factors have been examined. It will not be sufficient to only increase the number of NSPCs produced by administration of a mitogen. While this can indeed increase neurogenesis, the number of surviving and functionally integrated neurons (or oligodendrocytes) is still very limited. It would be prudent to combine use of mitogens with other factors that enhance neurogenesis or oligogliogenesis respectively, as well as factors that enhance their subsequent survival and integration. Which factors are chosen to combine will likely depend on the injury or disease model to be examined, whether neurons and/or oligodendrocytes need to be replaced and may also be species dependent to a certain extent, although the combination of a mitogen such as EGF and a survival factor, such as BDNF may be generally beneficial. Some factors, such as Epo, have multiple actions, including NSPC proliferation, neuronal differentiation, and oligodendrocyte maturation, as well as being neuroprotective, which makes them particularly attractive as potential therapeutics for treatment of the damaged brain. Most of the work described above was conducted in rodents and to date little work has been performed following neural injury in brains of gyrencephalic species, such as non-human primates (Tonchev et al., 2003a,b, 2005, 2007). Sheep models are also being developed to allow such questions to be answered in a model more relevant to human brain damage (e.g., Wells et al., 2012). Human clinical trials are also underway to test therapeutic effectiveness of a range of extrinsic factors, such as Epo, following brain injury. For a complete list see www.clinicaltrials.gov. Two decades since their discovery in mammalian brains, facilitation of the NSPC response to brain damage by administration of

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## **ACKNOWLEDGMENTS**

Ann M. Turnley is funded by a National Medical Health and Research Council of Australia Fellowship (#628344).

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into mature neurons in the poststroke adult striatum. *J. Neurosci.* 26, 6627–6636.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 October 2012; paper pending published: 30 November 2012; accepted: 30 December 2012; published online: 18 January 2013.*

*Citation: Christie KJ and Turnley AM (2013) Regulation of endogenous neural stem/progenitor cells for neural repair factors that promote neurogenesis and gliogenesis in the normal and damaged brain. Front. Cell. Neurosci. 6:70. doi: 10.3389/fncel.2012.00070*

*Copyright © 2013 Christie and Turnley. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## *Jonathan C. Niclis 1,2\*†, Anita Pinar 1†, John M. Haynes 3, Walaa Alsanie1, Robert Jenny1, Mirella Dottori <sup>4</sup> and David S. Cram1*

*<sup>1</sup> Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC, Australia*

*<sup>2</sup> The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia*

*<sup>3</sup> Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia*

*<sup>4</sup> Department of Anatomy and Neuroscience, Centre of Neuroscience Research, University of Melbourne, Parkville, VIC, Australia*

#### *Edited by:*

*Clare Parish, Florey Neuroscience Institute, Australia*

#### *Reviewed by:*

*Robert F. Halliwell, University of the Pacific, USA Joohyung Lee, Prince Henry's Institute, Australia*

#### *\*Correspondence:*

*Jonathan C. Niclis, The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Genetics Lane, Royal Parade, Parkville, VIC 3010, Australia.*

*e-mail<EMAIL_ADDRESS>†These authors have contributed equally to this work.*

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG37 and CAG51 typical late-onset repeat expansions in comparison to wildtype control lines during undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate-evoked responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.

#### **Keywords: Huntington's disease, human embryonic stem cells, neuronal differentiation, GABAergic neurons**

### **INTRODUCTION**

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat tract in exon 1 of the *HTT* gene (Group, 1993). Disease alleles (CAG*>*35) exhibit an age-dependent penetrance, with the lowest disease range (CAG36−39) associated with a later onset (McNeil et al., 1997). CAG40<sup>+</sup> alleles are associated with full penetrance, while larger expansions (CAG60+) result in juvenile or infantile onset (Andrew et al., 1993; Squitieri et al., 2006). Substantial correlation of ∼50% exists between CAG repeat length and age of onset, with secondary contributions from known and unknown genetic and environmental factors (Andrew et al., 1993; Wexler et al., 2004). HD affects approximately 1 in 10,000 individuals worldwide, with onset of disease usually in the fourth or fifth decade of life. HD culminates in death 15–20 years after persistent, irreversible and debilitating clinical symptoms (Naarding et al., 2001). To date, there is no cure for HD and consequently, there is an unmet clinical need for more effective therapeutics.

The *HTT* gene comprises 67 exons and encodes a ubiquitously expressed protein of approximately 350 kDa called Huntingtin (HTT). HTT plays a critical role in early embryogenesis with homozygous *HTT* null mouse embryos exhibiting incomplete neural development and lethality at embryonic day 8.5 and 10.5 of gestation (Zeitlin et al., 1995). HTT has been extensively studied in relation to its interacting partners, sub-cellular localization and effects on gene expression, and acts in a variety of cellular systems (Zuccato et al., 2010). The clinical symptoms of HD seen in patients can largely be classified as either neural, such as behavior and cognitive alterations, or motor, such as involuntary movements and abnormalities of voluntary movements. Underlying neurodegeneration is most prominent within the GABAergic medium spiny neurons of the striatum although widespread neuronal loss occurs with disease progression (Graveland et al., 1985; Vonsattel et al., 1985).

Understanding the molecular nature of HD is critical to the development of novel and efficacious therapies. Numerous animal models such as the R6/2 transgenic mouse (Mangiarini et al., 1996) together with post mortem patient tissues have proven valuable resources for identifying and elucidating major CNS cellular mechanisms and hallmarks that contribute to HD pathology; these are comprehensively reviewed in Zuccato et al. (2010) and include transcriptional dysregulation, CAG repeat expansion, excitotoxic stress, autophagy-lysosomal, and proteasome-ubiquitin system perturbation, anterograde and retrograde transport interference, mitochondrial dysfunction and cholesterol biosynthesis alteration.

The hierarchical relationship between the disparate mechanisms/hallmarks and their degree of individual contribution to the overall HD phenotype still remains uncertain. New human HD models such as pluripotent stem cells (PSCs) may provide an alternative system to shed light on this etiological question. Studies investigating hPSC lines, specifically human embryonic stem cell (hESC) lines, carrying pathogenic CAG repeat mutations have provided limited data in relation to the consequences of disease allele expression (Niclis et al., 2009; Bradley et al., 2011; Seriola et al., 2011). More recently, mouse and human induced pluripotent stem cell (iPSC) lines have been analysed for the appearance of HD hallmarks (Camnasio et al., 2012; Castiglioni et al., 2012; HDIPSCC, 2012; Jeon et al., 2012) and several phenotypes have been observed, including transcriptional dysregulation, CAG repeat instability, mutant HTT aggregates, cholesterol biosynthesis perturbation, lysosomal dysfunction and neuronal vulnerability. However, phenotypes reported from the various HD iPSC studies differ significantly and rarely correlate, for example CAG repeat instability is seen in human but not R6/2 iPSCs. A further caveat is that the HD iPSC lines studied to date either carry rare homozygous HD mutations or alleles with egregious repeat tracts (CAG60−180) indicative of juvenile and infantile onset and may represent atypical disease scenarios.

Limited studies assess the appearance of HD phenotypes in hPSC lines with clinically relevant trinucleotide expansions (CAG35−60) that correlate with late ages of onset. To this end, we have extended an earlier study by Niclis et al. (2009) using two HD hESC lines SI-187 (CAG51*/*19) and SI-186 (CAG37*/*15) that were generated from embryos identified with pre-implantation genetic detection (PGD). Here we show that the HD hESC lines are indistinguishable from control hESCs with respect to pluripotent characteristics, mitochondrial function, forebrain neural differentiation capabilities, relevant cellular phenotypes and with no evidence of a dysregulated transcriptome in key HTT associated genes. However, the larger of the two CAG repeat cell lines showed an altered responsiveness to the neurotransmitter glutamate. Overall, these results are informative for establishing a human neuronal cellular model of HD.

### **MATERIALS AND METHODS**

#### **CULTURE AND NEURAL DIFFERENTIATION OF hESC LINES**

Two HD hESC lines SI-186 (CAG37*/*15) and SI-187 (CAG51*/*19) (Niclis et al., 2009) as well as two wildtype control hESC lines, HES3 and H9 (Thomson et al., 1998; Reubinoff et al., 2000) were used. All hESC lines were screened for karyotypic abnormalities using Geisma staining and found to maintain euploid 46 XX karyotypes (data not shown). All four hESC lines were grown as bulk cultures on γ-irradiated mouse embryonic fibroblasts (MEFs) according to previously described conditions (Costa et al., 2008); briefly, cells were grown in DMEM/ F-12, supplemented with 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 1% Glutamax, 25 U/ml penicillin, 25 U/ml streptomycin, and 20% knockout serum replacement (all Invitrogen).

hESC neural differentiation was performed as previously described (Song et al., 2011a). Briefly, ∼3000 hESCs were distributed to each well of a round-bottom ultra-low attachment 96-well plate (Corning) containing 100μl of Differentiation Medium (Neurobasal A, 5% ITS-X, 2.5% Penicillin/Streptomycin, 5% Glutamax, 5% B27 and 5% N2; Invitrogen). From d0–d21 cells were grown as neurospheres at 37◦C in 5% CO2 in air, with 0.125% polyvinyl alcohol and 10 μM Roh-Associated Coil Kinase (ROCK) inhibitor Y-27632 at d0–4. From d0–d21 media was supplemented with 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml fibroblast growth factor (FGFb) (R&D Systems), and 100 ng/ml of Noggin (R&D Systems) from d0–8. Neurospheres were passaged at d14 by manual sectioning in half. To promote neuronal differentiation neurospheres were plated at d21 in Differentiation Medium onto 24-well plates (BD Biosciences) coated with 20μg/ml poly-D-lysine and 20μg/ml laminin (Invitrogen) in the absence of growth factors and media changed every 4 days for up to 24 days.

## **FLOW CYTOMETRY**

Cells were harvested using TrypLE Select (Invitrogen), passed through a 70μm filter and 1 <sup>×</sup> 105 cells stained with various antibodies listed in **Table 1**. Samples were analysed using a BD Canto II flow cytometer with data analyses performed using GateLogic. All samples were gated to assess only single cells as determined by forward scatter area vs. height channels and live cells as determined by negative DAPI selection. Background fluorescence was subtracted using unlabeled cells. A total of 50,000 events (∼40,000 live cells) were recorded, compensations were performed using single label antibody controls; positive gates were designated on concentration-matched isotype control antibodies.

## **GENOTYPING**

Genotyping was performed as previously described (Niclis et al., 2009). Briefly, HU3 and HU4 primers (Sermon et al., 1998) were used to amplify the CAG repeat expansion region in exon 1 of *HTT*. The HU4 sense primer was labeled with 6-carboxyfluorescein (6-FAM) and sizing of amplicons performed with an ABI Prism 3100 DNA sequencer coupled with GeneScan Software according to previously described methods (Cram et al., 2000). The number of CAG repeats was accurately calculated to ±1 bp from GeneScan sized F-PCR products, using

the formula: CAG repeat number = (size of PCR product – size of non-CAG repeat region)/3.

## **IMMUNOCYTOCHEMISTRY**

Neurospheres were collected at d10 or 21 and fixed for 15 min at room temperature with 4% paraformaldehyde, cryoprotected in sucrose (20% w/v in 0.1 M PBS) and embedded in OCT compound (Tissue–Tek) before serial sectioning (6 × 10μm thick sections) on a cryostat (Leica). Neuronal cultures were fixed at d35 or 45 for 15 min at room temperature with 4% paraformaldehyde, permeabilized with 1% Triton X-100 (Ameresco), blocked using 10% normal donkey serum (Invitrogen) and incubated with primary antibodies described in **Table 1** at 4◦C overnight. Species-specific AlexaFluor (Invitrogen) or Dyelight (Jackson Laboratories) 488 and 555 secondary antibodies (all 1:200) were added for 2 h at room temperature and then counterstained with 0.1μg/ml DAPI (SIGMA). Coverslips were mounted onto glass slides using Fluorescent Mounting Media (DAKO). Bound fluorescence was detected using an Olympus BX51 microscope coupled to the ULH100HG fluorescence system. Images were captured using a DP70 camera.

## **CELL GROWTH AND VIABILITY ASSAYS**

Duplicate MEF coated T25 culture flasks were seeded with 1 × 106 hESCs for each line. Cells were harvested at 24 and 72 h. FACS analysis of DAPI, CD9, and TRA-1-60 staining was used to distinguish live hESCs (DAPI–/CD9+/TRA1–60+) and MEFs (DAPI–/CD9–/TRA1–60–). Doubling rates were calculated based on hESC and live cells only, according to the following formulae: [Log2(β/α)]/2; α = 24 h count and β = 72 h count. Viability assays were performed similarly on hESCs 24 h after passage but with DAPI live and dead cell gating performed by FACS after CD9 and TRA-1–60 gating. Neurosphere growth rates were determined by measuring area sizes using Adobe Photoshop CS5, the average value for 12 neurospheres equated a single replicate, with three averaged values analysed at each time point for each line. Data values at d21 were doubled to account for passaging in half of spheres at d14. Data was statistically analysed with PRISM, using a One-Way ANOVA with a Kruskal–Wallis Test and Dunn's post test. Means were graphed with the SEM and a *p*-value (∗) of *<* 0.05 considered significantly different.

## **NEURONAL FUNCTIONAL ANALYSIS**

Neurons were cultured as described above. For intracellular Ca2<sup>+</sup> [(Ca2+)i] imaging, neurons were loaded with Fluo4-AM (2 μM, for 45 min at 37◦C, as previously described (Watmuff et al., 2012). Media was then replaced with HEPES buffer (of composition, mM, NaCl 145; MgSO4 1; KCl 5; glucose 10; CaCl2 2.5; HEPES 10; pH 7.4) containing bovine serum albumin (0.3% w:v) and placed on a heated (37◦C) stage. Neurons were viewed using a Nikon A1R confocal microscope and excited with a 488 nm laser, emission was recorded at 525/50 nm at two image frames per second. After 2 min of baseline recording glutamate (30μM) or vehicle was added to each well and imaging continued for 90 s as previously described (Raye et al., 2007; Khaira et al., 2011; Watmuff et al., 2012). After this time KCl (30 mM) was added to each well and imaging continued for a further 60 s. For analysis,

background emission light was subtracted from analysis regions defined within each cell body and emission intensity within each region calculated using Nikon Elements software. The maximal elevation of intracellular [Ca2+]i in response to vehicle or glutamate is expressed as the net fraction of the KCl-induced maximal increase in [Ca2+]i.

## **QUANTITATIVE REAL-TIME PCR**

Total RNA was extracted from <sup>∼</sup> <sup>1</sup> <sup>×</sup> 106 cells using the commercially available RNeasy Mini Kit (Qiagen) as per the manufacturer's instructions and treated with RNase-Free DNase (Qiagen). RNA was quantitated using a ND-1000 Spectrophotometer (NanoDrop Technologies). Purified RNA (0.4–2μg) was reverse transcribed to single stranded cDNA with SuperScript III reverse transcriptase using the First Strand Synthesis System Kit (Invitrogen) and treated with RNase H to remove contaminating RNA.

cDNA products were subjected to qRT-PCR of selected genes (**Table 2**) using TaqMan® Universal PCR Master Mix and the inventoried TaqMan® Gene Expression Assay Kit (Applied Biosystems) according to manufacturer's recommendations. Glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*) was used for normalization of RNA input, two independent reactions were prepared for each sample. Reaction plates were run on the 7500 Real Time PCR System (Applied Biosystems) at the following thermocycler conditions: stage 1, 50◦C for 2 min; stage 2, 95◦C for 10 min and stage 3, 40 cycles of 95◦C for 15 s and 60◦C for 1 min. The relative quantification (RQ) of gene expression in each sample was analysed using SDS Software version 1.3 (Applied Biosystems). The Ct value for each sample was measured in duplicate and the average normalized against the endogenous control *GAPDH* to determine the *-*Ct value. The *-*Ct values were then standardized against the calibrator's *-*Ct (wildtype HES3 line) to yield the *--*Ct. The RQ was then calculated as 2−*--*Ct. Statistical analysis for qRT-PCR data was performed using GraphPad Prism with a one-way ANOVA analysis of each gene at each specific time point. A Kruskal–Wallis test was performed on the non-matched, non-parametric data. The *p*-value was set to *<*0.05 and a Dunn's post-test performed with RQ data from three culture replicates plotted as a mean and scale bars denoting the mean ± SEM.

*All primers were purchased from Applied Biosystems.*

## **WESTERN BLOTTING**

<sup>1</sup> <sup>×</sup> 106 cells were lysed in 500μl of Cell Lysis Buffer (Cell Signaling) and protein concentrations determined using a BCA assay (Pierce, Thermo Scientific). Protein samples (10μg) in 1× SDS sample buffer containing 10% β-mercaptoethanol were electrophoresed on 4–12% NuPAGE gels (Invitrogen). Proteins were transferred to immobilon PVDF transfer membranes (Millipore), blocked in TBS-T containing 5% non-fat dry milk powder and then immunoblotted with either monoclonal antibody 1HU-4C8 (Millipore, 1:2000) specific for epitopes within amino acids 181–810 on wildtype and mutant HTT or monoclonal antibody 5TF1-1C2 (Millipore, 1:5000) which has a high avidity for long polyglutamine epitopes (*>*15 polyglutamine residues). A secondary anti-mouse HRP antibody (Calbiochem) in combination with an ECL kit was finally used to illuminate HTT protein.

## **JC-1 MITOCHONDRIAL STAINING**

JC-1 a cationic dye that undergoes a fluorescence emission shift from green (529 nm) to red (590 nm) upon accumulation in the mitochondrial matrix (Simeonova et al., 2004), was employed to measure changes in the mitochondrial membrane potential (MMP). Undifferentiated hESCs (1 <sup>×</sup> 106) prepared from bulk cultures were incubated in media containing 2μM of JC-1 for 30 min, washed twice in PBS and then analysed using a BD FACS Canto II flow cytometer 488 nm excitation and emission detection dually from both FITC and PE channels. Compensations were performed with hESC specific CD9 and EpCAM antibodies, conjugated to FITC and PE respectively. Data analysis was conducted using FACS Diva software (BD).

## **RESULTS**

## **CHARACTERIZATION OF PLURIPOTENT HD hESC LINES**

HD and control hESC lines were originally derived as colony cultures that intrinsically comprise significant heterogeneity with differential degrees of pluripotency and differentiation capacities across a gradient of isolatable sub-fractions (Laslett et al., 2007; Hough et al., 2009; Kolle et al., 2009). To eliminate this stochasticity, HD hESC lines were adapted to an enzymatically passaged bulk culture system to provide homogenous cultures amenable to stringent molecular and functional comparative evaluations. Of note, adaptation rates of HD lines were commensurate with controls, requiring ∼3–5 passages, and produced morphologically identical bulk culture monolayers of hESCs with low cytoplasmic-to-nuclear ratios (**Figures 1A,B**). Robust homogeneity was achieved with hESC bulk culture conditions, evidenced by a tightly clustered population of TRA-1-60+/CD9+ pluripotent cells and an absence of single positive cells indicating that the hESCs were maintained in a pluripotent state (**Figure 1C**). Both HD hESC lines exhibited FACS profiles with pluripotent markers similar to control lines verifying stable undifferentiated states (**Figure 1C**). Further, no significant differences in cell growth rates (doublings/48 h) were observed between HD and control hESC lines (SI-187 2.24, SI-186 2.8, HES3 2.36, and H9 2.52, *p >* 0*.*05; **Figure 1H**) or cell viability during enzymatic

#### **FIGURE 1 | Characterization of pluripotent HD hESC lines demonstrates identical characteristics to control lines. (A,B)** Brightfield morphology of hESC bulk cultures. **(C)** Flow cytometry analysis of hESCs seen as a TRA-1-60+/CD9+ population; feeder MEFs are a distinct CD9–/TRA-1-60 sub-population. **(D)** FL-PCR representation of HD genotypes. **(E)** qRT-PCR of

HTT mRNA expression levels. **(F)** Western blot of an N-terminal epitope antibody on the 350 kDa HTT protein. **(G)** Western blot of hESC lines immunoprobed with the IC2 monoclonal antibody. **(H)** Growth rates of hESC lines (One-Way ANOVA, *n* = 3, *p* ≥ 0*.*05) and **(I)** cell viability comparisons (One-Way ANOVA, *n* = 3, *p* ≥ 0*.*05).

passaging (SI-187 79.3%, SI-186 78.0%, HES3 84.7%, and H9 87.3%, *p >* 0*.*05; **Figure 1I**).

Analysis of FL-PCR products confirmed correct CAG repeat genotypes for HD lines SI-187 (CAG51*/*19) and SI-186 (CAG37*/*15) and control lines HES3 (CAG15*/*15) and H9 (CAG23*/*15) (**Figure 1D**). qRT-PCR using primers specific for conserved sequences on both wildtype and mutant alleles confirmed *HTT* mRNA was expressed at similar levels in HD and control hESC lines (**Figure 1E**). Western blot analysis using a polyclonal antibody reactive to an N-terminal epitope on the 350 kDa HTT protein (**Figure 1F**) confirmed HTT protein expression in all four hESC lines. Further immunoprobing using the IC2 monoclonal antibody that specifically reacts with long polyglutamine epitopes *>*15–20 residues (Trottier et al., 1995) detected a ∼350 kDa band exclusively within SI-187 and SI-186 cells confirming mutant HTT expression in both HD lines (**Figure 1G**).

## **NEURAL DIFFERENTIATION IS UNPERTURBED BY AN EXPANDED CAG TRACT**

HD neurodegeneration emerges within the forebrain and progresses to whole-brain atrophy. Differentiation of HD hESC lines to a regionally relevant forebrain phenotype was achieved using the spin aggregation neurosphere system we have previously reported (Song et al., 2011a). Seeding 3000 hESCs per well resulted in morphologically similar neurospheres within 24 h and throughout the neurosphere differentiation stage between HD and control lines (**Figure 2A**). Neural growth rates were equivalent between HD and control lines indicating the presence of mHTT does not interfere with neural precursor division and differentiation (*p >* 0*.*05; **Figure 2B**).

Quantitative flow cytometric assessment throughout neurosphere stages demonstrated highly analogous expression profiles across HD and control lines. All lines progress early to a neural fate and near total expression (*>*90%) of the pan-neural marker CD56 is observed by d21 (**Figure 2C**). Neural differentiation exhibited an anterior identity with *>*50% of cells expressing the forebrain surface antigen FORSE-1 (**Figure 2C**). The forebrain population was confirmed as neural in character with *>*90% of the FORSE-1+ population co-staining with CD56 by d21 (**Figure 2D**), and all FORSE-1+ cells separating from minor CD9+ fractions (**Figure 2E**). All hESCs rapidly exit a pluripotent state with low levels of the hESC marker CD9 d10 which falls to ∼2% by d21 (**Figure 2E**). Intriguingly, a delay in CD9 downregulation is observed in the fully penetrant SI-187 line at d10, although by d21 CD9 expression is consistent across all lines (**Figure 2E**). Overall, these findings suggest that pathogenic HD mutations do not perturb neural progenitor generation, expansion or specification to an anterior identity.

Control and HD neurospheres exhibited equivalent progenitor phenotypes at d10 (**Figures 3A–H**). Strong expression of the multipotent neural stem cell transcription factor SOX2 was observed in control (85.8%) and HD (88.2%) cells equally (**Figure 3D**, data not significant *p* = 0*.*05). This was concomitant with a rostral

**(D)** co-expression scatter plots for CD56 and FORSE-1 and**(E)**CD9 and FORSE-1.

Neurosphere growth rate analysis across all HD and control lines (One-Way

fate seen by the robust expression of the telencephalic marker OTX2 in comparable proportions between control (88.0%) and HD (92.4%) cells (**Figure 3H**, data not significant *p* = 0*.*05), and by co-localization of both proteins (**Figures 3C,G**). Importantly, continued differentiation to d21 facilitated robust expression of the forebrain specific transcription factor FOXG1 within the majority of neurosphere cells in both control and HD sections (**Figures 3I–O**). Expression levels of FOXG1 were not significantly different between control (71.9%) and HD (79.4%) neurospheres (**Figure 3L**, *p* = 0*.*05). Limited numbers of neuronal cells were seen at the end of neurosphere culture, detected by β-III-TUBULIN expression (**Figures 3J,N**). FOXG1 expressing regions preferentially co-localize with β-III-TUBULIN expression indicating maturation of anterior cells to a post-mitotic neuronal fate (**Figures 3K,O**).

progenitor marker SOX2 (red). Merged images demonstrate co-expression of OTX2 and SOX2 and quantitative evaluation shows no significant difference

> Subsequent to neurosphere plating on extracellular matrices, neuronal differentiation disseminated throughout cultures in control (**Figure 4A**) and HD neurons (**Figure 4D**) as shown by cytoskeletal β-III-TUBULIN immunostaining. Quantitative validation was made of these observations during late neurospheres (d21) and neuronal (d35) differentiation stages by β*-III-TUBULIN* transcript expression comparisons (**Figure 4G**). Antibody staining against an N-terminal HTT epitope revealed cytoplasmic and nuclear protein localization (**Figures 4B,E**) and specifically preferential nuclear reactivity within the nuclei of neurons with no discernable differences between control (**Figure 4C**) and HD neurons (**Figure 4F**). Further, quantitative *HTT* transcript expression levels were equal across all HD and control lines during neural precursor and neuronal differentiation stages (**Figure 4H**). Crucially, neuronal differentiation

with no quantitative differences between control and HD neurospheres

(**L**, paired *t*-test, *n* = 8 *p* ≥ 0*.*05).

of anterior neurosphere cells culminated in robust detection of the neurotransmitter GABA, which was observed in equal proportions throughout the elaborate neuronal networks produced from control HES3 (**Figures 5A–C**) and H9 (**Figures 5D–F**) as well as HD SI-186 (**Figures 5G–I**) and SI-187 (**Figures 5J–L**) cell lines. Co-staining for β-III-TUBULIN and GABA demonstrate the majority of neuronal cells possess a GABAergic identity (**Figures 5C,F,I,L**).

## **FUNCTIONAL RESPONSES OF NEURONAL CULTURES**

Neurons derived from this culture protocol were compared to their respective vehicle control wells with all four hESC derived neuronal cultures responding to glutamate (30 μM) with a significant elevations of [Ca2+]i, **Figure 5M** for a typical raw trace after background subtraction and **Figure 5N** for a typical response profile (One-Way ANOVA with *post-hoc* Tukey test). Interestingly, neurons derived from the fully penetrant SI-187 cell line responded to glutamate with a significantly greater elevation of [Ca2+]i than neurons from the wildtype HES3 and H9 lines, or the partially penetrant SI-186 line (*p <* 0*.*05, One-Way ANOVA with *post-hoc* Tukey test, **Figure 5O**).

## **TRANSCRIPTIONAL ANALYSIS OF MUTANT HTT MODULATED GENES**

Recent studies of HD human embryonic carcinoma cells (hECCs) and iPSC lines demonstrate *in vitro* gene expression alterations (Gaughwin et al., 2011; Castiglioni et al., 2012). Whether similar transcriptome disruptions are evident in late-onset CAG length hESC lines remains uncertain. Expression levels of critical transcripts identified in previous studies and those with direct paths from mHTT expression to gene disruption were assessed by qRT-PCR in undifferentiated HD and control lines (day 0), within neurospheres derivatives at day 14 and at neuronal promoting stages (day 35) (**Figure 6**). Selected for assessment were neural-specific genes Brain-Derived Neurotrophic Factor (*BDNF;* Zuccato and Cattaneo, 2007, 2009), dopamine receptor D2 (*DRD2*) and proenkephalin (*PENK*) (Luthi-Carter et al., 2000; Dunah et al., 2002; Hodges et al., 2006).  At day 0 and 14 significant downregulation of *PENK* mRNA expression was observed in SI-187 cells compared to HES3 cells (*p <* 0*.*05; **Figure 6C**) and *SREBP1* was significantly up-regulated in SI-187 compared to HES3 at day 0 (*p <* 0*.*05; **Figure 6D**). Nevertheless, significant gene expression changes in *PENK* and *SREBP1* mRNA of HD SI-187 cells were not corroborated when compared to the second control line H9 (*p >* 0*.*05), suggesting these discrepancies are a consequence of interline variation as opposed to CAG repeat length. mRNA expression levels of the remaining genes *BDNF, DRD2, 7-DHCR7*, and *TFEB*

were similar across HD and control lines at days 0, 14, and 35 (*p >* 0*.*05) (**Figures 6A,B,E,F**).

**(D)** H9, as well as HD lines **(G)** SI-186 and **(J)** SI-187. **(C,F,I,L)** Merged

## **ASSESSMENT OF MITOCHONDRIAL GENE EXPRESSION AND FUNCTION**

Mitochondrial dysfunction comprises a robust hallmark of HD pathology, measurable by cellular dysregulation of key transcripts, deficits in mitochondrial energy capacities, and fusion/fission events (Cantuti-Castelvetri et al., 2005; Cui et al., 2006; Knott et al., 2008; Song et al., 2011b). qRT-PCR analysis of mitochondrial genes *PGC-1*α, a master regulator of mitochondrial biogenesis and energy metabolism and *DRP1*, a regulator of mitochondrial fusion and fission showed no consistent differences in transcript levels between both control and HD hESCs or neural differentiated derivatives (**Figures 7A,B** respectively). Intriguingly, significant (*p <* 0*.*05) downregulation of SI-187 *PGC-1*α mRNA levels compared to H9 samples were observed at d14 and a similar yet non-significant trend was seen with SI-186 at d14 and both HD lines at d21 compared to wildtype controls (**Figure 6B**). Mitochondrial functional JC-1 assays that directly measure MMP found all hESC lines demonstrated similar populations of cells exhibiting dual red and green fluorescence (HES3 95%, H9 68%, SI-186 86%, and SI-187 81%; **Figure 7C**), indicative of an active MMP. Remaining green fluorescent cells from each line represented a sub-population of cells with less active mitochondria.

## **DISCUSSION**

neuronal cultures.

Human PSCs provide a unique window to investigate the ramifications of mutant HTT within the intracellular milieu from the earliest stages of human development until the specification of relevant neuronal subtypes. This study assesses two HD hESCs carrying expanded CAG repeat lengths representative of those harbored by individuals with archetypal late clinical onset. For the first time, we report detailed quantitative evaluations of the pluripotent properties and neural differentiation capabilities of these HD hESC lines using two wildtype control hESC lines as biological comparisons. Our studies of undifferentiated hESCs show expanded CAG repeat tracts have no adverse effects on stem cell parameters that were assessed, such as viability, mitochondrial

function, proliferation, and pluripotency markers. These findings are in agreement with recent pluripotent HD studies that observe no disturbance to similar parameters (Gaughwin et al., 2011; Camnasio et al., 2012; Castiglioni et al., 2012; HDIPSCC, 2012; Jeon et al., 2012).

Further, we demonstrate for the first time that neural differentiation of hESCs with HD mutations is equivalent to wildtype controls. Expression of neural markers CD56 and SOX2 and equivalent growth rates of neurospheres show unaltered differentiation in the presence of mutant HTT. Transient lag in the downregulation of the pluripotency marker CD9 was however, observed in one HD line, and corroborates findings of HD hECCs (Gaughwin et al., 2011). Crucially anterior specification assessed by key markers OTX2, FOXG1, and FORSE-1 were equivalent across all lines irrespective of CAG repeat length, and further extended differentiation produced consistent neuronal networks with similar acquisition of GABAergic identities upon *in vitro* maturation. These findings are consistent with mouse and human HD iPSC studies (Camnasio et al., 2012; Castiglioni et al., 2012; HDIPSCC, 2012).

HD patients and animal models are characterized by corticostriatal synaptic dysfunction and profligate excitotoxicity (Raymond, 2003; Estrada Sanchez et al., 2008). A major component of this pathological mechanism is glutamate receptor hypersensitivity on postsynaptic GABAergic striatal neurons. Striatal neurons receive essential glutamate signaling from cortical axons and mutant HTT is directly responsible for excessive Ca2<sup>+</sup> influx downstream of glutamate stimulation, as seen in YAC128 (Tang et al., 2005) and R6/2 (Cepeda et al., 2001) transgenic models. Mutant HTT aberrantly binds endoplasmic reticulum InsP3R1 receptors that are critical for Ca2<sup>+</sup> release. This in turn increases the responsiveness of InsP3R1 receptors to upstream glutamate receptor agonists and induces abnormal Ca2<sup>+</sup> release (Tang et al., 2003, 2005).

In our studies we identified increased neuronal Ca2<sup>+</sup> elevation in the fully penetrant HD line SI-187 upon glutamate stimulation that suggests late-onset HD hESCs are be capable of recapitulating some neuronal pathologies that characterize established HD model systems. Detectable alterations in Ca2<sup>+</sup> glutamate responses are not unexpected with evidence of this phenotype in GABAergic neurons from pre-onset HD mice (Cepeda et al., 2001; Laforet et al., 2001) and even within neurons isolated as early as post-natal day 0-1 (Zeron et al., 2004). Together, these findings suggest increased Ca2<sup>+</sup> responses may occur in early human stages of HD and thus represent a potentially valuable therapeutic target. The absence of such observations in the partially penetrant SI-186 line may correspond with the lesser pathological intensity of mutant proteins comprising 35–39 polyglutamine tracts and prolonged neuronal culture may be necessary to manifest this functional state.

Extensive mutant HTT dysregulation of the cellular transcriptomes is a dominant hallmark seen in post-mortem brain tissue from HD patients and across animal models, reportedly affecting ∼1–2% of total cellular transcripts (Cha, 2000; Luthi-Carter et al., 2002a,b; Hodges et al., 2006). Transcript alteration has emerged as a focal point across HD pluripotent cell studies of aggressive HD subclasses, though little consensus appears to date. Exemplifying this are contrary observations of *BDNF* transcript stability and downregulation compared to controls in generated neural cultures (Gaughwin et al., 2011; Castiglioni et al., 2012; HDIPSCC, 2012). This contradiction is surprising considering clear evidence from model systems of the process by which mutant HTT instigates reduced BDNF expression via nuclear translocation of the transcriptional repressor REST (Zuccato and Cattaneo, 2007; Zuccato et al., 2007). Our study found BDNF is not altered in neural and neuronal derivatives of HD hESCs carrying typical late-onset ranges, suggesting BDNF may not be dysregulated unless significant pathology has progressed.

None of the remaining transcripts analysed in the HD hESC lines were significantly changed at either pluripotent or neural differentiated time points when compared to both control hESC lines. *SREBP1*, a cholesterol biosynthesis regulator, was significantly upregulated in pluripotent SI-187 cells compared to one control and approached significance to the second control line; a finding consistent with overt cholesterol biosynthesis disruption in HD models (Valenza et al., 2007a,b), patients (Markianos et al., 2008) and undifferentiated HD iPSC lines (Castiglioni et al., 2012). Further, there was a transient downregulation of the mitochondrial gene *PGC-1*α at d14 in SI-187 neural derivatives which continued to d21 although the trend was not statistically significant at the latter time-point. To corroborate this finding, we undertook functional studies of MMP activity, which is reduced when *PGC-1*α is down-regulated. In both undifferentiated cells and in d14 neurospheres from SI-186 and SI-187 (data not shown) we found no evidence of altered MMP. Together these findings in conjunction with those of other pluripotent systems imply transcript disturbances in this class of *in vitro* models may be a phenomenon that predominates in lines equivalent to juvenile and infantile disease subclasses, warranting array based studies to scrutinize cellular transcriptomes across a range of lines carrying different CAG repeat lengths.

In summary we have provided detailed characterization of HD hESCs and also those that harbor typical CAG repeat expansions. We demonstrate for the first time that pluripotent dynamics and forebrain neuronal differentiation are unhindered but that glutamate signaling perturbations indicative of Huntington's pathology are observable subsequent to extended *in vitro* culture and cellular acquisition of a forebrain GABAergic neuron identity. These late-onset hESC lines demonstrate the potential of such model systems to provide a platform to interrogate disease mechanisms and hallmarks. Advances describing the efficient generation of DARPP32+ medium spiny neurons provide an opportunity for probing HD etiology within a more relevant cell type (Ma et al., 2012) and could be coupled with proteasomal inhibition or oxidative stress to exacerbate disease phenotypes as demonstrated recently (HDIPSCC, 2012; Jeon et al., 2012).

Emergent studies of HD iPSCs describe lines that possess atypical alleles representative of rare juvenile/infantile (CAG60+) or homozygous subclasses that are poorly studied in human patients (Camnasio et al., 2012; Castiglioni et al., 2012; HDIPSCC, 2012; Jeon et al., 2012). The observation of some HD specific phenotypes in these lines demonstrate their potential value as *in vitro* human models, however, such phenotypes rarely correlate across lines and some are seen as contradictory. Existing HD iPSC lines may not faithfully represent the molecular perturbations of late-onset HD since they potentially exhibit unknown consequences by the reprogramming somatic cells exposed to mutant HTT proteins for considerable durations, and hence PGD isolated hESCs described in this study provide a more "natural" cellular alternative.

Late-onset HD alleles appear to provide a unique system to probe pre-onset changes at the molecular level and within a neurological context, as attested by observations of elevated Ca2<sup>+</sup> glutamate responses. Understanding pre-onset HD alterations could elucidate the hierarchical relationship between various HD hallmarks, yet is presently restricted primarily to non-invasive imaging methodologies (Tabrizi et al., 2009; Nopoulos et al.,

## **REFERENCES**

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2011) and peripheral blood analysis (Borovecki et al., 2005; Runne et al., 2007; Bjorkqvist et al., 2008). The application of late-onset hESC to long-term *in vitro* neuronal cultures or cellular challenges that mimic *in vivo* conditions (i.e., oxidative or proteasomal stressors) may provide a system to delineate the thresholds that mark clinical onset that is otherwise difficult in extreme CAG repeat variants. Nevertheless, future work must simultaneously evaluate differing model systems to identify those which most faithfully recapitulate human HD, to steer prospective *in vitro* therapeutic activity toward models with the highest potential.

## **ACKNOWLEDGMENTS**

We are grateful for the intellectual guidance and technical expertise provided by Professors Andrew Elefanty and Ed Stanley, and financial and intellectual support received from Dr Clare Parish. This project was supported by funding from Monash Immunology and Stem Cell Laboratories. Antibodies obtained from the Developmental Studies Hybridoma Bank (DSHB) were developed under the auspices of the NICHD and maintained by The Uni- versity of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Research studies using HD hESC lines stem cell lines was approved by the Monash University Standing Committee on Ethics in Research Involving Humans (2006/063EA).

offspring conceived after ICSI. *Mol. Hum. Reprod.* 6, 861–866.

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derived from human blastocysts. *Science* 282, 1145–1147.

in YAC transgenic mouse model of Huntington's disease. *Mol. Cell Neurosci.* 25, 469–479.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 November 2012; paper pending published: 06 December 2012; accepted: 20 March 2013; published online: 05 April 2013.*

*Citation: Niclis JC, Pinar A, Haynes JM, Alsanie W, Jenny R, Dottori M and Cram DS (2013) Characterization of forebrain neurons derived from late-onset* *Huntington's disease human embryonic stem cell lines. Front. Cell. Neurosci. 7:37. doi: 10.3389/fncel.2013.00037*

*Copyright © 2013 Niclis, Pinar, Haynes, Alsanie, Jenny, Dottori and Cram. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Patient-derived stem cells: pathways to drug discovery for brain diseases

## *Alan Mackay-Sim\**

*National Centre for Adult Stem Cell Research, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, QLD, Australia*

#### *Edited by:*

*Clare Parish, Florey Neuroscience Institute, Australia*

### *Reviewed by:*

*Rheinallt Parri, Aston University, UK Lachlan Thompson, Florey Neuroscience Institute, Australia*

#### *\*Correspondence:*

*Alan Mackay-Sim, National Centre for Adult Stem Cell Research, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, QLD 4111, Australia. e-mail<EMAIL_ADDRESS>
The concept of drug discovery through stem cell biology is based on technological developments whose genesis is now coincident. The first is automated cell microscopy with concurrent advances in image acquisition and analysis, known as high content screening (HCS). The second is patient-derived stem cells for modeling the cell biology of brain diseases. HCS has developed from the requirements of the pharmaceutical industry for high throughput assays to screen thousands of chemical compounds in the search for new drugs. HCS combines new fluorescent probes with automated microscopy and computational power to quantify the effects of compounds on cell functions. Stem cell biology has advanced greatly since the discovery of genetic reprograming of somatic cells into induced pluripotent stem cells (iPSCs). There is now a rush of papers describing their generation from patients with various diseases of the nervous system. Although the majority of these have been genetic diseases, iPSCs have been generated from patients with complex diseases (schizophrenia and sporadic Parkinson's disease). Some genetic diseases are also modeled in embryonic stem cells (ESCs) generated from blastocysts rejected during *in vitro* fertilization. Neural stem cells have been isolated from postmortem brain of Alzheimer's patients and neural stem cells generated from biopsies of the olfactory organ of patients is another approach. These "olfactory neurosphere-derived" cells demonstrate robust disease-specific phenotypes in patients with schizophrenia and Parkinson's disease. HCS is already in use to find small molecules for the generation and differentiation of ESCs and iPSCs. The challenges for using stem cells for drug discovery are to develop robust stem cell culture methods that meet the rigorous requirements for repeatable, consistent quantities of defined cell types at the industrial scale necessary for HCS.

**Keywords: embryonic stem cells, induced pluripotent stem cells, olfactory stem cells, olfactory neurosphere-derived cells, high content screening**

## **INTRODUCTION**

The last decades have seen development and applications in cellular neuroscience of DNA, RNA, and protein analysis technologies that provide large volumes of information, getting away from the traditional methods that follow the activities of single genes or single cell types. The application of DNA sequencing, RNA microarrays and higher throughput protein mass spectrometry is allowing examination of biological systems in all their complexity. What has been missing for cell biologists is the ability to interrogate cell functions at the same scale and level of complexity. This is being addressed by the developments in automated technologies developed by pharmaceutical companies to screen massive compound libraries for the discovery of new drug leads. Until recently this high throughput screening, with robotic control and automated data capture and analysis of experiments in 96-, 384-, and 1,536-well plates, centered on enzyme and receptor assays but increasingly, interest is turning to cell-based assays that capture the complexity of the environment in which drugs will actually operate. There has been a move to drug screening based on cellular outcomes (e.g., cancer cell apoptosis, inhibition of growth) rather than predicted mechanism (e.g., enzyme

inhibition, receptor antagonism). This led to the concept and development of "high content screening" (HCS) that combines advances in fluorescence labeling of cells, robotic and automated microscopy, and automated image analysis that brings the analysis of cell functions to the high throughput formats of multiwell plates (Zanella et al., 2010). For the neuroscientist this technology opens up new frontiers in the ability to manipulate many experimental variables simultaneously in highly controlled experiments. For example, the effects of multiple drugs at multiple doses could be tested simultaneously on several cell types. With the many fluorescent reporter methods now available it is possible to follow three or four cellular events in the same experiment in hundreds of cells per well. With many well established platforms available, HCS opens the door to neuroscience for large scale, high throughput cell function analyses for understanding cell functions in health and disease.

In neuroscience, the discovery of the genetic causes of familial diseases has driven understanding of the functions of individual genes and proteins in cell function in the nervous system and the effects of mutations on brain function. Identification of a candidate gene is followed by genetically modified cell and

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mouse models to identify the functions of the identified gene at cell and systems level. Mouse models have been important for elucidating protein and gene functions but they often do not recapitulate human disease because the mice lack the human genetic background and the introduced human genes are acting in a non-human cellular context. Immortalized human cell lines have a more relevant genetic background but, being derived from tumors, they may not reflect a normal cellular context. Patientderived primary cells might be a solution for both drawbacks but they cannot usually be maintained for very long in culture and finding an accessible cell type for brain diseases is problematic. Stem cell technologies may have the solution to these drawbacks.

There is an emerging interest in using stem cells to understand the cellular bases of human diseases. There is an imperative here, especially for neurological diseases and conditions. Large pharmaceutical companies are withdrawing from investing in neuroscience research because of the failure of the current paradigms to convert findings in animal models to drugs for human disease (Schnabel, 2008). There are obviously many reasons why animal models, genetic or otherwise, are not proving useful for predicting human responses to drugs. On the other hand, there are good reasons to expect that human stem cells might be useful, if they can be derived from patients with a disease and, if they are the cell types that are affected by the disease. This is clearly a niche that stem cells have the developmental abilities to occupy. Patient-derived stem cells could be used to identify cell functions that are altered by disease and thereby provide a target for drug discovery. Assays can then be developed for HCS to use the patientderived stem cells to screen large drug libraries for therapeutic activity.

## **HIGH THROUGHPUT SCREENING**

Increasingly in the last 20 years pharmaceutical companies have developed high throughput technologies to screen large chemical libraries of natural products and synthetic compounds for activities against selected enzymes and receptors, candidate targets for diseases of interest (Macarron et al., 2011; **Figure 1**). These high throughput technologies arose from the desire of pharmaceutical companies to test all theoretical chemical compounds (∼100 million) against all theoretical human biological targets, estimated from the number genes (∼20,000) or proteins (∼200,000), indicating that as many as 10<sup>12</sup> assays would be needed to identify all interactions between chemicals and targets (Sundberg, 2000). These technologies are pharmacology on a large scale, initially using 96-well plates and now routinely 384- and 1,536-well plates. The imperative is to provide a means to screen very large compound libraries in order to find the very small percentage of lead compounds that are active in a selected assay. High throughput screening is historically based on solution based enzyme and receptor assays using scintillation, absorbance, fluorescence, and chemiluminescence with the aim of finding highly specific chemical interactions with individual biological targets (Sundberg, 2000). High throughput screening is also used in cell-based assays, typically to monitor activation of cell surface receptors through subsequent transduction pathways, transcription events, cell proliferation, or cell death. These assays use multiwell plates and monitor colorimetric, fluorescence, luminance, or absorbance within each well. With developments in genetic technologies and the understanding provided by the Human Genome Project, intracellular events are available for high throughput screening through the development genetic fluorescent reporter assays. Cell lines have genes for luminescent or fluorescent proteins, like luciferase or green fluorescent protein (GFP), spliced into reporter systems to read out activation or inhibition of specific genes or proteins of interest (Sundberg, 2000). High throughput technologies have advanced the development of robotic automation for cell culture, assays, and compound library storage; automated and multipurpose plate readers; fluorescence dyes and reporter systems; computational power, and automated data storage and analysis. It currently takes about 1 week to screen 10,000 compounds against a target and 1–3 months to screen a library of one million compounds (Macarron et al., 2011). High throughput screening has been successful in delivering numerous drugs from discovery through to clinical use and the market starting from chemical libraries of 200,000–500,000 compounds (Macarron et al., 2011).

## **HIGH CONTENT SCREENING**

High content screening is a further development in which the principles of high throughput screening are applied to the analysis of individual cells through the use of automated microscopy and image analysis (Zanella et al., 2010; **Figure 2**). This allows quantitative analyses of components of cells such as spatio-temporal distributions of individual proteins, cytoskeletal structures, vesicles, and organelles when challenged with chemical compounds. HCS can be used to monitor activation or inhibition of individual proteins and protein–protein interactions as well as allowing analysis of broader changes in biological processes and cell functions. Recent advances in the range of fluorescent probes for biological processes, functions, and cell components have combined with developments in fluorescence microscopy to give the cell biologists many new ways to understand cell functions in health and disease. These cell-centric developments have converged with high throughput concepts and with developments in automated microscopy and image analysis to evolve into the new technological synthesis of high throughput screening in which cell-based assays are conducted in multiwell plate formats. High content technologies are now used to screen chemical libraries for drug discovery as well as genome-wide RNA interference libraries to probe gene functions (Zanella et al., 2010). One of the advantages of HCS is the ability to apply different criteria to selected cells in the population to account for the heterogeneity of the cell population. For example, a chemical compound of interfering RNA may only act on cells in a particular stage of the cell cycle. Well-based assays provide a readout from the whole cell population, a mixture of cells in different phases of the cell cycle, whereas with cell-based HCS and appropriate markers, cells at identified phases of the cell cycle could be assessed independently. Thus HCS allows quantitative analysis of complex and heterogeneous cell cultures containing multiple cell types. Another advantage of HCS technologies is the ability to make multiple independent and quantitative measurements from single cells of interest. For example, transmitted light might be used to assess morphology; three or more fluorophores

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assays to identify active molecules modulating a biomolecular activity. Assays are performed in multiwell plates with robotics for cell and liquid handling and other aspects such as moving plates from incubator to drug delivery to plate reader. An assay for a cell function of interest must be validated for multiwell plate readers, such as fluorescence, absorbance, or luminescence. Here the assay is a fluorescence assay (Cyquant) for cell number. High levels of quality assurance and quality control are required to reduce the variance of the measurement so that the assay can be reliably scaled to multiple plates. Typically, thousands of compounds are screened for activity, with "hits" being defined as those compounds whose activity on the assay falls outside three

screens are then used to validate the initial hits. These screens may include dose–response curves or assays of related functions of interest, resulting in a selection of "validated hits," which may comprise fewer than 1% of a typical library of compounds of varied structure. Here the validated hits are the numbered dots above the red line. Validated hits are then further assayed and modified chemically in an iterative process to improve potency, selectivity, pharmacological properties, and toxicological profile to produce a drug lead compound that will go into "preclinical" testing in animal models. High throughput screening is applied to large libraries of any potentially bioactive molecules including natural products, small molecule drugs, RNAs, and DNAs.

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**FIGURE 2 | High content screening: industrial scale microscopy.** High content screening is the automated microscopic analysis of fluorescence in cells of a multiwell plate. Most high throughput screening assays read activity per well (e.g., fluorescence, absorbance, and luminescence). In high content screening fluorescence in individual cells is quantified with automated image analysis. Typically, algorithms find the cells in the image and then measure fluorescence intensity and spatial distribution. The example shows tubulin fluorescence in cells (green) and cell nuclei (blue) at different concentrations of taxol, a tubulin-binding drug. The drug effect is difficult to see by eye but the analysis algorithm identifies each cell and quantifies tubulin fluorescence intensity, plotted here as a dose–response curve to two drugs, taxol (blue) and vinblastine (red) in two cell types: control-derived ONS cells (solid lines) and HSP patient-derived ONS cells (dashed lines; Abrahamsen et al., 2013). With high content screening thousands of cells can be analyzed to reveal small but significant differences between drugs, doses, and cell types.

might be used to identify molecular components, structures, or organelles; and image analysis might be used to quantify spatial relationships between the fluorescent reporters under control conditions or when challenged with chemical compounds. With assays performed in multiwell plates there is opportunity to scale up experiments to include replicates, concentration– response curves, and parallel assessments of different cells or compounds. Modern instruments equipped with incubators and confocal optics have the ability to investigate cells over time in three spatial dimensions. These technologies will advance further and become cheaper allowing HCS principles to be applied increasingly by academic labs and not restricted to large pharmaceutical companies.

In the neurosciences, there is now an opportunity and challenge to combine patient-derived, disease-specific stem cells with HCS technologies with the aim of finding new drugs for brain diseases and conditions. This is not a simple aspiration because the majority of brain diseases are a result of complex genetic and environmental risk factors. Furthermore, brain diseases are usually just that, "brain" diseases and not "cell" diseases in the sense that cancers are. Nonetheless, it is possible that most brain diseases result from identifiable cellular dysfunctions such as those identified in monogenic disease. Such mutations tell us that specific brain dysfunctions can be manifest in specific cell types and pathways, despite universal genetic mutation. This gives hope that cellular models will shed light on the molecular and cellular mechanisms of emergent properties (e.g., cognition, emotion) evident when the brain functions as a whole.

## **DISEASE-SPECIFIC PLURIPOTENT STEM CELL MODELS OF NEUROLOGICAL DISEASE**

The analysis of gene function through gain- or loss-of-function in cell, fly, and mouse models has been very instructive in elucidating functions of genes and proteins but less successful in providing models that predict drug efficacy in human diseases. An example is the failure of the superoxide dismutase transgenic mouse model for amyotrophic lateral sclerosis that has yielded multiple compounds that are therapeutic in mouse but not humans (e.g., creatine; Klivenyi et al., 1999; Groeneveld et al., 2003). These problems arise because of species and model differences which do not mimic gene dosage, gene mutation variability or genetic background, or the complex of other contributing genetic factors that will be present in individuals with a disease. Patient-derived cells provide a potential solution to some of these problems because they reflect the genetic background and variation of the human population, arising from individuals with natural mutations. Sources of patient-derived cells have included easily accessible fibroblasts and lymphocytes and lymphoblastoid cell lines but there is concern that these cells will not manifest the same disease-associated properties as the cells specifically dysfunctional in the disease of interest, for example dopaminergic neurons of the substantia nigra in Parkinson's disease or oligodendrocytes in multiple sclerosis. For such cell type-specificity the field holds high hopes of pluripotent stem cells which can theoretically be induced to differentiate into any cell type of interest.

Disease-specific pluripotent stem cells include human embryonic stem cells (ESCs) with genetic or chromosomal disorders

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derived from surplus blastocysts during *in vitro* fertilization and pre-implantation genetic diagnosis (Stefanova et al., 2012). Although these are not strictly "patient-derived" they carry specific genetic mutations or deletions that would normally lead to disease. Induced pluripotent stem cells (iPSCs) from patients have become the dominant choice for patient-derived pluripotent stem cells. A recent review lists 18 diseases for which ESCs have been derived, compared with 40 for which iPSCs have been derived (Grskovic et al., 2011). At this time many of the publications in this field are mainly demonstrations that pluripotent stem cells have been derived, often without demonstrating a disease-phenotype. Some show that the pluripotent cells can be differentiated into specific cell types of interest and some demonstrate deficits in cellular functions compared to control cells, as proof-of-principle for disease modeling (Grskovic et al., 2011; Maury et al., 2012). No doubt the numbers of diseases for which iPSCs are available will increase greatly in the next few years and deeper analyses of their functions will be forthcoming. It is a major challenge for the field to move beyond the proof-of-principle stage to discovery of new aspects of disease biology and new targets for therapeutic intervention.

The list of neurological diseases and conditions for which ESCs or iPSCs have been derived is largely limited to monogenic diseases including Charcot–Marie–Tooth disease type 1A, Down syndrome-trisomy 21,familial amyotrophic lateral sclerosis,familial dysautonomia,familial Parkinson's disease, Fragile X syndrome, Friedreich ataxia, Gaucher's disease, Huntington's disease, Rett syndrome, Spinal muscular atrophy, spinocerebellar ataxia types 2 and 7, and X-linked adrenoleukodystrophy (Grskovic et al., 2011; Maury et al., 2012; Rajamohan et al., 2013). It is thought that diseases of complex genetics and environmental risk factors may be harder to model with pluripotent stem cells but patient-derived iPSCs have been generated from patients with Parkinson's disease (Soldner et al., 2009) and schizophrenia (Brennand et al., 2011; Pedrosa et al., 2011). Patient-derived iPSCs from people with sporadic Parkinson's disease were differentiated into dopaminergic neurons but failed to show an obvious difference in phenotype compared to control cells (Soldner et al., 2009). Similarly, a disease-associated phenotype could not be demonstrated in iPSCs from two cases of sporadic Alzheimer's disease (Israel et al., 2012). In one study, iPSCs from schizophrenia patients were differentiated into neurons and gene expression profiling identified a cluster of differentially expressed genes involved in neurogenesis, neuronal differentiation, axon guidance, and adhesion with another cluster of differentially expressed genes involved in cell cycle regulation (Pedrosa et al., 2011). A second study in schizophrenia showed that neurons differentiated from patient-derived iPSCs had reduced neurite number and reduced connectivity with other neurons*in vitro* and reduced glutamate receptor expression (Brennand et al., 2011). These studies of patient-derived iPSCs from schizophrenia patients demonstrate that such models can reveal disease-associated cellular deficits in a disease of complex genetics, although the patients were all from families with psychosis, rather than sporadic cases.

It is a challenge to translate pluripotent cells into robust disease models (Maury et al., 2012). For example, ESCs are limited by the availability of genetic testing and pre-implantation genetic diagnosis. For their part, iPSCs are potentially compromised by the methods of their generation; most cell lines published to date were produced by integrating vectors, although this will change as the efficiency and predictability of non-integrating methods improves. There are other technical challenges for disease modeling such as the current lack of robust and efficient protocols for differentiating ESCs and iPSCs into disease-associated cells of interest. Attention is drawn to the importance of selecting appropriate matched controls because case–control cell differences could arise from sampling bias and "disease-associated" differences may result from particular comparator control cells (Zhu et al., 2011; Maury et al., 2012). This difficulty is compounded by the large cost of generation and maintenance of ESCs and iPSCs that will limit sample sizes for most laboratories. One preferred method would be to use each patient-derived cell line as its own control by correcting its genetic defect (Zhu et al., 2011; Maury et al., 2012) but this is possible only in the monogenic disease cases of known mutations that can be selectively corrected. It is expected that many of these challenges will be overcome as new methodologies develop for efficient production of patient-derived iPSCs that are generated with non-integrating reprograming methods (Grskovic et al., 2011) and efficient methods are devised for differentiation into desired cell types: specific neurons, astrocytes, and oligodendrocytes.

## **PATIENT-DERIVED OLFACTORY STEM CELLS AS MODELS FOR NEUROLOGICAL DISEASES**

Published studies of ESCs and iPSCs as disease models are all confined to small numbers of cell lines from patients and controls. This makes it difficult to generalize from these case–control studies to the general population. Variability in the reprograming process and epigenetic status makes it essential that several clones from several individuals are compared to confirm that a "disease-phenotype" is not confounded by individual differences among case or control cell lines (Vitale et al.,2012). Ideally, patientand control-derived cells should be sampled from large populations to be certain that differences between the samples are representative of the population. In the case of genetic diseases with high penetrance of the clinical phenotype, the case–control design may be robust to small sample size but this may not be true of lower penetrance phenotypes, or of sporadic diseases of complex genetics. This sampling problem can be overcome with accessible cells that can be derived easily from larger numbers of individuals, such as patient-derived fibroblasts and lymphocytes, which have been used to identify potential cellular mechanisms or biomarkers of neurological diseases including Alzheimer's disease (Takahashi et al., 1999; Moreira et al., 2007), Parkinson's disease (Martin et al.,1996; Hoepken et al.,2008), and schizophrenia (Ilani et al., 2001; Wang et al., 2010). Although these cell types have identified cellular and molecular differences between patients and controls, their relevance to diseases of the nervous system is moot and the patient–control differences can be non-existent (Matigian et al., 2008).

Another approach to modeling diseases is to sample patientderived adult stem cells. Neural progenitor cells were isolated from post-mortem brain from Alzheimer's patients and healthy controls (Lovell et al., 2006). Mesenchymal stem cells were isolated from

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controls and patients with amyotrophic lateral sclerosis (Ferrero et al., 2008) and Parkinson's disease (Zhang et al., 2008). None of these studies reported disease-associated differences, on the contrary all stated that patient and control cells were of similar phenotype.

There is a multipotent adult stem cell resident in the olfactory mucosa, the organ of smell in the nose (Murrell et al.,2005). A stem cell in the olfactory epithelium maintains a continual neurogenesis throughout life that regenerates the sensory neurons (Mackay-Sim and Kittel, 1991a,b; Murrell et al., 1996). Olfactory epithelium is one of the few places in the nervous system that contains a neural stem cell but, unlike the other sites in the brain, it is accessible under local anesthetic (Feron et al., 1998) and several studies have shown disease-associated differences in cell biology in this tissue. Olfactory neuroblasts isolated from post-mortem epithelial biopsies from controls and individuals with Alzheimer's disease demonstrated differences in amyloid precursor protein processing (Wolozin et al., 1993) and oxidative stress (Ghanbari et al., 2004). Histological analysis of olfactory epithelium indicates disturbed neural differentiation in schizophrenia (Arnold et al., 2001) and Rett syndrome (Ronnett et al., 2003). Cultures of olfactory mucosa show increased cell proliferation and reduced tissue adhesion in schizophrenia compared to healthy controls (Feron et al., 1999; McCurdy et al., 2006). Clearly the olfactory organ demonstrates disease-associated differences in cell biology in several brain diseases including a monogenic disease (Rett syndrome) and complex genetic diseases of no known genetic cause (Alzheimer's disease and schizophrenia). This led to the development of an olfactory stem cell-based system for investigating brain diseases based on olfactory neurosphere-derived cells (ONS cells; Matigian et al., 2010).

The olfactory mucosa comprises the superficial epithelium and the underlying lamina propria separated by a basement membrane. Within the epithelium are basal cells among which are the multipotent stem cells that can regenerate all the cell types of the epithelium including the sensory neurons as well as other non-neural supporting and gland cells (Leung et al., 2007; Packard et al., 2011). Within the human lamina propria is a multipotent stem cell with characteristics both of neural and mesenchymal stem cells, an ectomesenchymal stem cell (Delorme et al., 2010). The lineage relation between these stem cells, if any, is not known. When biopsies of human olfactory mucosa are dissociated and grown in a serum-free medium containing epidermal growth factor (EGF) and basic fibroblast growth factor 2 (FGF2), neurospheres form that can differentiate into neurons and glia and cells of non-ectodermal lineage, including developing cardiac and skeletal muscle, kidney and liver, and blood (Murrell et al., 2005). Neurospheres are dissociated and when the cells are grown as adherent cultures in a serumcontaining medium, these"neurosphere-derived" cells (ONS cells) have a flattened, undifferentiated appearance with a marker phenotype similar to the ectomesenchymal cells derived from primary cultures of olfactory mucosa (Delorme et al., 2010; Matigian et al., 2010), that is, they express markers of both neural and mesenchymal lineages. Patient-derived ONS cells show robust disease-specific differences compared to ONS cells derived from healthy controls (Mackay-Sim, 2012). Gene expression profiling of ONS cells from healthy controls and patients with Parkinson's disease or schizophrenia showed disease-specific differences in gene expression, protein expression, and cell functions (Matigian et al., 2010). Disease-associated gene expression differences were quite different in schizophrenia and Parkinson's disease, with alterations in neurodevelopmental pathways in schizophrenia and in oxidative stress and metabolic function in Parkinson's disease (Matigian et al., 2010). Deeper analysis of the gene expression showed disease-specific differences in the variance of gene expression in the major signaling pathways. Overall schizophrenia patient-derived ONS cells were less variant in their gene expression compared to controls whereas Parkinson's patient-derived ONS cells were more variant than controls, indicating another dimension along which diseases can affect cell functions (Mar et al., 2011).

Parkinson's patient-derived ONS cells showed gene expression and functional differences indicating dysfunctions in pathways involved mitochondrial metabolism and oxidative stress (Matigian et al., 2010). Pathway analysis of the gene expression differences found that the antioxidative nuclear factor (erythroid-derived 2) like 2 (NRF2) signaling pathway was overrepresented among the dysregulated genes (Matigian et al., 2010). NRF2 is a transcription factor that ameliorates the effects of oxidative stress. Deeper analysis of the ONS cells demonstrated that the NRF2 signaling pathway was dysfunctional: although the NRF2 protein was equally expressed in patient and control cells the downstream targets of NRF2 were reduced, suggesting lesser activation of NRF2 signaling in the patient cells (Cook et al., 2011). In contrast, schizophrenia patient-derived ONS cells showed gene expression and functional differences indicating dysfunctions in pathways involved neurodevelopment (Matigian et al., 2010). Pathway analysis of the gene expression differences identified aspects of the cell cycle that were dysregulated, in particular G1/S phase transition, the check point in cells that controls the beginning of DNA synthesis (Matigian et al., 2010). Functional analysis demonstrated that patient-derived cells proliferated faster than control-derived cells in accord with previous observations in olfactory biopsy cultures (Feron et al., 1999; McCurdy et al., 2006). This faster rate of cell proliferation was due to a 2-h shorter cell cycle period in patient cells (Fan et al., 2012). G1/S phase transition is dependent on the intracellular levels of cyclin D1, a cell cycle control protein; cyclin D1 levels were elevated in patient cells and reached higher levels more quickly than control cells (Fan et al., 2012). In schizophrenia, ONS cells gene expression was significantly dysregulated in the focal adhesion kinase signaling pathway, which is involved in regulating attachment to the extracellular matrix through cell surface integrin receptors (Fan et al., 2013). Functional analyses of the patient-derived cells showed they were less adhesive and more motile than control-derived cells with smaller and fewer sites of adhesion that disassembled more quickly than in control-derived cells (Fan et al., 2013). These studies demonstrate that patientderived ONS cells can show robust disease-specific differences in cell biology even in sporadic diseases of complex genetics.

Olfactory neurosphere-derived cells are also proving useful for understanding monogenic diseases. Hereditary spastic paraplegia (HSP) is an autosomal dominant disease affecting the long spinal axons from the motor cortex to the lower motor neurons

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in the spinal cord. ONS cells from patients with HSP were similar in many basic cell functions to ONS cells from healthy controls despite dysregulation of expression of 60% of the genome, indicating a high level of homeostatic regulation in response to dominant mutations in *SPAST*, which codes for a microtubule severing protein (Abrahamsen et al., 2013). Closer inspection of cell functions using HCS revealed significant reductions in stable microtubules and in the intracellular distributions of peroxisomes and mitochondria (Abrahamsen et al., 2013). Live-cell time-lapse imaging revealed that peroxisomes traveled more slowly in HSP patientderived cells, consistent with the corticospinal axon pathology in HSP (Abrahamsen et al., 2013). Ataxia telangiectasia (AT) is a fatal autosomal recessive disease characterized by radiation sensitivity, cancer, and cerebellar dysfunction. ONS cells from children with AT had radiation sensitivity and DNA-repair deficits that were corrected by insertion of the full-length gene (Stewart et al., 2013). Immature neurons from AT patient-derived ONS cells showed evidence of impaired differentiation (Stewart et al., 2013). Olfactory ectomesenchymal stem cells, similar to ONS cells, demonstrated a disease-associated effect of mis-splicing of the *IKBKAP* gene leading to reduced levels of IKBKAP protein and altered cell migration in patient-derived cells in familial dysautonomia (Boone et al., 2010).

## **PATIENT-DERIVED OLFACTORY STEM CELLS FOR DRUG DISCOVERY**

Patient-derived ONS cells have several advantages for HCS for drug lead identification. They are cheap to grow and maintain, growing in standard cell culture conditions with no expensive growth factors after the neurosphere-forming stage. As ONS cells they can be grown for at least 16 passages without significant change in gene expression thus demonstrating minimal phenotypic change and without change in karyotype (unpublished observations). ONS cells are derived from neural tissue and can obviously show disease-specific phenotypes relevant to the neurological diseases from which the donors suffer. Proof-of-principle analyses have shown that brain diseases "in a dish" can be ameliorated by drug treatment. For example, Parkinson's patient-derived ONS cell functions were restored to control-derived cell levels by treatment with L-sulforaphane, an agonist of NRF2 (Cook et al., 2011). Similarly, a small molecule drug kinetin was able to reverse the mis-splicing of the *IKBKAP* gene in familial dysautonomia patient-derived ectomesenchymal cells (Boone et al., 2010) as it has in iPSCs from these patients (Lee et al., 2009). HCS was used to test the differential sensitivity of HSP patient-derived and control-derived ONS cells to the tubulin-binding drugs, taxol and vinblastine (Abrahamsen et al., 2013). HSP patient-derived cells had 50% the control level of spastin, a tubulin-severing enzyme, 150% of the control level of stathmin, a tubulin depolymerizing enzyme, and 50% of the control level of acetylated a-tubulin, an indicator of stabilized microtubules (Abrahamsen et al., 2013). HCS showed that patient-derived and control-derived ONS cells were highly sensitive to the drugs, with effects at 0.1 nM (**Figure 2**). Both drugs increased acetylated a-tubulin, but with different dose–response curves, and low doses of both drugs (∼0.3 nM) restored patient cell acetylated a-tubulin to the control cell level (Abrahamsen et al., 2013).

These experiments show that disease-associated dysfunctions in olfactory cells can be ameliorated by candidate chemical compounds acting on targets known to be disrupted in the patientderived cells compared to controls. The next challenge is to see whether ONS cells are useful for screening libraries of compounds. They have some of the necessary characteristics such as ease of generation, low cost, robust and repeatable growth characteristics and predictable phenotype. These properties make them useful for building up banks of cells that will allow assessment of variability of cell biology across a wider population of patients and controls, to discriminate disease-specific differences from individual differences in complex diseases like Parkinson's disease and schizophrenia.

## **FUTURE PROSPECTS**

One of the challenges for the field is to develop robust and repeatable protocols for producing the large quantities of specified neurons or glia that are required for high throughput screening. For ESCs and iPSCs differentiating protocols exist for making different types of neurons, such as dopaminergic neurons, cortical neurons, and motor neurons (Chambers et al., 2009) but the yield is generally low and variable. This is not necessarily limiting for low-throughput investigations of disease-associated cell phenotypes and lead validation but is certainly limiting for primary drug screens. ONS cell production is not limiting for drug discovery but they currently also lack robust and repeatable protocols for differentiating them on demand into neurons and glia *in vitro*. In growth factor-free medium 50% of the cells were "astrocytes" [glial fibrillary acidic protein (GFAP)-positive cells], a proportion elevated by ciliary neurotrophic factor (Murrell et al., 2005). "Neurons" (β-tubulin-III-positive cells) were less frequent but rose to 25% of the population with nerve growth factor in the medium. (Murrell et al.,2005). Retinoic acid induced a majority (50%) of O4-positive "oligodendrocytes" (Murrell et al., 2005). ONS cell differentiation was defined in terms of morphology and immunofluorescence and so lacks the definitive demonstration of differentiation shown repeatedly for ESCs and IPSCs.

The concept of drug discovery through patient-derived stem cell models of brain diseases is attractive but has many other challenges apart from the practical issues of cost and reliable production. Concerns are raised about the epigenetic status of iPSCs and ESCs – epigenetic status is variably altered by reprograming and by culture methods (Stadtfeld and Hochedlinger, 2010) hence diseases in which epigenetics plays a role may not be modeled well by iPSCs and ESCs (Zhu et al., 2011; Maury et al., 2012). There is not yet a consensus about which cell type is the best for reprograming (Stadtfeld and Hochedlinger, 2010) or which method of reprograming is the most robust and reliable (Grskovic et al., 2011). Differentiation of human iPSCs is not yet routine and predictable and can take many weeks (Grskovic et al., 2011). There is discussion about the applicability of iPSCs to model late-onset diseases like Parkinson's disease or Alzheimer's disease, with the inference that iPSCs may be better suited to neurodevelopmental disease modeling (Juopperi et al., 2011). Olfactory stem cell models appear to be suited to late-onset and sporadic diseases perhaps because they are not subject to the genetic and epigenetic changes due to reprograming. Olfactory stem cells are relatively

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inexpensive and can be reliably grown in large quantities but they also suffer from difficulties in robust and routine differentiation protocols. As more diseases get modeled by iPSCs and ONS cells the strengths and weaknesses of each model will be determined for specific applications and the relevance of each model to the disease of interest will be determined. The use of particular cell models for drug discovery will depend on many variables including relevance to the disease, ease and cost of use, and stage of the drug discovery process (**Figure 3**). These questions and challenges are all signs of a field at the very beginning of its genesis and many will undoubtedly be resolved in the coming years.

Other developments in reprograming will affect this future. It is now possible to generate neurons directly from skin fibroblasts (Vierbuchen et al., 2010). This may greatly reduce the cost of producing patient-derived neurons for drug discovery because fibroblasts, like ONS cells are cheaper to generate, expand, and maintain than iPSCs. Through similar direct reprograming it may be possible to on demand specific classes of neurons, astrocytes, and oligodendrocytes. Ultimately whether any of these new technologies reach routine application in drug discovery will depend on cost, robustness, and industrial scalability as well as the biological validity of the cells as disease models.

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## **ACKNOWLEDGMENTS**

This work was funded in part by grants from the Hereditary Spastic Paraplegia Research Foundation Inc., the

## **REFERENCES**

National Health and Medical Research Council of Australia, and the Australian Government Department of Health and Ageing.

Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis. *Nat. Med.* 5, 347–350.

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S., et al. (2010). Expression profiling of fibroblasts identifies cell cycle abnormalities in schizophrenia. *J. Proteome Res.* 9, 521–527.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 November 2012; paper pending published: 07 December 2012; accepted: 06 March 2013; published online: 27 March 2013.*

*Citation: Mackay-Sim A (2013) Patientderived stem cells: pathways to drug discovery for brain diseases. Front. Cell. Neurosci. 7:29. doi: 10.3389/fncel.2013. 00029*

*Copyright © 2013 Mackay-Sim. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

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## Generating regionalized neuronal cells from pluripotency, a step-by-step protocol

## *Agnete Kirkeby\*, Jenny Nelander and Malin Parmar*

*Department of Experimental Medical Science and Lund Stem Cell Center, Lund University, Lund, Sweden*

#### *Edited by:*

*Clare Parish, Florey Neuroscience Institute, Australia*

#### *Reviewed by:*

*Chang-Hwan Park, Hanyang University, South Korea Sonja Kriks, Memorial Sloan-Kettering Cancer Center, USA*

#### *\*Correspondence:*

*Agnete Kirkeby, Department of Experimental Medical Science and Lund Stem Cell Center, Lund University, Lund, SE-221 84, Sweden.*

*e-mail<EMAIL_ADDRESS>
Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling. Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion. Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain. Here, we present a step-by-step protocol for neuralization and regionalization of human pluripotent cells for transplantation studies or *in vitro* analysis.

**Keywords: human embryonic stem cells, pluripotent stem cells, differentiation, protocol, neuronal subtypes, regionalization, GSK3, brain development**

## **INTRODUCTION**

Given that the human brain is far more complex than brains of rodents, chicks, or invertebrate model organisms, it is clear that knowledge of human brain development cannot be fully extrapolated only from animal models. Therefore, studies on human fetal tissue in combination with a reliable human *in vitro* modeling system are required. Due to very limited access to fetal tissue at different developmental time points, human cell sources such as pluripotent cells are necessary to allow for comprehensive, dynamic analysis of human neural development, and to generate sufficient amounts of cells for future regenerative therapies (Kirkeby and Parmar, 2012).

Based on previously published methods for neural differentiation (Zhang et al., 2001; Perrier et al., 2004; Nat et al., 2007; Chambers et al., 2009; Fasano et al., 2010), we have developed and refined a standardized protocol for rapid neural conversion of pluripotent cells using defined culture settings free of feeder cells, matrigel, knockout serum replacement (KSR), and manual picking steps (Kirkeby et al., 2012). By mimicking developmental cues important for neural tube patterning in mammals, the cells can be efficiently patterned to regionalized cultures of a high purity. The protocol presented here is based on rapid neural conversion through dual SMAD inhibition (Chambers et al., 2009) and regionalization of the cells in this protocol constitutes a flexible system controlled by dose-dependent chemical inhibition of glycogen synthase kinase 3 (GSK3) for rostro-caudal patterning and dose-dependent activation of sonic hedgehog (SHH) and bone morphogenetic protein (BMP) signaling for dorso-ventral patterning (Kirkeby et al., 2012).

## **DIFFERENTIATION PROCEDURE**

The protocol presented here will produce neural cells of a telencephalic fate in the absence of any patterning factors. To control rostro-caudal and dorso-ventral patterning, see separate section below. This protocol is preferably started on a Monday to avoid medium changes during the weekends (see overview of differentiation procedure in **Figure 1**). It is important to start the differentiation with healthy, pluripotent cells. All differentiated colonies should be removed from the culture before initiating differentiation.

**Optional**: If pluripotent cells are cultured on mouse embryonic fibroblasts (MEFs), colonies can be passaged onto matrigel in conditioned media or onto vitronectin in Essential 8 medium for 1 passage (3–7 days) before starting differentiation. This step will remove MEFs from the culture. Differentiation may also be initiated from colonies on MEFs, but if too many MEFs are present in the culture, this may result in attachment of embryoid bodies (EBs) to the bottom of the culture dish.

**NOTE**: When dissociating cells with EDTA, a washing step is not necessary. Here, cells are spun down to avoid dilution of the differentiation medium with EDTA solution. Alternatively, colonies can be enzymatically dissociated with dispase, but this requires additional washing steps and may result in a lower cell yield.

3. To start differentiation, re-suspend the cells in differentiation medium: NIM + Y-27632 (10μM) + SB431542 (10μM) + noggin (100 ng/ml). The volume of differentiation medium will depend on the amount of starting material. In general, the volume of differentiation medium should be equivalent

to 1–1.5× the volume of growth medium used for the undifferentiated cells. That is, if starting with cells from 1 well of a 6-well plate, the differentiation volume should be 1.5–2.5 ml depending on the density of the cells. Plate cell suspension in non-treated cell culture plates for EB formation (i.e., 0.5 ml/well in a 24-well plate or 1 ml/well in a 12-well plate). Optional patterning factors can be added to the medium as needed (see section "Rostro-caudal and Dorsoventral patterning of neural progenitors").

**NOTE**: ROCK inhibitor is added to the differentiation medium from day 0–2 to increase survival of EBs. If Fcconjugated noggin is used, add 200 ng/ml noggin instead of 100 ng/ml. For some cell lines, 10μM SB431542 may appear toxic, and the concentration should then be lowered to 2–5μM. Start coating with poly-ornithine (PO) on this day.

4. **Day 2**: Transfer EB suspensions to a tube and spin down gently at 80× g for 5 min. Aspirate the medium and re-suspend the EBs in new NIM medium + SB431542 (10 μM) + noggin (100 ng/ml) + optional patterning factors using the same volume as on day 0. Return EBs to the wells they came from.

**NOTE**: If necessary, wash wells with medium to retrieve any EBs that have attached to the well bottom. If big aggregates of EBs are present, these should be broken up with a pipette. If medium is very yellow by day 2, increase the volume of differentiation medium. Coat plates with fibronectin and laminin (FN/lam) on this day.