Human pluripotent stem cells as tools for neurodegenerative and neurodevelopmental disease modeling and drug discovery.

Review

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Introduction

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General concepts on cell disease modeling

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Generation of neuronal subtypes relevant for

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neurologic diseases modeling and drug development

Human pluripotent stem cells as tools for neurodegenerative and neurodevelopmental disease modeling and drug discovery Stefania Corti*, Irene Faravelli, Marina Cardano & Luciano Conti† †

Universit a degli Studi di Trento, Centre for Integrative Biology - CIBIO, Trento, Italy and *University of Milan, Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation, Neurology Unit, IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

platforms 4.

Human INs

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hPSCs-based models of neurodegenerative and neurodevelopmental diseases

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Conclusion

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Expert opinion

Introduction: Although intensive efforts have been made, effective treatments for neurodegenerative and neurodevelopmental diseases have not been yet discovered. Possible reasons for this include the lack of appropriate disease models of human neurons and a limited understanding of the etiological and neurobiological mechanisms. Recent advances in pluripotent stem cell (PSC) research have now opened the path to the generation of induced pluripotent stem cells (iPSCs) starting from somatic cells, thus offering an unlimited source of patient-specific disease-relevant neuronal cells. Areas covered: In this review, the authors focus on the use of human PSCderived cells in modeling neurological disorders and discovering of new drugs and provide their expert perspectives on the field. Expert opinion: The advent of human iPSC-based disease models has fuelled renewed enthusiasm and enormous expectations for insights of disease mechanisms and identification of more disease-relevant and novel molecular targets. Human PSCs offer a unique tool that is being profitably exploited for high-throughput screening (HTS) platforms. This process can lead to the identification and optimization of molecules/drugs and thus move forward new pharmacological therapies for a wide range of neurodegenerative and neurodevelopmental conditions. It is predicted that improvements in the production of mature neuronal subtypes, from patient-specific human-induced pluripotent stem cells and their adaptation to culture, to HTS platforms will allow the increased exploitation of human pluripotent stem cells in drug discovery programs. Keywords: cell-based drug screening, disease modeling, drug discovery, human ES cells, human iPS cells, neurodegenerative diseases, neurodevelopmental disorders, neuronal subtypes, screening platforms Expert Opin. Drug Discov. [Early Online]

1.

Introduction

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have revolutionized prospects in many sectors of biology and biomedicine fields. hESCs are derived from the inner cell mass of blastocyst stage human embryos and are characterized by an intrinsic capacity for self-renewal and the ability to generate all cell types derived from the three embryonic germ layers (pluripotency) [1,2]. They are commonly characterized by the expression of a range of pluripotency markers (i.e., OCT4, SOX2, NANOG, SSEA-3 and SSEA-4) and high levels of 10.1517/17460441.2015.1037737 © 2015 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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Human pluripotent stem cells (hPSCs) and their neuronal products hold great potential for facilitating technology for the discovery and development of conventional small-molecule drugs for the treatment of neurodegenerative and neurodevelopmental diseases. Knowledge from neurodevelopmental biology field has made possible the optimization for efficient production of disease-relevant neuronal subtypes. Diverse neurodevelopmental and neurodegenerative human diseases have been successfully modeled in vitro using patient-derived hPSC-based systems. The use of patient-derived hPSCs has already identified disease-relevant cellular phenotypes and served for drug testing. Most of the currently available hPSC-based neuronal systems need improved adaptation to high-throughput screening platforms in terms of costs, miniaturization and adaptability to robust cell-based assay.

human adult tissues (i.e., neurons, cardiomyocites). In the last few years, hiPSC-based models for several monogenic and polygenic central nervous system (CNS) pathologies have been reported, such as Alzheimer’s disease (AD) [7], Parkinson’s disease [8,9], amyotrophic lateral sclerosis (ALS) [10,11], spinal muscular atrophy [12], RETT syndrome (RTT) [13] and schizophrenia (SCZ) [14]. These in vitro models are characterized by a distinct competence to reproduce the in vivo etiopathological environment, so that some authors coined the term ‘in vivitro’, in order to point out the great translational power of hiPSCs [15]. This process could not only lead to the development of various cellular therapies, but also push the boundaries of personalized medicine. Here, we review the recent developments in the use of hPSCs, mostly focusing on patient-derived hiPSCs, to model neurodegenerative and neurodevelopmental disorders and discuss the major challenges in this moving field, which has opened new opportunities for the drug discovery sector.

This box summarizes key points contained in the article.

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telomerase. hESCs can be maintained in vitro through several passages with a stable genetic identity, thus representing a valid source for in vitro studies on disease-relevant human cells and for cell-based therapies [3]. However, it is worthy to consider that the availability of a large number use of hESCs for cell-based therapies is limited both by ethical issues and by restrictions in the currently available genetic variants. In the last years, the advent of iPSC technology has been a breakthrough into the ‘pluripotency’ field, avoiding the requirement of embryos as the source of rodent and, most importantly, of human PSCs. Moreover, the use of hiPSCs opens new possibilities for studies of human development and disorders, further increasing the potential biomedical applications of this type of cells [4]. iPSCs are the product of a reprogramming procedure based on the transient expression of key pluripotency-associated transcription factors able to ignite a process driving the full conversion of somatic cells directly into pluripotent cells [5,6]. hiPSCs closely resemble hESCs with respect to expression of pluripotency markers, self-renewal potential, and multilineage differentiation competence. This technology, originally based on the retroviral transduction of OCT4, SOX2, cMyc and KLF4, has been shown to be straightforward, and since its discovery has been implemented in terms of efficiency, safety and robustness. The opportunity to setback the molecular clock converting terminally differentiated cells into unspecialized and pluripotent ones, which can be differentiated toward different cell types, has opened myriads of prospective in regenerative medicine without ethical implications. Beside their potential applications for cell-based therapies, hiPSCs have attracted a lot of interest as a unique tool for human disease in vitro modeling due to the possibility to obtain patients’ specific cells otherwise hard to obtain from 2

General concepts on cell disease modeling

hPSCs technology has opened the path to a series of studies aiming to uncover various aspects of human development and pathology. Several research groups have published differentiation protocols that, starting from pluripotent cells, allow obtaining a wide range of fully mature cells [16]. Through this process, the investigation of otherwise unexplored in vitro basic cell mechanisms peculiar of human development is finally achievable. Regarding neural differentiation, many efforts have been spent to set up protocols that generate defined neuronal populations [3,17-19]. This step is crucial to investigate specific mechanisms underlying the onset of brain disorders. This is especially true referring to neurodegenerative diseases, such as ALS and AD, in which symptoms become overt many years after the onset of the disease process. The discovery and development of reprogramming techniques leading to the generation of hiPSCs provided further advancement in the field of cell-based disease modeling. The possibility to have access to the genetic code of a single patient has allowed recreating reliable in vitro models of the mechanisms leading to disease in a specific individual [11,12,20]. It is also worthy to consider that animal models are not always reliable and recapitulating all the aspects of human pathology. Recent advances in cell culture techniques have led to the setting up of complex co-culture systems to study the interactions between patients’ hiPSC-derived neuronal/glial cells and healthy cells, investigating the role of specific cell-to-cell interactions in disease development and in particular noncell autonomous mechanisms [21,22]. Microfluidics technology has recently allowed creating 3D environment for cell growth and differentiation. Microfluidic supports based on oxygen permeable devices can be exploited for long-term cell cultures, offering the significant advantage to build microenvironment more similar to in vivo cytoarchicture [23]. A series of studies has tested the feasibility of this

Expert Opin. Drug Discov. (2015) 10(6)


Human PSCs as tools for neurodegenerative and neurodevelopmental disease modeling and drug discovery

technology for reliable in vitro modeling [23-25]. Indeed, the proper combination of biomaterials with specific physical devices permits us to compartmentalize different cellular subtypes in complex and dynamic organizations for disease modeling and developmental studies [23,26]. Moreover, the assessment of human cell platforms has provided a precious source for screening potential therapeutic compounds [27]. However, the realization of reliable in vitro human cell platforms for disease modeling and drug discovery requires the optimization of standardized protocols for cell reprogramming and differentiation and a careful analysis of obtained cells in terms of genetic code, morphology, expression markers and electrophysiological activity. Future advances in the field of developmental biology will provide further data to implement differentiation techniques aiming to mimic the physiological steps of human development and pathology.

Generation of neuronal subtypes relevant for neurologic diseases modeling and drug development platforms

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The research on neurological diseases has particularly benefited from the advent of hPSC cultures since their high flexibility to the in vitro model such pathologies coupled to the enormous potentialities for drug discovery. As neurons are post-mitotic cells that cannot be expanded to obtain enough material for high-throughput screening (HTS), hPSCs represent a precious source of mature human neurons. However, the intrinsic plasticity of hPSCs is strictly connected to the difficulty to obtain homogeneous differentiated neuronal cultures [28] and new procedures are necessary both to limit the undesired culture heterogeneity and to improve differentiation efficiency and yields. So far, many studies involving the induction of various types of neurons from hPSCs cells have been developed by mimicking in vitro the developmental process. The first step in producing neuronal subtypes from hPSCs is the generation of neuroepithelial cells (NEPs, Figure 1), efficiently achieved by dual SMAD signal inhibition by Noggin and SB431542, leading to inhibition of transforming growth factor-b (TGF-b) and bone morphogenetic protein (BMP) pathways, respectively [18]. These NEPs have the potential to maturate into defined region-specific CNS neuronal subtypes by means of sequential exposure to appropriate in vitro environmental cues. Below are described some of the recent studies providing promising strategies for controlled generation of specific neuronal subtypes useful to model neurological disorders with hPSCs. Human dopaminergic neurons Dopaminergic (DA) neurons located in the Substantia Nigra Pars Compacta play a critical role in regulating postural reflexes and represent the neuronal subtype selectively affected in Parkinson’s Disease (PD). 3.1

Neurons with DA identity have been obtained from hESCs after a first step of neuroectodermal induction on stromal feeder cell, a second step in which cells were instructed to acquire the ventral midbrain/hindbrain fate by exposure to fibroblast growth gactor 8 (FGF-8) and Sonic hedgehog (SHH), and a terminal DA differentiation in the presence of ascorbic acid, brain-derived neurotrophic factor, glial-cell derived neurotrohic factor, TGFb3 and 2’-O-dibutyryladenosine 3’:5’ cyclic monophosphate (Figure 1) [29]. Kriks and coauthors reported a protocol that allows the proficient obtainment of DA neurons with great engraftment potential. By using a modified dual-SMAD inhibition protocol and strong activators of WNT and SHH pathways, the authors promoted the conversion of hPSCs in midbrain floor-plate precursors. These cells were further in vitro differentiated, giving rise to mature and functional DA neurons able to survive in vivo in PD animal models, and to determine a striking behavioral rescue [30]. More recently, it has been reported the efficient conversion of neuralized hESCs in DA neurons through a protein-transduction based method, by the direct delivery of a trans-activator of transcription (TAT)-LMX1A fusion recombinant protein, as an alternative to genetic modification (Figure 1) [31]. The simultaneous treatment with SHH and (TAT)-LMX1A particularly enhanced the expression of the DA markers TH and PITX3, underlining the role of this two molecules as major regulators in DA neurons differentiation [31]. Primitive neural stem cells (pNSCs) have been isolated from hESCs by inhibiting glycogen synthase kinase 3 (GSK3), TGF-b and Notch signaling pathways. This cell population can be maintained in self-renewing conditions by Leukemia Inhibitory Factor, GSK3 inhibitor (CHIR99021), and TGF-b receptor inhibitor (SB431542) [32] and has been shown to efficiently differentiate into DA neurons (Figure 1). Following this procedure, PD patient-specific hiPSCs carrying a mutation in the leucine-rich repeat kinase 2 (LRRK2) gene (G2019S), known to be associated with familiar and sporadic PD, were differentiated in pNSCs [33]. Mutant pNSCs exhibited a morphological phenotype recapitulating several aspects of the human disease, characterized by an abnormal differentiation capability and aberrant nuclear morphology. Although these cells might represent a good tool for in vitro modeling studies, the use of cytokines might render the system too expensive for large-scale exploitation in HTS platforms. Some researchers recently reported the so-called small-molecule neural progenitor (smNP). In this system, cells were differentiated from hPSCs by using small molecules able to inhibit both BMP and TGF-b signaling and to stimulate WNT and SHH pathways. These smNP cells can efficiently generate DA neurons under specific stimuli (Figure 1) [34]. The features of these neural progenitors make them really suitable for largescale disease modeling drug screening.


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RA, SHH

NSC spheres

pNSC FGF-8, SHH IGF1, BDNF, GDNF, TGFβ3, dbcAMP

WNT agonist, Notch inhibitors

GSK3, TGFβ, Notch inhibition

EBs

Small molecule cocktail

Motor neuron [19,45,46]

PSC


smNPC

Dual SMAD SFEBq inhibition EGF, FGF2, N2B27 DA neuron BDNF LCP [29–32,34] AA, BDNF, GDNF, TGFβ3, dbcAMP -FGF2 NEP SHH, (TAT)-LMX1A Ventralizing FGF8, SHH molecules SHH

Cortical neuron [36–38] MGE-like progenitors

Interneuron [50–53]

Figure 1. Schematic representation of the in vitro differentiation protocols used to obtain specific neuronal subtypes from hPSCs. DA neurons can be originated from NEPs [29-31], smNPCs ([34]) or pNSCs [33] by exposure to specific stimuli. Cortical neurons can be obtained from NEPs [37], NEP-derived LCP [38], or three-dimensional aggregates called SFEBq [36]. Motor neurons have been differentiated from hPSC-derived NSC spheres [46] or from EBs [19,45]. Interneurons can be differentiated from hPSC-derived MGE-like progenitors [52], but also directly from NEPs [51,53]. DA: Dopaminergic; EB: Embryoid bodies; hPSC: Human pluripotent stem cell; LCP: Late cortical progenitors; MEG: Medial ganglionic eminence; NEP: Neuroepithelial cell; pNSC: Primitive neural stem cell; smNEP: Small molecule neural progenitor cell.

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Human cortical neurons

Cortical neurons, involved in the higher cognitive functions, are drastically affected in several psychiatric disorders, characterized by alteration in differentiation, migration and synaptogenesis processes [35]. Using a three-dimensional aggregation culture (SFEBq culture), hPSCs have been induced to generate self-organized polarized cortical neurons, according to a spatially and temporally controlled pattern (Figure 1) [36]. Using a different approach, Shi et al. have exploited hPSCs to generate NEPs specifically instructed to originate cortical projection neurons in a fixed temporal order (Figure 1) [37]. However, although the elegance and the innovativeness of these studies, the application of such protocols to HTS technologies is associated to several limitations in terms of differentiation time and homogeneity of the resulting neuronal cultures. A more recent study by Boissart et al. has reported a new differentiation protocol partially able to overcome these difficulties. The authors described the generation of late cortical progenitors (LCP) from human hPSC-derived NEPs, which 4

can efficiently generate mature glutamatergic neurons of the cortical superficial layers (Figure 1) [38]. In particular, this rapid and efficient differentiation appears particularly well suited for HTS approaches. LCP-differentiated neurons can be directly cultivated in 384-well plate and differentiated in post-mitotic neurons, allowing screening of small molecules able to modulate fundamental neuronal properties [38]. Human motor neurons Human motor neuron (hMN) differentiation in vitro can be pursued by simulating the steps of MNs development in vivo. Retinoic acid (RA) and SHH play crucial roles during MNs development through the rostrocaudal and dorsoventral axis of the neural tube [39,40]. By adapting an MN differentiation protocol previously developed for mouse ESCs [41], Eggan’s group published in 2008 a study focused on the generation of spinal MNs derived from hESCs (Figure 1) [19]. Generated hMNs could be plated with murine glia derived from SOD1G93A mice, a reliable animal model of ALS. This study paved the way to subsequent experiments on human cell-based disease modeling. Many research groups 3.3




have reported multiple combinations of differentiation cocktails involving RA, Shh or their agonists [42,43]. It is crucial to identify the proper time point for adding and stopping their administration: early exposure to Shh leads to interneurons (INs) differentiation, whereas a protracted use pushes cells toward oligodendrocytes differentiation. Shh exposure when cells manifest the expression of OLIG2 is necessary for MN commitment (Figure 1) [44]. Maury et al. experimented a combination of a Wnt signaling agonist with three g-secretase inhibitors that were shown to significantly speed up and increasing MNs generation starting from hPSCs (Figure 1) [45]. Moreover, the careful administration of the small molecules in a time-dependent manner was essential to obtain MN progenitors committed towards a hindbrain identity compared to a spinal cord specification. Indeed, the possibility to generate specific subtypes of MNs holds the premises to study and model human diseases characterized by a selective loss of MN subpopulations, such as ALS, where different MN subtypes present a selective vulnerability to the disease process. Protocols implying the use of viral vectors might limit direct clinical applications but represent one of the most efficient and rapid method of differentiation. Established differentiation protocols for hESCs could effectively be applied to hiPSCs as well. IPSC-derived MNs have been generated from spinal muscular atrophy (SMA) patients [42,46] and ALS patients [43,47]. Obtained MNs recapitulated the morphological and biological features of ALS/ SMA MNs thus providing the basis for the construction of human MN platforms for disease modeling and drug screening. 4.

Human INs

INs account for 20% of the cells within the human cortex. Several IN subtypes can be distinguished based on their morphology, connection target, cortical layer position, branching asset and electrophysiological activity [48]. Regarding the expression of specific calcium binding proteins, INs have also been classified into Somatostatin, Calretinin and Parvalbumin populations [48]. As INs aberrant development and dysfunction have been involved in a wide spectrum of human pathologies, from autism to epilepsy and SCZ, many efforts have been spent in recreating in vitro cell models of these disorders [48]. The first step usually includes conversion of hPSCs into NEPs through the inhibition of Wnt, BMP and Nodal signaling [18,49] and exposure to some ventralizing molecules (Figure 1) [50]. A recent report by Maroof et al. describes and optimized protocol of IN differentiation starting from hPSCs [51] based on the sequential use of small molecules. Following a neural induction step performed through the dual SMAD inhibition, they tested a combination of a tankyrase inhibitor (XAV939) with the administration of Shh activators in a time-dependent manner, showing that Shh treatment during day 10 -- 18 is

crucial for INs specification (Figure 1). These human INs were able to form synapses in vitro and to migrate in vivo [51]. Nicholas et al. investigated whether hPSC-derived INs could be effectively compared to endogenous human INs in terms of neural development and maturation [52]. INs mature phenotype in culture was reached with a protracted timeline (> 7 months), thus resembling human fetal IN development. This study provided the basis for in vitro modeling developmental diseases involving IN aberrant maturation and function, even if it could be necessary to further maintain cells in culture for mimicking those diseases involving the adult stage. The protocols previously reported are based on the use of a rich combination of small molecules carefully dosed in terms of timing and concentration. These variables could lead to a risk of increasing costs and hampering standardization. To overcome these limits, Liu et al. recently developed a differentiation protocol exploiting just high concentration of recombinant Shh or its agonist purmorphamine with a culture medium lacking nerve growth factor, which can induce cholinergic neurons (Figure 1) [53]. After 2 weeks of differentiation, they could obtain a cell population highly enriched of GABA INs starting from hPSCs, providing an essential step towards the study of developmental and psychiatric human disorders.

hPSCs-based models of neurodegenerative and neurodevelopmental diseases

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Great strides have been made in the advancement of hPSC technology and their ability to generate pluripotent-derived cellular models that are genetically identical to disease patients remains an attractive feature (Table 1). However, the recapitulation of neuropathologic phenotypes in vitro and the unveiling of new disease-relevant molecular mechanisms remain challenges in the field. Parkinson’s disease PD is a neurodegenerative age-related disorder characterized by several motor impairments, such as tremor, rigidity, bradikinesia, postural instability, and other non-motor symptoms, as psychiatric manifestations, sleep disturbance and hyposmia [54]. Motor alteration is essentially due to the loss of DA neurons residing in the ventral part of the Pars compacta of the Substantia nigra [55]. PD etiology is still unclear, even though genetic and environmental factors can be strictly associated with the illness onset. Mutations affecting the LRRK2 [56], a-synuclein (SNCA) [57] and vacuolar protein sorting 35 (VPS35) [58] genes have been correlated to autosomal dominant forms of PD. The current therapeutic strategy is essentially focused on improving patients’ quality of life and producing symptomatic relief of the motor functions. Unfortunately, long-term use of Levodopa, the most effective compound for PD symptomatic treatment, has been associated to 5.1


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Table 1. Recent reported studies using human pluripotent stem cells for neurological and neurodevelopmental disease modeling and drug discovery. Pathology AD

Study Yagi et al. (2011) [71]


Israel et al. (2012) [70]

ALS

Starting cells

Derived cells

Conclusions

hiPSCs carrying mutations in PS1 (A246E) and PS2 (N141I) hiPSCs from sporadic and familial APPDp patients

Glutamatergic, GABAergic and cholinergic neurons Glutamatergic, GABAergic and cholinergic neurons

g-secretase inhibitors were able to reduce levels of amyloid b in hiPSC-derived neurons

Kondo et al. (2013) [7]

hiPSCs carrying E693D Glutamatergic, and V717L APP mutations GABAergic and plus sporadic AD hiPSCs cholinergic neurons

Bilican et al. (2012) [75] Egawa et al. (2012) [77]

hiPSCs carrying TDP43 M337V mutation hiPSCs carrying Q343R, M337V, and G298S mutations in TDP43

Motor neurons

Sareen et al. (2013) [47]

hiPSCs carrying C9ORF72 hexanucleotide expansion

Motor neurons

Yang et al. (2013) [27]

hiPSCs carrying mutations Motor neurons in either SOD1 or TDP-43 gene

Chen et al. (2014) [76] Kiskinis et al. (2014) [43]

hiPSCs carrying A4V or Motor neurons D90A SOD1 mutation hiPSCs carrying SOD1A4V Motor neurons mutation

Familial Lee et al. (2009) hiPSCs carrying IKBKAP mutations dysautonomia [90] PD

Motor neurons

Neural crest lineages

Devine et al. (2011) [8]

hiPSCs carrying asynuclein triplication

Nguyen et al. (2011) [61]

hiPSCs carrying p.G2019S Dopaminergic mutation in the LRRK2 neurons gene

Seibler et al. (2011) [62] SanchezDanes et al. (2012) [65] Liu et al. (2012)

hiPSCs carrying mutant PINK1 hiPSCs from familial and sporadic PD patients

Dopaminergic neurons Dopaminergic neurons

hiPSCs carrying LRRK2 dominant mutation hiPSCs carrying the LRRK2 mutation G2019S hiPSCs carrying A53T SNCA mutation

Neural stem cells

[33]

Reinhardt et al. (2013) [64] Ryan et al. (2013) [63]

Dopaminergic neurons

Dopaminergic neurons Dopaminergic neurons

Accumulation of amyloid b, aGSK-3b and p-tau/ total tau was shown in hiPSCs-derived neuronal cells derived from both familial and sporadic AD patients Ab oligomer aggregation accumulation was related with ER and oxidative stress. HiPSC-derived neurons from sporadic AD patients showed no response to docosahexaenoic acid (an omega-3 fatty acid) treatment Specific vulnerability of ALS MNs was shown to the inhibition of the PI3K pathways Anacardic acid (a histone acetyltransferase inhibitor) was effective in ameliorating cell phenotype, probably through the downregulation of TDP43 mRNA and other genes linked to the oxidative stress A potential correlation between C9ORF72 and TDP43 ALS form was reported as well a mechanism of gain of function linked to C9ORF72 expansion A GSK-3b inhibitor (kenpaullone) increased MNs survival in culture in comparison with two compounds that failed in clinical trials (dexpramimpexole and olesoxime) An impairment of neurofilament turnover was shown in mutated cells Mitochondrial motility impairment and an intrinsic hyper excitability related to ER stress-characterized human SOD1 MNs Neurogenesis, splicing and migration deficits were observed, which were suitable to treatment with kinetin PD hiPSCs did not exhibit any qualitative and quantitative impairment in the competence to generate DA neurons. A certain grade of intrinsic variability was shown between different clones DA neurons displayed manifest differences in vulnerability to toxic agents affecting mitochondrial function, oxidative stress and protein aggregation Mutant PINK1 in DA neurons affected Parkin recruitment to mitochondria Many genes appeared dysegulated within mutated neurons, including CADPS2, CPNE8, and UHRF2. These defects were correlated with ERK activity Derived neural stem cells showed age-associated cellular deterioration and increased proteasomal stress Mutant cells phenotype recapitulated several aspects of in vivo human disease Mutated cells showed decreased mitochondrial function with augmented steady levels of ROS/ RNS. This resulted in a reduction of MEF2C activity, which decreased PGC1a expression

AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; APP: Amyloid precursor protein; GSK: Glycogen synthase kinase; hiPSC: Human induced pluripotent stem cell; PD: Parkinson’s disease; RTT: Rett syndrome; SMA: Spinal muscular atrophy.

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Table 1. Recent reported studies using human pluripotent stem cells for neurological and neurodevelopmental disease modeling and drug discovery (continued). Pathology


RTT

Study

Starting cells

Marchetto et al. (2010) [13]

hiPSCs carrying different MeCP2 mutations

Glutamatergic neurons

Cheung et al. (2011) [85]

hiPSCs carrying D3 -- 4 MeCP2 mutation

Glutamatergic neurons

Kim et al. (2011) hiPSCs carrying mutation [87] within different domains of MeCP2 hiPSCs carrying CDKL5 Ricciardi et al. pathogenic mutations (2012) [88] (R59X, L220P) hiPSCs carrying Djuric et al. MeCP2e1 mutation (2015) [86] Schizophrenia Brennand et al. hiPSCs from patients with childhood onset or (2011) [14] familial form of the disease hiPSCs from four Wen et al. members of a family in (2014) [96] which a frameshift mutation of DISC1 hiPSCs carrying Yoon et al. 15q11.2 microdeletion (2014) [97] SMA

Derived cells

Glutamatergic neurons Glutamatergic neurons Glutamatergic and GABAergic neurons Glutamatergic, GABAergic and dopaminergic neurons Forebrain neurons

Neural precursors cells

Ebert et al. (2009) [12] Sareen et al. (2012) [46]

hiPSC line from a SMA I Motor neurons patient hiPSCs from fibroblasts of Motor neurons a SMA1 patient

Corti et al. (2012) [42]

hiPSCs from a SMA I patient.

Motor neurons

Garbes et al. (2013) [82] Wang et al. (2013) [81]

hiPSCs from SMA patients Human embryonic stem cells with SMN-FL knockdown

GABAergic neurons Spinal motor neurons

Conclusions Derived cells showed morphological defects in neuronal soma size, spine and synapse number, impaired electrophysiological activity. IFG1 and gentamicin treatment ameliorated the phenotype Female hiPSCs maintained an inactive X-chromosome in a nonrandom X-chromosome inactivation pattern Mutant RTT-iPSCs showed a defect in neuronal maturation with a noncell autonomous effect of MeCP2 mutations on neuronal maturation Synaptic-related defects have been observed in hiPSCs derived from atypical RTT patients carrying mutations in the X-linked CDKL5 gene MECP2e1 mutation affected morphology and function of human iPSC-derived neurons Glutamatergic neurons exhibited reduced neuronal connectivity associated to decreased neurite number, PSD95 protein levels, and glutamate receptor expression, partially rescued by loxapine Mutant DISC1 caused synaptic vesicle release deficits in patient hiPSC-derived neurons together with altered expression of many genes related to synapses and psychiatric disorders Deficits in adherens junctions and apical polarity were shown due to the haploinsufficiency of CYFIP1, which regulates cytoskeletal dynamics SMN mutation affected late phases of MN maturation rather than early development A pathogenetic role of apoptosis in SMA was demonstrated. The addition of an antibody directed against the Fas-receptor (involved in caspase 8 signaling) was able to prolong survival of SMA MNs The genetic correction of SMN2 exploiting oligodeoxynuleotides resulted in significant amelioration of the SMA phenotype CD36 could represent a biomarker for valproic acid therapeutic response A key role of apoptosis in SMA development was reported

AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; APP: Amyloid precursor protein; GSK: Glycogen synthase kinase; hiPSC: Human induced pluripotent stem cell; PD: Parkinson’s disease; RTT: Rett syndrome; SMA: Spinal muscular atrophy.

several limitations concerning motor and neuropsychiatric complications [59]. Different groups have reported the derivation of hiPSC lines from familial PD patients with a-synuclein [8], Parkin [60] LRRK2 [61] and PINK1 [62] mutations. Interestingly, although these PD hiPSCs did not exhibit any qualitative and quantitative impairment in the competence to generate DA neurons with respect to control hiPSCs, they displayed manifest differences in vulnerability to toxic agents affecting mitochondrial function, oxidative stress and in other biochemical properties. These observations corroborated such hiPSC lines as bona fide PD models. Two independent studies have shown

accumulation of a-synuclein and increased susceptibility to oxidative stress in DA neurons derived from hiPSC lines from patients carrying genomic triplication of the SCNA locus [8]. These studies also enlightened a certain degree of variability when considering different PD hiPSC lines, issue that has been elegantly solved by genome editing allowing us to create isogenic control cells lines [63]. Derivation of hiPSCs from a PD patient homozygous for a mutation in G2019S in LRRK2 revealed accumulation of a-synuclein in LRRK2 mutant DA neurons compared to controls [61]. Also, mutant LRRK2 DA neurons exhibited increased susceptibility to oxidative stress and 6-OHDA-induced cell death.


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Interestingly, neural progenitors derived from hiPSCs carrying the same G2019S mutation showed age-associated cellular deterioration and increased proteasomal stress [33]. Serial passaging of these neural progenitors resulted in a decreased differentiation potential. Validation of the specificity of these LRRK2 phenotypes was demonstrated by genetic correction of the G2019S mutation in hiPSCs. A more recent study has analyzed the transcriptomic profiles of LRRK2 mutant neurons and their isogenic controls showing that several genes are deregulated, including CADPS2, CPNE8, and UHRF2, by the presence of G2019S mutation. These defects were specifically dependent on the extracellular signal-regulated kinases activity [64]. Knockdown experiments demonstrated that dysregulation of these genes specifically contributed to DA neurons degeneration in vitro and that PD-associated phenotypes can be ameliorated by inhibition of Extracellular signalregulated kinases. These results provide new insights into the pathogenic mechanisms driven by mutant LRRK2 and open to the development of potential new therapeutics. Differently from familial PD caused by specific genetic mutations, hiPSC-based modeling can be more challenging for sporadic PD forms, in which both environmental and genetic risk factors contribute to the disease. Nevertheless, a recent study by Sanchez-Danes et al. successfully recapitulated some traits of the sporadic PD and LRRK2 PD by using DA neurons derived from a selection of patient-specific hiPSC lines [65]. The authors showed that DA neurons derived both from LRRK2-PD and sporadic PD-hiPSCs exhibited defects in autophagic clearance and morphological alterations, including a reduced number of neurites and neurite arborization. This study demonstrates that overlapping phenotypes are present in familiar and sporadic neurons from hPSC lines from PD patients, indicating that hiPSC-based in vitro models might be useful to capture the patients’ genetic complexity. Alzheimer’s disease AD is a chronic neurodegenerative disorder associated to dementia and progressive deficiency of cognitive, memory and language abilities, altered behavior, a variety of neuropsychiatric symptoms and impaired movement coordination. Although its etiology is still unknown, specific common tracts characterize this pathology, such as the accumulation in the brain of toxic aggregates composed of protein fragments called b-amyloid (Ab) and neurofibrillary tangles deriving from tau protein. Genetically, AD can be classified in familial (FAD) or sporadic forms. Generally, FAD have an early onset and are associated to mutations of amyloid precursor protein (APP), presenilin-1 (PS-1) or presenilin-2 (PS-2) [66]. Differently, sporadic AD usually arises after 60 years and are connected to environmental factors and some risk genes. Apolipoprotein allele variant "4 seems to be the most important [67]. APP is sequentially cleaved by a- and g-secretase, a multiprotein complex which includes PS-1 and PS-2 proteins, generating a smaller and soluble peptide, whereas in AD patients 5.2

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it is converted in Ab 4-kilodalton insoluble oligomer, generated by b- and g-secretase processing [68]. This aberrant protein accumulates in aggregates, creating cytotoxic plaques, which impair synaptic and neuronal function, ultimately leading to neuronal death. The anomalous hyperphosphorylation of Tau, a neuronal axon microtubule associated protein, occurs in AD, impairing the microtubule interaction and determining the accumulation of intracellular neurofibrillary tangles [69]. Recently, Israel et al. have reported the accumulation of Ab in neuronal cells derived from the hiPSCs from sporadic AD patients [70]. These findings confirmed a phenotype previously observed in neurons derived from hiPSCs from familial AD patients with mutations in PS-1, PS-2 or APP, thus denoting that hiPSCderived neurons from sporadic and familiar AD forms develop overlapping phenotypes [71]. In these studies, it was also shown that g-secretase and b-secretase inhibitors have differential effects in reducing levels of Ab in hiPSC-derived neurons depending on the underlying AD mutations [70,71]. Also, there was not consistency in the response of hiPSCderived neurons from distinct sporadic AD patients to docosahexaenoic acid (an omega-3 fatty acid) treatment, in terms of reduction of stress response and accumulation of Ab oligomers [7]. Accordingly, although these observations reveal the remarkable competence of AD hiPSC-derived neuronal models to reveal potential pathological processes and to test the clinical efficacy of drugs, both in sporadic as well as genetic AD forms, they enlighten also an intrinsic variability. In this view, the use of a larger number of hiPSC lines might greatly enhance the development of new strategies and approaches for ad treatments. Amyotrophic lateral sclerosis ALS is a neurodegenerative disease of the adulthood that primarily affects upper and lower MNs. The clinical phenotype is characterized by progressive muscle paralysis leading to respiratory failure and early death, on average 3 -- 5 years from the onset of symptoms [72]. Riluzole is currently the only therapy available on the market and it is able to prolong the survival for only 4 -- 5 months [73]. Both sporadic and familial forms of ALS have been described [74]. Available animal models often only partially recapitulate the complex genetic and phenotypic background of human pathology. The ability to manipulate hPSCs in vitro has provided the basis for revolutionizing the study of this disease. It has been possible for the first time to study in vitro the steps of development of human ALS neurons in order to identify the molecular mechanisms that occur in vivo, probably much earlier than the symptomatic onset. Sareen et al. generated a hiPSC line carrying C9ORF72 hexanucleotide expansion, responsible for the most common genetic form of the disease. They analyzed the role of C9ORF72 expansion in hiPSCderived MNs identifying a potential connection between C9ORF72 and TDP43 ALS form as well a mechanism of gain of function linked to the mutation [47]. Bilican et al. 5.3




focused their investigation on hiPSCs derived from a patientcarrying TDP43 M337V mutation. They discovered a specific vulnerability of ALS MNs to the inhibition of the PI3K pathways, thus providing a precious basis to future studies involving the use of neurotrophins targeting this pathway to extend MN survival [75]. Other groups focused their attention on hiPSC-derived MNs from patients carrying SOD1 mutation. Chen et al. showed an impairment of neurofilament turnover in SOD1-MNs [76] whereas Kiskinis et al. [43] found mitochondrial motility impairment and intrinsic hyper excitability related to endoplasmic reticulum stress characterized human SOD1 MNs. The possibility to identify precise molecular mechanisms of MN death offers the possibility of designing molecules that are able to target them specifically in order to obtain a rescue of the phenotype. Yang et al. tested a wide series of potential therapeutic candidates for ALS in vitro and validated their results exploiting platforms of human MNs obtained from two ALS patients carrying mutations in either SOD1 or TDP-43 gene [27]. They identified a GSK-3b inhibitor (kenpaullone), which actually increased MNs survival in culture in comparison with two compounds that failed in clinical trials (dexpramimpexole and olesoxime). Egawa et al. derived multiple hiPSC lines from TDP43-mutated ALS patients and tested four chemical compounds in order to rescue ALS MN phenotype. They actually found that anacardic acid (a histone acetyltransferase inhibitor) was effective in ameliorating the diseased phenotype, probably through the downregulation of TDP43 mRNA and other genes linked to the oxidative stress [77]. Spinal muscular atrophy SMA is a neuromuscular disorder that represents the most common genetic cause of death during childhood. The clinical phenotype is featured by progressive symmetric muscular paralysis and children usually present hypotonic and atrophic limbs [78]. The disease is usually stratified based on the severity ranging from type I to type IV with SMA I patients having a life expectancy of < 2 years [78]. The clinical phenotype also correlates with the genetic background. SMA patients present a genetic mutation in the survival MN (SMN) gene [79]. The telomeric gene SMN1 is responsible for the production of the full-length SMN protein whereas its paralogous SMN2 presents a C to T replacement within exon 7, which causes an alternative splicing of SMN2 and the subsequent production of just 10% of full-length protein [80]. The number of copies of SMN influences the clinical phenotype of SMA patients. Ebert’s group published in 2009 the first generation of hiPSC line from an SMA I patient. RT-PCR analyses confirmed the decrease of SMN full-length expression and the presence of transcripts without exon 7, resulting from the alternative splicing [12]. Derived cells were differentiated into MNs and showed a diseased phenotype, in terms of morphology and survival, after 10 weeks in culture. This data could suggest that alterations due to SMN mutation affect late phases of MN maturation rather than early development. 5.4

Moreover, this work assessed for the first time the reliability of hiPSC-based platforms in modeling SMA in vitro. This possibility paved the way to a series of studies investigating SMA pathogenetic mechanisms. Wang et al. exploited SMN full-length knockdown MNs derived from hESCs to model and study the role of mitochondrial dysfunction in determining disease development [81]. They found that the oxidative damage in mitochondria leading to the activation of apoptotic cascade actually precedes the phenotypic alterations in SMN-MNs. Moreover, MN survival was increased by the supplement of N-acetylcystein, which preserves cells from the oxidative stress. This study suggested a key role of apoptosis in SMA development and the possibility to exploit antioxidant compounds in SMA therapy. Sareen et al. further assessed the involvement of apoptosis in SMA pathogenesis exploiting hiPSC lines derived from SMA I patients [46]. Differentiated MNs showed a decrease in size and survival compared with healthy controls. High levels of apoptotic markers (cleaved caspases 3 and 8) were detected in culture after 8 weeks of differentiation thus confirming a pathogenetic role of apoptosis in SMA degeneration. Indeed, the addition of an antibody directed against the Fas-receptor (involved in caspase 8 signaling) was able to prolong survival of SMN MNs. The assessment of reliable human cell platforms has allowed both to test in vitro therapeutic strategies targeting specific pathogenetic mechanisms and to screen a wide series of potential effective compounds. Our group [42] has generated and differentiated with a multistage protocol towards MNs multiple hiPSC lines derived from SMA patients. SMA MNs recapitulated in vitro the features of the diseased phenotype showing a reduced soma size and axonal length and impaired ability to form a neuromuscular junction. The genetic correction of SMN2 exploiting oligodeoxynuleotides resulted in significant amelioration of the SMA phenotype and provided further data on the feasibility of the molecular approach in rescuing SMA MNs. Other research groups exploited hiPSC-based cell models to screen molecular compounds such as valproic acid and tobramycin, which have been shown to increase SMN protein expression [12,82]. Taken together, these results suggest that hPSCs represent a precious tool to investigate specific aspects of SMA pathology and test molecules targeting revealed its pathogenetic mechanisms. Neurodevelopmental disorders Neurodevelopmental disorders represent a wide and heterogeneous class of diseases characterized by impairment of neural/ neuronal processes occurring during CNS development. They have a strong genetic component. Although they can result from a single mutation, they are more frequently multigenic. Neurodevelopmental disorders are particularly challenging to in vivo and in vitro models because the development of functional brain structures depends on local growth signals coupled to local and distant connectivity. In this view, slight perturbations in local activity-dependent processes during 5.5


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early developmental stages result in global connectivity impairments in postnatal life [83]. Thus, it can be challenging to ascertain the causal pathology that generates impairments in CNS development because the secondary consequences of the initial causative event can be extremely widespread. This difficulty is further complicated by the restraints in human neural pathology investigation, limited to post-mortem analyses of brain tissue, genetic association studies and imaging techniques, none of which can truthfully address the primary mechanisms of disease etiology. In this scenario, hPSCs systems provide an unparalleled opportunity to explore human neuronal development in an accessible an experimentally flexible system. Conceivably, the most experimentally accessible neurodevelopmental diseases are those characterized highly or completely penetrant single genetic mutations. Among them, RTT has attracted much attention for hPSC-based modeling. RTT represents an extraordinary paradigm of a monogenic autism spectrum disorder (ASD). More than 95% of RTT cases are caused by mutations in the X chromosome-linked MECP2 gene [84]. Because of this, males with MECP2 mutations die early in development whereas females are generally viable although the severity of symptoms is highly variable due to the mosaicism of X chromosome inactivation. Different groups have generated hiPSCs lines from RTT patients. Marchetto et al. reported specific phenotypes in hiPSC-derived neurons from MECP2 mutant female patients [13]. Most relevant phenotypes involved morphological defects in neuronal soma size, spine density and synapse number as well as impaired calcium signaling and electrical evoked activity [13]. IGF1 treatment on these neuronal cultures resulted in an increase in the synapse number leading to a partial functional rescue. Because of the heterogeneity in X-chromosome inactivation described in these neuronal cultures, this and other independent studies [85-87] indicate the existence of non-cell autonomous effects of MECP2 deficiency. Commonly, MECP2 expression in patients is disrupted due to the occurrence of nonsense mutations. Treatment of the RTT hiPSC-derived neurons with gentamicin, an antibiotic that impairs the ribosomal proofreading process, induced an increase in MECP2 expression levels and reestablished the normal glutamatergic synaptic transmission function. Importantly, similar synaptic-related defects have been observed also in hiPSCs derived from atypical RTT patients carrying mutations in the X-linked CDKL5 gene [88]. Together these functional rescue experiments demonstrated the reliability of RTT hiPSCs to test existing drugs and develop new pharmacological approaches. Another monogenic neurodevelopmental disorder that has been modeled in vitro by generating patient-specific hiPSC lines is familial dysautonomia (FD). FD is a rare disease primarily occurring due to a point mutation in the I-k-B kinase complex associated protein (IKBKAP) gene. This mutation is associated to tissue-specific splice expression of the IKBKAP gene in FD patients and results in severe autonomic nervous 10

system (ANS) dysfunctions [89]. Lee et al. have reported the generation of hiPSCs from FD patients and showed the occurrence of some degree of differential splicing in hiPSCderived cell types derived from patient-specific hiPSCs [90]. By using this FD hiPSC-based system, Lee and co-workers screened a small set of candidate compounds previously demonstrated to be effective in restoring IKBKAP expression and showed that among them, kinetin, a vegetal hormone, was able of preventing splice variation and deficits in ASN neuronal derivatives [90]. For most neurodevelopmental disorders, including the vast majority of ASD and SCZ, no single gene has been shown to be sufficient to produce disease symptomatology. Genomewide association studies performed on large cohorts of ASD patients and matching controls have identified several hundred of ‘candidate risk or susceptibility genes’ that are not directly causative to the disease but may have a contribution to its onset and/or progression together with environmental and epigenetic components. This extremely high complexity emphasizes the potential utility of using patient-specific hPSC models to investigate these various possibilities. Among the different ASDs, SCZ is attracting a growing interest in hPSC-based modeling. Notwithstanding the late onset of SCZ, typically occurring in late adolescence, the pathophysiology underlying this disease is considered to be developmentally regulated [91]. To date several groups have reported the generation of hiPSC lines from SCZ patients carrying mutations in specific susceptibility genes. One such gene is Disrupted-in-SCZ 1 (DISC1), shown to play essential roles in early cortical development and in adult neurogenesis in rodents [92]. Human DISC1 function remains elusive, as the mechanisms through which its deficiency might participate to SCZ pathology [93-95]. To evaluate potential genetic interactions and signaling pathways related to DISC1, a recent study has reported the generation hiPSC lines (and their isogenic control by means of gene editing) from four members of a family in which a frameshift mutation results in the disrupted DISC1 function and strongly associated with increased risk for SCZ and major depressive disorders [96]. The authors found that mutant DISC1 causes synaptic vesicle release deficits in patient hiPSC-derived neurons together with altered expression of many genes related to synapses and psychiatric disorders. This study showed that a psychiatric-relevant mutation could provoke synapse deficits and transcriptional dysregulation in human neurons, providing new insight into the molecular and synaptic etiopathology of psychiatric disorders. One of the first studies that have reported the generation of SCZ patient-specific iPSCs was from Brennand et al. They generated hiPSC lines from four SCZ patients with either childhood onset or a familial form of the disease but no identified specific genetic link among them [14]. Glutamatergic neurons differentiated from these hiPSC lines exhibited reduced neuronal connectivity coupled to decreased neurite number, PSD95 protein levels, and glutamate receptor




expression. Additionally, altered gene expressions of components of the cyclic AMP and WNT signaling pathways were described. Although the synaptic transmission defects of the SCZ iPSC-derived neurons were partly rescued by administration of the antipsychotic drug loxapine for 3 weeks, no synaptic functional improvement was observed. Notably, four other antipsychotic drugs failed to rescue the neuronal connectivity, raising questions about the significance of the positive effects elicited by loxapine in these hiPSC lines. Another study reported the generation of hiPSC from three individuals carrying a 15q11.2 microdeletion linked to SCZ [97]. Notably, conversion of these hiPSCs into neural progenitors revealed deficits in adherens junctions and apical polarity due to the haploinsufficiency of CYFIP1, a gene within 15q11.2 region that encodes a subunit of the WAVE complex, which regulates cytoskeletal dynamics. This study provides new insight to understand how the susceptibility genes might contribute to neurodevelopmental alterations leading to neuropsychiatric disorders. On the whole, although these studies involved only small and heterogeneous groups of SCZ patients, on a larger scale the approach to evaluate gene expression in an unbiased screen can lead to the identification of novel genetic interactions and key signaling pathways that are altered in SCZ neurons. 6.

Conclusion

In the last few years, an increasing number of groups have reported the derivation of patient-specific hPSC lines for the study of various CNS monogenic and polygenic disorders. These hPSCs have been shown to be consistently differentiated into disease-relevant neuronal subtypes allowing almost limitless access to the cellular sources from potential large heterogeneous patients’ population carrying different genetic mutations or risk-associated genetic variants. These advancements provide exceptional opportunities for dissecting disease mechanisms and develop novel drug candidates for therapy. Yet, a number of limitations and challenges in this approach have to be tackled before this technology to become a wide effective and consistent resource. Better-quality methodology and more sensitive analysis will be required to enable the detection of subtle yet coherent and important phenotypes between controls and patients’ cell lines. Additional investigation of the effects of reprogramming methodology and differentiation protocols, on the genetics and function of the hPSC-derived neurons compared with natural brain cells is needed in order to have uniform and more coherent results. Improved technologies are being generated at an increasing pace in the recent years and it is expected that they should increase the knowledge gained from other research fields, including whole-genomic analysis, different proteomics and transcriptomic platforms and bioinformatics. It is expected that further studies in monogenic neurological disorders that display highly penetrant and early onset

syndromes will improve the overall understanding of these syndromes and offer beneficial insight into the complex nature of these brain disorders. 7.

Expert opinion

For many big pharmaceutical industries, developing drugs for the CNS diseases is increasingly seen as a high-risk therapeutic area with most candidates failing, thus resulting into a severe scaling back of pharmaceutical industry research in the neuroscience field. Contrast this with the persisting and growing unmet clinical need of psychiatric disorders imposing the largest disease burden globally, and an approaching dramatic increase in the health care costs for neurodegenerative diseases due to an aging population. One serious weakness that may have contributed to the delayed improvement in innovation in the CNS therapeutics is the widespread use of rodent cellular animal models of CNS disorders in both academia and industry, which might have a poor relevance for the human conditions targeted. Also, possible toxic effects of candidate drugs are often missed in rodent cells and animal models in general due to specific interactions with human biological processes that cannot be accurately recapitulated in these assay systems. With a growing number of patient-specific hPSCs available to the scientific community, increasing attempts to use hPSCs for disease modeling and drug screening are being explored. Success in this area requires the development and characterization of reliable disease phenotypes from both hPSCs and their derivatives. Initial results indicate that the approach to test candidate drugs for human neurological diseases in vitro using patientspecific hiPSCs can be successful and is not surprisingly that most major pharmaceutical companies have now started to develop in-house stem cell programs, and collaborate with academic groups. Yet many efforts are required for hPSCs derivatives to be used for drug screening at an industrial level. A sufficient number of cells need to be produced in a cost-effective manner. However, the cost of the reagents for large-scale hPSC cultures is prohibitive because of the reliance of expensive culture media and, similarly, scaled production of differentiated mature neuronal subtypes tends to rely on costly growth factors. In time, it is hoped hPSC-derived neurons to be produced in large quantities at low cost. The field is investing many efforts in this direction and in the last years costs for efficiently generating and cultivating hPSCs have been consistently lowered. Also, the transpositions of protocols for the obtainment of hPSC-derived mature neurons to high-throughput technologies, including drug screening, face the key limitations of the protracted time course of these procedures. Several weeks of differentiation are necessary to obtain late-born neurons and this is not compatible with HTS platforms. More rapid


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and less complex procedures are required and the field would benefit also from the development of reliable assays on earlier stages of maturation, that is, neural progenitors. Additionally, in an industrial setting, drug discovery and safety evaluation relies on high content imaging of many thousands of wells in 96-, 384- and 1536-well plates. Such automatized HTS platforms are starting to find use in phenotypic and functional assays on differentiated hPSC-derived cells and future more optimized integration will help accelerate the use of these cells by the pharmaceutical industry. For the next generation of drug development it will also be important to establish global hiPSC banks collecting large numbers of patient-specific hPSCs carrying causative mutations and wider genetic variations. Indeed, using panels of hPSCs from broad patient populations, compounds could potentially be profiled in these cells, representing ‘clinical trials in test tubes’. Also, testing drug candidates in hPSCs carrying distinct genetic mutations of certain diseases might help to choose the appropriate patient populations for the lead molecules. Effective patient stratification is expected to reduce clinical trial cost and also attrition rates. A valuable example of hPSC lines banking is the academic--industry partnership ‘StemBANCC project’, a consortium of 35 partners aiming to generate and make available to the academic and industrial institutions a wide panel of high-quality hiPSC lines. Beside these considerations, an important development of hPSC technology will be to facilitate a deeper assessment of Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Declaration of interest The authors are supported by the Associazione Amici del Centro Dino Ferrari and Associazione Amici del Moyamoya. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Affiliation Stefania Corti*1, Irene Faravelli1, Marina Cardano2 & Luciano Conti†2 †, *Authors for correspondence 1 University of Milan, Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation, Neurology Unit, IRCCS Foundation Ca’Granda Ospedale Maggiore Policlinico, via Francesco Sforza 35, Milan 20122, Italy Tel: +39 02 55033817; E-mail: [email protected] 2 Universita degli Studi di Trento, Centre for Integrative Biology - CIBIO, Via Sommarive 9, 38123 Povo, Trento, Italy Tel: +39 0461 285216; E-mail: [email protected]

15

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