Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases

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1 Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases Maria C.N. Marchetto, Beate Winner and Fred H. Gage Laboratory of Genetics (LOG-G), The Salk Institute for Biological Studies, North Torrey Pines Road, La Jolla, CA 92037, USA Received February 15, 2010; Revised and Accepted April 19, 2010 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 doi: /hmg/ddq159 Advance Access published on April 23, 2010 R71 R76 Most of our current knowledge about cellular phenotypes in neurodevelopmental and neurodegenerative diseases in humans was gathered from studies in postmortem brain tissues. These samples often represent the end-stage of the disease and therefore are not always a fair representation of how the disease developed. Moreover, under these circumstances, the pathology observed could be a secondary effect rather than the authentic disease cellular phenotype. Likewise, the rodent models available do not always recapitulate the pathology from human diseases. In this review, we will examine recent literature on the use of induced pluripotent stem cells to model neurodegenerative and neurodevelopmental diseases. We highlight the characteristics of diseases like spinal muscular atrophy and familial dysautonomia that allowed partial modeling of the disease phenotype. We review human stem cell literature on common neurodegenerative late-onset diseases such as Parkinson s disease and amyotrophic lateral sclerosis where patients cells have been successfully reprogrammed but a disease phenotype has not yet been described. So far, the technique is of great interest for early onset monogenetic neurodevelopmental diseases. We speculate about potential further experimental requirements and settings for reprogrammed neurons for in vitro disease modeling and drug discovery. INTRODUCTION In neurology, nerve biopsies are feasible and performed to investigate diseases of the peripheral nervous system. However, owing to the invasiveness of this procedure, neurons in the central nervous system (CNS) are only taken for biopsy under rare conditions. This inability to sample live brain cells limits our knowledge of human neuropathological abnormalities during the course of diseases. Currently, our understanding about disease-related neuronal phenotypes in humans is generated from analyzing postmortem brain tissues that are not always well preserved. In addition, these samples often represent the end-stage of the disease. Mouse models provide a means to mimic human genetic forms of neurodegenerative diseases, and great insights into mechanisms have been made using transgenic/knockout technologies. However, this approach is limited to monogenetic disorders and thus can only represent a minority of diseases. Owing to technical challenges, species differences and genetic background, even neurologic disorders with defined genes in some cases cannot be adequately modeled by mouse transgenic technology, indicating the need for advancement toward humanized models. Yamanaka s original reprogramming experiment surprised the scientific community by overturning the dogma that specialized cells in the body retain an immutable identity (1). A set of transcriptional factors has the ability to jump start a specific cell fate from a differentiated cell type, in a remarkable demonstration of cell flexibility. In this review, we will examine the recent literature on the use of induced pluripotent stem cells (ipsc) to model neurodegenerative and neurodevelopmental diseases. Although patient-specific ipsc have the promise of being less immunogenic for potential future cell transplantation therapies, our main focus here will be on the future clinical relevance of these cells for in vitro neurologic disease modeling and drug discovery. IPSC DERIVED FROM HUMAN NEUROLOGIC DISEASES Diseases that have been modeled for reprogramming can be divided into rare, monocausal genetic diseases and the large group of sporadic and multifactorial diseases. No large-scale disease modeling is currently available for the latter group. Downloaded from hmg.oxfordjournals.org at University of California, San Diego on March 21, 2011 To whom correspondence should be addressed. Tel: extn 1012; Fax: ; gage@salk.edu # The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 R72 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 It has been more difficult and challenging to obtain conclusive results from this group due to the complexity of the different genetic backgrounds and environmental clues involved in these diseases. However, even patients with monogenetic diseases within families display large genotype phenotype variability (2), likely due to environmental influence. It will be interesting to determine whether the same variability can be reproduced in ipsc-derived neural cells or if reprogramming in culture eliminates environmental noise. Reprogramming of fibroblasts for several neurologic diseases has been reported (Table 1), but few studies have actually recapitulated the phenotype of diseases in the ipsc-derived neuronal population. Successful generation of ipsc-derived neurons has been reported for sporadic middle- or late-age onset neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and Parkinson s disease (PD) (3 5). From the reprogramming point of view, it is remarkable that aged fibroblasts (up to 85 years old) from ALS and PD patients could still be reset with similar efficiency as fibroblasts from younger patients. Nonetheless, demonstration of disease-related phenotypes in the relevant cell type (i.e. the specific cells that are affected in the disease) has been a major challenge during the past 2 years. It may well be that ipsc-derived neural cells from agedependent neurodegenerative diseases will not show a phenotypic difference compared with normal control cells with regard to morphology, differentiation and survival. Test assays for challenging these cells for characterization of disease phenotypes could be required. For example, culturing cells under increased oxidative stress may reveal and/or accelerate aberrant neuronal phenotypes in late-onset diseases. Partial disease modeling with spontaneous induction of a disease phenotype was reported in two young childhood-onset monogenetic diseases: spinal muscular atrophy (SMA) and familial dysautonomia (FD) (6,7). Both diseases are autosomal recessive and share the common trait of rapid disease progression within the first years of life. In addition, both diseases are associated with loss of function of the gene as well as a role in RNA processing. SMA is a group of autosomal recessive diseases caused by large deletions or point mutations in the survival motor neuron (SMN) genes, leading to loss of function of the survival motor neuron protein. SMN1 gene encodes a 20 kb protein, spans 9 exons and has a role in RNA processing (8 11). SMA type 1 is characterized by mutations in the SMN1 gene and promotes fast progressing degeneration of motor neurons, inducing muscular atrophy and symmetric proximal lower limb weakness. SMA type 1 onset occurs around the sixth month of life, and death often occurs due to respiratory failure before the age of 2. Ebert et al. (6) were able to derive ipsc from fibroblasts from a single SMA patient and showed a decrease in ipsc-derived motor neuron survival after 6 weeks of differentiation, compared with ipsc-derived neurons from the patient s unaffected mother. It remains to be shown whether the neuronal cells derived were indeed functional (i.e. able to fire action potentials or make neuromuscular junctions). Moreover, authors detected an increase in the nuclear number of SMA gems protein aggregates that correlate with disease intensity in fibroblasts and ipsc derived from the SMA patient. Interestingly, this deficiency could be reversed in fibroblasts or SMA-iPSC by increasing Table 1. ipsc for modeling human neurological diseases Disease Neuronal pathology References Type 1 SMA Decrease in motor neuron number after (6) long-term culture FD Neuronal differentiation and migration (7) impaired PD (sporadic) Not shown (3,4) ALS (SOD1 mutation) Not shown (5) Huntington s disease Not shown (3) Rett syndrome Not shown (31) (MeCP2 mutation) Down syndrome Not shown (3,55) wild-type SMA protein levels using non-specific SMN-inducing compounds (6). This work showed for the first time a proof-of-principle for a potential future drugscreening platform using the ipsc technology. However, initial enthusiasm was decreased by the absence of other SMA patients and controls. Incorporation of additional control and patient cells would have reduced the concern that the observed phenotype is a consequence of the intrinsic ipsc variability system (discussed in what follows). FD is an autosomal recessive disease mostly occurring in persons of Ashkenazi Jewish descent (12). The disease is characterized by degeneration of sensory and autonomic neurons, leading to severe and often lethal autonomic dysfunction. Common clinical features include alacrima, hypoactivity and relative indifference to pain and temperature. A splicing defect in the IkB kinase complex-associated protein (IKBKAP) gene results in a tissue-specific splicing defect, inducing a loss of function or reduced levels of the IKAP protein (13). IPSC derived from three patients with FD revealed that neural crest precursors, specifically, had low levels of IKBKAP expression. In addition, a defect in neuronal differentiation and migration was reported. A drug candidate, kinetin, was able to reduce the levels of mutant IKBKAP splice forms and improved neuronal differentiation, but not cell migration, in ipsc-derived neural crest precursors, suggesting incomplete phenotype complementation (7). Drug screening using kinetin-like variations could be performed using recovery of both of neuronal differentiation and cell migration phenotypes as readout in future studies. PD is the second most common neurodegenerative disease. Prominent clinical features are motor symptoms (bradykinesia, tremor, rigidity and postural instability) and non-motor-related PD symptoms (olfactory deficits, autonomic dysfunction, depression and sleep disorders). PD is a synucleinopathy, with accumulation of misfolded alpha-synuclein, that forms intracellular inclusions: Lewy bodies and Lewy neurites. Loss of dopaminergic (DA) neurons in the substantia nigra of the midbrain and in other brain regions is a characteristic neuropathological hallmark (14,15). Several different techniques to produce DA neurons in culture from human embryonic stem cells (HESC) are currently available. They include co-culture systems [such as mesencephalic astrocytes (16) and stromal cell-derived inducing activity (17)] and direct differentiation protocols (18). When these cells were transplanted into animal models of PD, functional integration was observed, although technical issues were reported (19). Downloaded from hmg.oxfordjournals.org at University of California, San Diego on March 21, 2011

3 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R73 Disease modeling in the dish using embryonic stem cells (ESC) is still limited. Even though specific toxicity and cell death of a-synuclein overexpression were shown in mouse ESC-derived DA neurons (20,21), a-synuclein overexpressed in human neural embryonic cells resulted in patterns of degeneration that recapitulate PD features only in some cases (22). Mouse ipsc-derived precursors were differentiated into DA neurons and transplanted into 6-OHDA-lesioned rats, a rat model of DA depletion. The authors showed that a striatal graft of ipsc-derived neurons expressed midbrain DA markers and functionally integrated after transplantation (23). Primary fibroblasts from sporadic PD patients were successfully reprogrammed and differentiated into DA neurons as efficiently as those from healthy individuals (3,4). These results are promising, as age does not seem to interfere with reprogramming. Phenotypic differences were not reported, indicating that more subtle analysis or even strong stressors or toxins will be necessary to reveal phenotypes of diseases with late onset. ALS or Lou Gehrig s disease is a progressive fatal neurodegenerative disease affecting mainly motor neurons. The most common clinical features of ALS are degeneration of motor neurons producing fasciculation, muscle wasting and hyperreflexia. Respiratory complications usually develop in patients with advanced disease and the cause of death is generally paralysis of the respiratory muscles and diaphragm. With a projected lifetime risk of 1/2000, ALS is considered one of the most common motor neuron diseases (24,25). ALS is universally fatal, with a median age of onset of 55 years and survival of 2 5 years from symptoms onset. Although the exact pathophysiological mechanisms underlying neurodegeneration in ALS remain uncertain, the presence of a persistent inflammatory reaction prompted researchers to study the involvement of a non-cell-autonomous component in motor neuron death. HESC have been used for modeling both the autonomous and the non-cell-autonomous effects of ALS in vitro, using a gene that is mutated in 20% of the familial cases, superoxide dismutase 1 (SOD1) (26 28). IPSC technology allows for the unprecedented opportunity to also include patient genetic background in the cell-modeling system. Dimos et al. (5) successfully reprogrammed cells from two familial ALS patients. The ipsc generated were able to differentiate in motor neurons (the affected neuronal subtype in ALS pathology), but no phenotype has yet been observed or reported. It remains to be determined whether ipsc-derived neurons have the potential to recapitulate ALS late-onset pathology in vitro and if both familial and sporadic (vast majority of ALS) cases share common phenotypic traits in culture. MODELING NEURODEVELOPMENTAL DISORDERS We anticipate that ipsc technology could be used for modeling complex neurodevelopmental disorders such as autism and schizophrenia. However, initial results would probably emerge from modeling monogenetic, early-onset occurrences of these diseases. An example of a neurodevelopmental disease with potential for disease modeling by ipsc technology is Rett syndrome. Rett syndrome is characterized by arrested development in early childhood, regression of acquired skills, loss of speech, stereotypical movements, microcephaly, seizures, autistic characteristics and mental retardation (29). Rett patients have mutations in the X-linked gene, MeCP2, a gene that binds to methylated cytosines in the DNA and is believed to epigenetically regulate global expression of genes (30). In fact, Rett patients ipsc have been recently generated, but neither their X-inactivation/reactivation status nor their differentiation potential has been extensively studied (31). Studying the in vitro phenotypic consequences of the mutation in specific genes can help to identify a molecular mechanism responsible for subtle alterations in the nervous system, perhaps pointing to common mechanisms for more complex, multigenetic diseases. A future challenge for neurodevelopmental disorders is the contribution of genetic background and environmental clues. New gene-targeting techniques in human pluripotent cells such as homologous recombination and zinc-finger nucleases may help to eliminate background noise and individual variability (32 37). Effective gene targeting in HESC could disrupt a specific disease-related gene and the resulting neuronal behavior could be compared with the patient s neuron containing the mutation. As an alternative for revealing complex neuronal phenotypes and niche-specific behaviors, patient-derived cells could be transplanted on the CNS of animal models. In fact, HESC were shown to integrate and form functional connections with host cells when transplanted in the ventricles of embryonic mice (38). Such a chimeric model was recently proposed as an in vivo model to study environmental contributions to complex neurological diseases (39). USING NEURONAL CELLS FOR DRUG SCREENING In the past, drug screening was performed in human cell lines and they have represented a major step forward in medical therapy progress. A prime example is the development of vaccines for polio, which were originally made by in vitro studies on cell lines like HeLa cells (40). ipsc derived from patients seem to offer a significant advantage as they take into consideration the patient s background, the affected cell type and the developmental time. In addition, they allow the generation of both genetic and sporadic forms of the disease. One of the great benefits of reprogramming cells is the possibility of studying the developmental steps from human neural cells before they are differentiated into mature neurons. Neural progenitor cells in culture can give rise to glial and neuronal populations (41). These populations can further differentiate into subtypes of glia as well as subtypes of neurons with distinct properties (i.e. DA or cholinergic). It is not unlikely that some neurodegenerative diseases have their origins in the neural progenitor population rather than in the mature neuron. In these cases, therapeutic interventions should be tested at an early stage of development. Finally, ipsc technology could help to unveil the changes in connectivity properties between neurons that are affected by a given neurologic disease. Downloaded from hmg.oxfordjournals.org at University of California, San Diego on March 21, 2011

4 R74 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 Figure 1. ipsc to model neurodegenerative and neurodevelopmental diseases. Human ipsc from neurologic patients and controls are generated after somatic tissue reprogramming (e.g. skin or blood cells). Neural progenitor cells (NPC) are generated and are further differentiated into neurons and/or glial cells. Neurons are then differentiated into subtypes of neurons such as dopaminergic, cholinergic, etc. Cellular phenotype is assessed by measuring neuronal morphology (i.e. process branching, spine density/size/maturation). Next, connectivity and circuitry integration can be analyzed by calcium influx transients, electrophysiology and transneuronal tracing with the rabies virus. In addition, the cross-talk between neurons and glia can be studied to tease out autonomous and non-autonomous aspects of the disease. Once a distinct disease-related phenotype is identified, drug-screening platforms can be developed to test compounds that improve cellular phenotype. Therapeutic compounds could emerge from the screenings, potentially benefiting neurologic patients. Stem cell biologists can borrow a number of wellestablished tools from the neurosciences to address neuronal maturation and circuitry integration in vitro. For evaluating single-cell properties in a disease context, one could analyze neuronal morphology, branching, spine density/size/maturation and neuronal polarity. Circuitry integration analysis could be assessed by calcium influx transients, and presynaptic connections could be revealed after rabies virus infection. Likewise, paired electrophysiological recordings could address synaptic strength between cells. Recent reports have used such tools in a disease context and have been able to find altered neuronal phenotypes in complex disorders (42 44). In addition, as different types of neural cells can be generated from ipsc, neuron-autonomous and nonautonomous effects (i.e. the interplay between neurons and glia) can be studied individually or in combination. Technology is already available to generate functionally active, cortical-like mouse neurons from differentiated cells without the need for complete reprogramming to ipsc (45,46). However, it remains to be seen whether this technology can generate the plethora of neuronal subtypes that ipscderived neurons can. Moreover, direct conversion from somatic cells to postmitotic neurons, without the step of pluripotency, will limit the amount of cells available to study the disease and will likely not recapitulate disease phenotypes that occur in the neural progenitor state. Once a consistent abnormal disease-related phenotype is identified, screening platforms can be developed to test compounds (proteins, small molecules, small hairpin RNAs) that revert or protect the cellular phenotype. After rigorous testing, therapeutic compounds will emerge from the screenings that could potentially benefit a large cohort of patients (Fig. 1). CAUTIONARY NOTES The available lines of HESC are notoriously variable with regard to epigenetic marks, expression profile and differentiation propensity (47,48). Even though the initial hope was that ipsc technology would generate pluripotent cells that were less variable than HESC cell lines, recent reports suggest that significant intrinsic variability remains in the ipsc lines generated to date (49). Pick et al. (49) detected Downloaded from hmg.oxfordjournals.org at University of California, San Diego on March 21, 2011

5 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R75 abnormal expression of imprinted genes in a significant number of ipsc lines. Those differences have been generally attributed to the introduction of reprogramming factors, using randomly integrating viral vectors and/or by persistent donor cell gene expression (4,50). It remains to be seen if ipsc generated in the absence of integrating factors still recapitulate intrinsic variability (51 53). Moreover, expression profile analysis on integration-free human ipsc has shown an expression signature in ipsc that is distinct from both the original population and standard HESC (51). An intriguing study recently performed by Hu et al. (54) showed that the neuronal differentiation competence of ipsc was highly variable when compared with HESC differentiation and surprisingly independent of transgene expression. More data are necessary to uncover the levels of variability between HESC and ipsc lines (generated with different methods) in both undifferentiated and differentiated states. Determining the variability levels between lines and clones will allow researchers to elucidate more robust phenotypes on cells derived from diseased ipsc. Moreover, dissecting the ipsc-intrinsic variability may provide clues as to which wild-type ipsc would be the most suitable experimental control and how many control lines should be derived for each experiment. For example, it is currently unclear whether the best controls for diseased ipsc lines should be ipsc derived from age/ gender-matched donors or from a family member regardless of age/gender. CONCLUDING REMARKS Scientists are now using the powerful ipsc technology to investigate early stages of human development and to model diseases. So far, this technique has been shown to be of specific interest for monogenetic neurodevelopmental diseases, providing an innovative way to understand disease pathology. Modeling late-onset neurodegenerative diseases and multifactorial neurodevelopmental diseases will require additional advances. For example, in the future, the switch from one cell type to another might help the direct conversion of astrocytes to motor neurons in spinal cord trauma patients or other neurodegenerative diseases, perhaps combining gene and cell therapy in vivo. On the other hand, the recapitulation of all stages of neural development by ipsc is an invaluable tool to depict the exact moment of the disease onset, optimizing therapeutical interventions. The potential of cellular reprogramming is limited only by human creativity and ethical guidelines. Neuroscientists in the past could not have imagined a scenario in which patient-derived neural cell types would be readily accessible to thousands of laboratories around the world, and researchers in the future will never imagine neuroscience without it. ACKNOWLEDGEMENTS The authors would like to thank J. Simon for illustrations and M.L. Gage for editorial comments. Conflict of Interest statement. None declared. FUNDING The authors are funded by the California Institute of Regenerative Medicine (CIRM) and National Institutes of Health (NIH). F.H.G. is supported by The Lookout Fund and the Mathers Foundation. M.C.N.M. is supported by Christopher and Dana Reeve Foundation (CDRF); B.W. is supported by Tom Wahlig Foundation and is a Feodor Lynen fellow of the Alexander von Humboldt Foundation. REFERENCES 1. Takahashi, K. and Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. 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7 A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells Maria C.N. Marchetto, 1,5 Cassiano Carromeu, 2,5 Allan Acab, 2 Diana Yu, 1 Gene W. Yeo, 3 Yangling Mu, 1 Gong Chen, 4 Fred H. Gage, 1 and Alysson R. Muotri 2, * 1 The Salk Institute for Biological Studies, North Torrey Pines Road, La Jolla, CA 92037, USA 2 University of California San Diego, School of Medicine, Department of Pediatrics, Rady Children s Hospital San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, 9500 Gilman Drive, La Jolla, CA 92093, USA 3 University of California San Diego, School of Medicine, Department of Cellular & Molecular Medicine, Stem Cell Program, 9500 Gilman Drive, La Jolla, CA 92093, USA 4 Pennylvania State University, Department of Biology. 201 Life Science Building, University Park, PA 6802, USA 5 These authors contributed equally to the work *Correspondence: muotri@ucsd.edu DOI /j.cell SUMMARY Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (ipscs) from RTT patients fibroblasts. RTT patients ipscs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment. INTRODUCTION Autism spectrum disorders (ASD) are complex neurodevelopmental diseases affecting 1 in 150 children in the United States (Autism and Developmental Disabilities Monitoring Network Surveillance Year 2000 Prinicipal Investigators; Centers for Disease Control and Prevention, 2007). Such diseases are mainly characterized by impaired social interaction and repetitive behavior. Family history and twin studies suggest that, in some cases, these disorders share genetic roots, but the degree to which environmental and genetic patterns account for individual differences within ASD is currently unknown (Piven et al., 1997; Ronald et al., 2006). A different combination of genetic mutations is likely to play a role in each individual. Nevertheless, the study of mutations in specific genes can help to identify molecular mechanisms responsible for subtle alterations in the nervous system, perhaps pointing to common mechanisms for ASD. Rett syndrome (RTT) is a progressive neurological disorder caused by mutations in the X-linked gene encoding MeCP2 protein (Amir et al., 1999). RTT patients have a large spectrum of autistic characteristics and are considered part of the ASD population (Hammer et al., 2002; Samaco et al., 2005, 2004; Zappella et al., 2003). These individuals undergo apparently normal development until 6 18 months of age, followed by impaired motor function, stagnation and then regression of developmental skills, hypotonia, seizures and autistic behavior (Amir et al., 1999). MeCP2 may be involved in the epigenetic regulation of target genes, by binding to methylated CpG dinucleotides within promoters, and may function as a transcriptional repressor, although this view has been challenged recently (Chahrour et al., 2008; Yasui et al., 2007). Pluripotent human embryonic stem cells (hescs) have been successfully generated from early stage human embryos and can differentiate into various cell types (Thomson et al., 1998). However, to develop cellular models of human disease, it is necessary to generate cell lines with genomes predisposed to diseases. Recently, reprogramming of somatic cells to a pluripotent state by overexpression of specific genes (induced pluripotent stem cells, ipscs) has been accomplished (Takahashi and Yamanaka, 2006; Yu et al., 2007). Isogenic pluripotent cells are attractive not only for their potential therapeutic use with lower risk of immune rejection but also for understanding complex diseases (Marchetto et al., 2010; Muotri, 2008). Although ipscs have been generated for several neurological diseases (Dimos et al., 2008; Ebert et al., 2009; Hotta et al., 2009; Lee et al., 2009; Park et al., 2008; Soldner et al., 2009), the demonstration Cell 143, , November 12, 2010 ª2010 Elsevier Inc. 527

8 of disease-specific pathogenesis and phenotypic rescue in relevant cell types is a current challenge in the field (Marchetto et al., 2010). We have developed a human model of RTT by generating ipscs from fibroblasts of RTT patients carrying different MeCP2 mutations and unaffected individuals. We show that RTT-iPSCs retained the capacity to generate proliferating neural progenitor cells (NPCs) and functional neurons that underwent X-inactivation. We observed a reduced number of dendritic spines and synapses in ipsc-derived neurons. Moreover, we detected an altered frequency of intracellular calcium spikes and electrophysiological defects in RTT-derived neuronal networks, revealing potential new biomarkers for RTT pathology. Gain and loss of function experiments in ipsc-derived neurons confirmed that some of the alterations observed were related to MeCP2 expression levels. Finally, we used the ipsc system to test candidate drugs to rescue synaptic deficiency in RTT neurons. Together, our results suggest that RTT and other complex CNS diseases can be modeled using the ipsc technology to investigate the cellular and molecular mechanisms underlying their abnormalities. RESULTS Generation of ipscs from RTT Patients and Normal Individuals Nonaffected control fibroblasts and cells carrying four distinct MeCP2 mutations (Figure 1A and Table S1 available online) isolated from clinically affected female patients with RTT symptoms were infected with retroviral reprogramming vectors (Sox2, Oct4, c-myc and Klf4), as described elsewhere (Takahashi et al., 2007). After 2 to 3 weeks, compact ipsc colonies emerged from a background of fibroblasts (Figures 1B and 1C). Colonies were manually picked and transferred to matrigel (Figures 1D and 1E). We obtained at least 10 clones from each control (WT)-iPSC and RTT-iPSC that continuously expressed pluripotent markers such as Nanog, Lin28, Tra-1-81 and Sox2 (Figures 1F and 1G and Figures S1A S1C). All ipsc clones used in this study maintained a normal karyotype (Figure 1H). Teratomas containing derivatives from all 3 embryonic germ layers confirmed that the ipscs were able to differentiate in vivo (Figure 1I). PCR fingerprinting confirmed their derivation from respective fibroblasts (data not shown). Next, we asked if the global molecular signatures of RTT-iPSC clones carrying the two distinct MeCP2 mutations (1155del32 and Q244X) and WT-iPSC clones (from AG09319) resembled those of available hesc lines (HUES6). Gene expression profiles measured using human genome Affymetrix Gene Chip arrays were grouped by hierarchical clustering, and correlation coefficients were computed for all pair-wise comparisons (GEO accession number GSE21037). We observed that the WT-iPSC and RTT-iPSC clones were almost indistinguishable. The results clearly revealed that the ipsc and hesc lines were more similar to each other than to the respective original fibroblasts (Figure S1D). These findings, combined with manual inspection of the gene expression of known pluripotent- and fibroblast-related genes (Figures S1E and S1F), indicated that the reprogramming was successful. In Table S2 we present a summary of all ipsc subjects and clones utilized for each experiment. Neural Induction of ipscs Our protocol for neuronal differentiation is outlined in Figure 2A. We initiated neural differentiation by plating embryoid bodies (EBs). After a week, EB-derived rosettes became apparent (Figure 2B). Rosettes were then manually collected, dissociated and re-plated. The NPCs derived from rosettes formed a homogeneous population after a couple of passages. NPCs were positive for early neural precursor markers, such as Nestin, Sox1-2 and Musashi1 (Figure 2C). To obtain mature neurons, EBs in the presence of retinoic acid (RA) were dissociated and re-plated (Figure 2B). At this stage, cells were positive for Tuj1 (b-iii-tubulin) and Map2 (Microtubule-associated protein 2) (Figure 2D). Moreover, we detected expression of GABA (g-amino butyric acid) and VGLUT1 (vesicular glutamate transpoter-1). We also observed synapsin puncta outlining Map2-positive neurites (Figure 2D). We did not detect a significant alteration in RTT neuronal survival when compared to controls, as measured by Map2 staining (Figure 2E and Figure S2A). In addition, infection with a lentivirus expressing the DsRed gene under the control of Synapsin promoter (Syn::DsRed) did not reveal any difference in neuronal survival between RTT and controls (Figure 2E and Figure S2B). Interestingly, the number of GABA-positive neurons was also not affected between RTT and controls (Figure 2F and Figure S2C). X-Inactivation during Neuronal Differentiation of RTT-iPSCs In female hescs, both chromosomes should be active, but one X chromosome becomes silenced upon differentiation (Dhara and Benvenisty, 2004). Similar to ESCs, female mouse ipscs have shown reactivation of a somatically silenced X chromosome and have undergone random X-inactivation upon differentiation (Maherali et al., 2007). Because MeCP2 is an X-linked gene, we examined the ability of our RTT-iPSCs clones to reset the X chromosome (i.e., to erase X-inactivation) and whether X-inactivation would take place again after neuronal differentiation (Figure 3A). We stained RTT-iPSCs clones and their respective fibroblasts with an antibody against trimethylated histone 3 Lysine 27 (me3h3k27), an epigenetic silencing marker present on the inactive X chromosome in interphase nuclei (Silva et al., 2003). Some, but not all, undifferentiated RTT-iPSCs clones displayed diffuse immunoreactivity throughout the nucleus, similar to some hescs, showing that the memory of the previous inactivation state had been erased (Figure 3B). For further analysis, we only selected clones that displayed a diffuse me3h3k27 pattern to differentiate into neurons. Upon neuronal differentiation, intense nuclear foci staining, a prominent diagnostic of the inactive X, was found in 80% of neurons labeled by the infection of a lentivirus carrying the neuron-specific Synapsin promoter driving the EGFP reporter (Syn::EGFP). Nuclear foci were also present in RTT fibroblasts before reprogramming (Figure 3B). We quantified the percentage of cells displaying either a diffuse or intense X-inactivation (nuclear foci) (Figure 3C). Our data suggest that the majority of cells in selected clones from both hescs (99%) and ipscs (95%) have a diffuse pattern. In contrast, 528 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

9 Figure 1. Generation of ipscs (A) Schematic representation of the MeCP2 gene structure and mutations used in this study. UTR, untranslated region; MBD, methyl-cpg binding domain; NLS, nuclear localization signal; Poly-A, polyadenylation signal; TRD, transcriptional repression domain; WW, domain-containing WW; X, stop codon. Respective cell-line codes are shown close to their mutations. (B) Morphology of human fibroblasts before retroviral infection. (C) Aspect of ipscs colonies 14 days after infection. (D and E) Representative images of established ipsc colonies. (F and G) Representative images of RTT-iPSCs showing expression of pluripotent markers. (H) No karyotypic abnormalities were observed. (I) Representative images of teratoma sections. The scale bar represents 100 mm. See also Figure S1. differentiated populations of fibroblasts and ipsc-derived neurons have me3h3k27 nuclear foci staining, indicating X-inactivation. We also used fluorescent in situ hybridization (FISH) to visualize Xist RNA, a noncoding transcript involved in X chromosome silencing that physically wraps the inactive X (Lucchesi et al., 2005). Before reprogramming, the majority of fibroblasts exhibit a clear Xist cloud. The signal is lost after reprogramming, indicating that selected ipsc clones have two active X chromosomes in our culture conditions. A Xist cloud is also observed in ipsc-derived neurons (Figure 3D). Fluorescent in situ hybridization (FISH) analysis using a centromeric X chromosome probe in ipsc-derived NPCs and neurons showed the presence of two X chromosomes (Figure 3E). As a consequence of both X-chromosomes activation after reprogramming, the MeCP2 protein can be detected in undifferentiated ipscs from RTT patients (Figure 3F). However, after differentiation, RTT-iPSC-derived neurons recapitulated X-inactivation and the population became mosaic regarding MeCP2 expression. Immunostaining was performed on several RTT-iPSC clones, and a representative example of MeCP2 expression after differentiation is shown in Figure 3F. Clones obtained from RTT fibroblasts carrying the 1155del32 MeCP2 mutation do not produce a fulllength MeCP2 protein (Traynor et al., 2002). Next, we selected one WT-iPSC clone (WT-33 C1) and one RTT-iPSC clone (1155del32 C15) to determine whether the RTT-iPSC-derived neuronal population showed reduced MeCP2 protein levels. As expected, we observed a reduction in the full-length MeCP2 protein amounts in both fibroblasts and neurons derived from the RTT-iPSC clone (Figure 3G). We tested the original fibroblasts and ipsc-derived neurons from this patient for X-inactivation using standard methodology for the androgen receptor locus (Allen et al., 1992). RTT fibroblasts carrying the 1155del32 MeCP2 mutation had a 55:45 distribution, but RTT-derived neurons showed highly skewed X-inactivation, with a 96:4 distribution (Figure S3). The outcome of the X-inactivation process, measured by the androgen receptor locus, seems to be consistent within the same clone. An independent differentiation of the same clone (RTT-1155del32 C15) yielded a 98:2 distribution. Unfortunately, androgen receptor locus analysis was not conclusive for the MeCP2 mutation Q244X Cell 143, , November 12, 2010 ª2010 Elsevier Inc. 529

10 Figure 2. Neural Differentiation of ipscs (A) Schematic view of the neural differentiation protocol. (B) Representative images depicting morphological changes during neuronal differentiation. The scale bar represents 100 mm. (C) NPCs are positive for neural precursor markers: Sox1, Sox2, Musashi1, and Nestin. The scale bar represents 50 mm. (D F) (D) Representative images of cells after neuronal differentiation. ipsc-derived neurons express mature neuronal markers: GABA, Map2 and Synapsin. The scale bar represents 20 mm. Similar numbers of Map2-positive and Syn::DsRed-positive (E) as well as GABA-positive (F) neurons from WT and RTT cultures. Data shown as mean ± SEM. See also Figure S2. Our data show that X-inactivation was erased in selected reprogrammed RTT-iPSCs clones and subsequently restored during neuronal differentiation. Importantly, the recapitulation of X-inactivation produces mosaic neuronal cultures with different ratios of cells expressing normal MeCP2 levels, mimicking what is observed in RTT patients brains. Our data do not preclude that partial reprogramming from a single fibroblast or retention of the X-inactivation would lead to clones with highly skewed X-inactivation, where neurons would express only the normal or mutant form of MePC2. In fact, we do observe WT and RTT-iPSC clones retaining X-inactivation after reprogramming. The RTT-T158M C3-derived neurons showed 100:0 distribution. The expression of the mutant MeCP2 allele was confirmed by sequencing. cells. However, a reduction of 50% in the amount of MeCP2 protein level (Figure S4E) is consistent with a random X-inactivation. We have not analyzed the distribution for RTT-R306C clones. Normal Cellular Proliferation from RTT-iPSC-Derived NPCs An increased incidence of large head size has been reported in autism (Piven et al., 1995). Other studies have suggested that the autistic brain is smaller at birth, followed by rapid head growth during early development and then a period of reduced brain growth (Courchesne et al., 2003). Head growth deceleration has also been reported for RTT patients (Hagberg et al., 2001). Since the cellular mechanism behind this phenomenon is unknown, we investigated whether a perturbed NPC replication cycle was affected in RTT. NPCs derived from RTT-iPSCs, WT-iPSCs and hescs (Cyth25 and HUES6) were generated and kept under proliferating conditions in the presence of FGF2. NPCs were derived using the same protocol described above, had identical passage numbers and were analyzed for cell cycle by flow cytometry. Our results showed no significant differences in any cycle phase between HESC-, WT-iPSC- and RTT-iPSC-derived NPCs (Figure 4A), though we cannot exclude the possibility that 530 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

11 altered head growth in RTT patients is caused by eventual abnormal NPC proliferation in another developmental stage. We then investigated potential phenotypic changes in RTT neurons compared to controls. Reduced Glutamatergic Synapse Number and Morphological Alterations in RTT Neurons Strong evidence implicates synapse alteration in ASD, including RTT (Zoghbi, 2003). Loss of MeCP2 and doubling of MeCP2 dosage in mice have opposite effects on excitatory synapse numbers in individual neurons (Chao et al., 2007). These results suggest that MeCP2 may be a rate-limiting factor in regulating glutamatergic synapse formation and indicate that changes in excitatory synaptic strength may underlie global network alterations in RTT. Therefore, we determined whether excitatory synapse numbers were reduced in human RTT neurons. After 8 weeks of differentiation, glutamatergic neurons were identified using antibodies against VGLUT1 (Takamori et al., 2000), and dendrites were labeled with Map2 (Figure 4B). To confirm the specificity of glutamatergic neurons in our cultures, we showed that VGLUT1 puncta were mostly adjacent to the postsynaptic density-95 (Psd95) protein (Niethammer et al., 1996) (Figure S4A). We found a reduction in the density of VGLUT1 puncta from RTT-iPSCs clones carrying 3 different MeCP2 mutations compared to HUES6 and distinct WT-iPSCs-derived Map2- positive neurons, suggesting a specific defect in glutamate transport in RTT cultures (Figure 4B and Figure S4B). Since neurons carrying different MeCP2 mutations showed reduced VGLUT1 puncta in our cultures, we tested whether loss of function of MeCP2 was directly related to the number of glutamatergic synapses in our neuronal cultures. We cloned an shrna against MeCP2 in a lentiviral vector that is able to knockdown both isoforms of MeCP2 (Figure S4C). Neurons derived from WT-iPSCs expressing the shmecp2 showed a similar reduction in VGLUT1 puncta when compared to control neurons expressing a scramble shrna (shcontrol) (Figure 4C and Figure S4B). Overexpression of MeCP2 using a lentiviral vector (Figure S4C) increased the number of VGLUT1 puncta in WT and RTT neurons (Figure 4D and Figure S4B). Our data strongly suggest that MeCP2 is a rate-limiting factor in regulating glutamatergic synapse number in human neurons. We also investigated whether RTT neurons displayed any morphological alteration when compared to controls. To visualize neuronal anatomy, we infected the cultures with the Syn::EGFP lentivirus. Morphological analysis of RTT neurons revealed that the number of spines in RTT neurites was reduced when compared to WT neurons and after ectopic expression of shmecp2 (Figure 4E). Consistent with this observation, the number of spines in dendrites of neurons from postmortem RTT patients brains was previously reported to be lower than that in normal individuals (Chapleau et al., 2009). Finally, we documented that the cell soma sizes from neurons derived from the RTT-iPSCs carrying different MeCP2 mutations were smaller when compared to controls (reduction of ± 4.83%). Similarly, loss of function using the shmecp2 knockdown strategy in WT neurons reduced soma size at levels comparable to RTT levels (reduction of ± 4.31%) (Figure 4F and Figure S4D). Rescuing a RTT Neuronal Phenotype Recent studies have shown that re-activation of MeCP2 expression knockout mice led to a prolonged life span and delayed onset or reversal of certain neurological symptoms (Giacometti et al., 2007; Guy et al., 2007). These reports suggest that some RTT phenotypes can be rescued in vivo. We used our model to analyze the effect of selected compounds that may revert the neuronal phenotype in culture as a validation for future highthroughput drug screening platforms. Administration of IGF1 was recently described to promote a partial reversal of the RTT-like symptoms in a mouse model (Tropea et al., 2009). We treated RTT-derived neurons carrying different MeCP2 mutations in culture with IGF1 and observed an increase in glutamatergic synapse number, suggesting that the drug treatment could correct the RTT neuronal phenotype (Figure 4B and Figure S4B). Around 60% of MeCP2 mutations in RTT are nonsense mutations (Laccone et al., 2001). Thus, we tested whether we could increase MeCP2 expression levels in affected neurons by suppressing the nonsense mutation (Q244X) with read-through of the premature stop codon using pharmacological treatments. High concentrations of aminoglycosides antibiotics, such as gentamicin, can bind to the 16S rrna, impairing ribosomal proofreading (Kellermayer, 2006). As a consequence, a fulllength protein is produced by incorporating a random amino acid at the stop codon position. We treated RTT-Q244X clones 3- and 4-derived neurons with two different doses of gentamicin and found that MeCP2 protein levels and glutamatergic synapse numbers were increased after 1 week (Figure 4G and Figure S4E). Treatment with a higher gentamicin dose (400ug/ml) for the same period did not rescue RTT neurons and lowered the number of VGLUT1 puncta in control neurons (Figure 4G). The finding that RTT patient-derived neurons displayed changes in neuronal morphology and in number of synapses prompted us to explore putative circuit alterations in vitro. Altered Activity-Dependent Calcium Transients in RTT-iPSC-Derived Cells Early in neural development, spontaneous electrical activity leads to increases in intracellular calcium levels and activation of signaling pathways that are important in regulating several neuronal processes (Spitzer et al., 2004). Recently, a disturbance in calcium homeostasis during early postnatal development was reported in a MeCP2 knockout model (Mironov et al., 2009). Moreover, several studies showed that functional mutations in genes encoding voltage-gated calcium channels and in genes whose activity is modulated by calcium, such as MeCP2, could lead to ASD (Splawski et al., 2006; Zhou et al., 2006). Neuronal activity-induced calcium influx can trigger the calcium/calmodulin-dependent protein kinase (CamK). CamK activation has been reported to induce phosphorylation of MeCP2, which was further postulated to regulate neuronal spine maturation (Tao et al., 2009; Zhou et al., 2006). Although these studies raised an interesting link between neuronal activity and spine maturation, the extent of cellular alteration in human ASD neurons was never characterized. To test if RTT-iPSCs-derived neuronal networks are affected in our system, we preloaded the cells with the calcium indicator fluo-4am and highlighted neurons using Cell 143, , November 12, 2010 ª2010 Elsevier Inc. 531

12 Figure 3. RTT-iPSC Clones Undergo X-Inactivation during Differentiation (A) Schematic representation of X-inactivation dynamics during reprogramming and further neural differentiation. RTT fibroblasts are mosaic for the MeCP2 WT gene expression. During reprogramming, X-inactivation is erased and ipscs express both MeCP2 alleles. Upon neuronal differentiation, X-inactivation is re-established and the resultant cells are mosaic for MeCP2 WT gene expression. (B) Immunofluorescence for me3h3k27 in fibroblasts, pluripotent cells (Nanog-positive) and after neuronal differentiation (Syn::EGFP-positive). Pluripotent cells (hescs and ipscs) show diffuse staining whereas differentiated cells (fibroblasts and neurons) exhibit prominent me3h3k27 nuclear foci (arrowheads). Cells were counterstained with Dapi. The scale bar represents 15 mm. 532 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

13 the Syn::DsRed vector. Cultures with similar cell density and numbers of DsRed-positive neurons were used (Figure S2B). Spontaneous calcium transients were analyzed from WT and RTT neuronal networks in several independent experiments over time (Figure 5). In our analyses, we only considered calcium transients generated by synaptic activity. Neurons were selected after confirmation that calcium transients were blocked with TTX or with the glutamate receptor antagonists CNQX (AMPA) and APV (NMDA) treatments, indicating neuronal signaling dependence on local synaptic connections (Figures S5A, S5B, and S5D). Gabazine, an antagonist of GABAa receptors, increased the number of calcium transients in the networks, indicating the presence of glutamatergic and gabaergic synapses in our system (Figure S5C, D). A representative example of calcium tracing in control and RTT neurons is depicted in Figure 5A and shows a sharp increase in amplitude followed by a decrease over time. The frequency of calcium oscillations in RTT neurons and in WT neurons expressing shmecp2 was abnormally decreased when compared to controls, suggesting a deficiency in the neuronal network connectivity and activity dynamics (Figures 5B and 5C and Figures S5E and S5F). The deficiency in connectivity was further corroborated by a decrease in the percentage of Syn::DsRed-positive neurons exhibiting calcium transients in the RTT cultures when compared to controls (Figure 5D and Figure S5F). Decreased Frequency of Spontaneous Postsynaptic Currents in RTT Neurons Next we determined the functional maturation of the ipsc-derived neurons using electrophysiological methods. Whole-cell recordings were performed from cells that had differentiated for at least 6 weeks in culture. Neurons were visualized by infection with the Syn::EGFP viral vector (Figure 6A). Both WT and RTT neurons showed similar transient sodium inward currents, sustained potassium outward currents in response to voltage step depolarizations, and action potentials evoked by somatic current injections (Figure 6B). Therefore, our data indicated that WT and RTT reprogramming did not affect the ability of WT-iPSC- and RTT-iPSC-derived neurons to mature and become electrophysiologically active. We also recorded spontaneous excitatory and inhibitory postsynaptic currents (sepscs and sipscs) as a way of measuring intercellular connectivity and network formation (Figures 6B and 6C). Cumulative probability plots of amplitudes and inter-event intervals of spontaneous postsynaptic currents revealed that RTT neurons have a significant decrease in frequency and amplitude when compared to WT neurons (Figures 6D and 6E). Together, our data suggest that the neuronal network is altered in RTT ipscderived cultures. DISCUSSION The lack of detectable symptoms in female RTT patients until 6 18 months of age and the apparent phenotypic reversibility of some RTT phenotypes in MeCP2 knockout animals indicate that MeCP2 is not essential for early wiring of the nervous system but instead may only be required at late stages. It is possible that RTT patients have aberrant excitatory synaptic strength at very early stages, when the disease phenotype is not yet clearly observed. In fact, increasing evidence from clinical studies and mouse models indicates the presence of alterations during the so-called presymptomatic developmental phase (Charman et al., 2002; De Filippis et al., 2009; Kerr et al., 1987; Picker et al., 2006; Santos et al., 2007). To study human RTT neurons in culture, we derived ipscs from RTT fibroblasts. RTT ipscs are pluripotent and able to recapitulate X-inactivation upon neuronal differentiation. Even though the ratio of neurons expressing mutant MeCP2 due to X-inactivation was variable, the phenotypes described here for all RTT-derived neurons are similar. One interpretation could be that astrocytes, or other nonneuronal cells, carrying MeCP2 mutations present in our cultures could also affect neurons expressing the normal MeCP2 protein. In fact, the non-cell-autonomous influence was recently described for RTT, indicating that glial cells carrying MeCP2 mutations can distress healthy neurons (Ballas et al., 2009; Kishi and Macklis, 2010; Maezawa et al., 2009). Using human neurons carrying MeCP2 mutations, we showed that RTT glutamatergic neurons have a reduced number of synapses and dendritic spines when compared to nonaffected controls. Moreover, electrophysiological recordings from RTT neurons showed a significant decrease in the frequency and amplitude of spontaneous synaptic currents compared to WT neurons. The reduced frequency in RTT neurons could reflect the presence of fewer release sites or a decreased release probability. The results of electrophysiology recordings are consistent with the decreased VGLUT1 puncta observed in Map2-positive dendrites from RTT neurons. Also consistent with these findings, the frequency of intracellular calcium transients was decreased in RTT neurons when compared to controls. Our data indicate a potential imbalance in the neuronal networks associated with RTT pathology. The observations described here provide valuable information for RTT and, potentially, ASD patients, since they suggest that presymptomatic defects may represent novel biomarkers to be exploited as diagnostic tools and that early intervention may be beneficial. Therapies aiming at earlier stages of development may attenuate the downstream consequences of MeCP2 mutations. Restoring protein levels may be challenging, since MeCP2 levels are tightly regulated and chronically overdosing neurons with the (C) Quantification of cells with diffused or foci me3h3k27 nuclear staining. Data shown as mean ± SEM. (D) RNA FISH shows that Xist RNA domains are present in the original fibroblasts before reprogramming. ipscs show no Xist expression. Neurons derived from normal and RTT ipscs show clear Xist clouds, indicating transcriptional silencing of the X chromosome (arrows). The scale bar represents 5 mm. (E) Two DNA FISH signals are evident in the nuclei of ipsc-derived NPCs and neurons, revealing the presence of two X chromosomes. The scale bar represents 10 mm. (F) RTT-iPSCs (1155del32) expressed WT MeCP2 but derived neurons displayed mosaicism regarding WT (arrowhead) and mutant (arrow) MeCP2 forms. The scale bar represents 50 mm. (G) RTT-derived fibroblasts and neurons have reduced levels of WT MeCP2 protein by Western blot. See also Figure S3. 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14 Figure 4. Alterations in RTT Neurons (A) Proliferating RTT NPCs displayed no signal of aberrant cell cycle when compared to controls. (B) Representative images of neurons showing VGLUT1 puncta on Map2 neurites. Bar graphs show synaptic density in RTT and WT neurons. IGF1 treatment increased VGLUT1 puncta number in RTT-derived neurons. The scale bar represents 5 mm. (C) Reduction of MeCP2 expression decreased the number of glutamatergic synapses in WT neurons. (D) Overexpression of MeCP2 increased the number of glutamatergic synapses. (E) Representative images of neurites of different genetic backgrounds. Bar graph shows the spine density from independent experiments using different RTT backgrounds and controls and after expression of shmecp2. The scale bar represents 5 mm. (F) Representative images of neuronal cell body size. Bar graph shows the percentage of soma size decrease in RTT compared to WT neurons. Neuronal morphology was visualized using the Syn::EGFP lentiviral vector. The scale bar represents 50 mm. (G) A lower dose of gentamicin was able to rescue glutamatergic synapses in RTT neurons. Numbers of neurons analyzed (n) are shown within the bars in graphs (E) and (G). For all clones and mutations used refer to Figure S4 and Table S2. Data shown as mean ± SEM. 534 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

15 WT allele can be as harmful as a loss of expression (Collins et al., 2004; Ramocki et al., 2009; Van Esch et al., 2005). Thus, we tested pharmacological treatment as a way to recover the RTT neuronal phenotype. We investigated the use of IGF1 in human neuronal cultures. Although it likely acts in a nonspecific manner, IGF1 is considered to be a candidate for pharmacological treatment of RTT and potentially other CNS disorders in a future clinical trial (Tropea et al., 2009). While IGF1 treatment increased synapse number in some clones, it stimulated glutamatergic RTT neurons above normal levels. Our data indicate that the IGF1 dose and timing parameters need to be precisely tuned in future clinical trials to avoid side effects. In a different approach, we tested a read-through drug (gentamicin) to rescue neurons derived from ipscs carrying a nonsense MeCP2 mutation. A lower dosage of gentamicin was enough to increase full-length MeCP2 levels in RTT neurons, rescuing glutamatergic synapses. New drugs with reduced toxicity and enhanced suppression of premature stop codon mutations might be good therapeutic candidates (Nudelman et al., 2009; Welch et al., 2007). Control of glutamatergic synapse number and the other neuronal phenotypes analyzed here may be caused by loss of MeCP2 function in the cell. Alternatively, significant experimental and genomic variability in our system could be directly responsible for the RTT differences displayed in our data. Our gain and loss of function data strongly suggest that MeCP2 is indeed the causative agent of the cellular phenotypes reported here that might be relevant to the clinical features of RTT. Our data indicate that ipscs not only can recapitulate some aspects of a genetic disease but also can be used to better design and anticipate results from translational medicine. This cellular model has the potential to lead to the discovery of new compounds to treat RTT and other forms of ASD. Finally, other CNS diseases may be modeled in vitro using a similar approach. EXPERIMENTAL PROCEDURES Figure 5. Altered Activity-Dependent Calcium Transients in RTT- Derived Neurons (A) Representative examples of WT and RTT calcium signal traces. Red traces correspond to the calcium rise phase detected by the algorithm used (see Extended Experimental Procedures). (B) Fluorescence intensity changes reflecting intracellular calcium fluctuations in RTT and WT neurons in different Regions of Interest (ROI). (C) RTT neurons show a lower average of calcium spikes when compared to WT control neurons. (D) The percentage of Syn::DsRed-positive neurons signaling in the RTT neuronal network is significantly reduced when compared to controls. Data shown as mean ± SEM. See also Figure S5. Cell Culture and Retrovirus Infection Female RTT and control fibroblasts were generated from explants of dermal biopsies following informed consent under protocols approved by the University of California San Diego. The Syn::EGFP or DsRed reporter vector was obtained by cloning the Synapsin-1 promoter (a gift from Dr. G. Thiel, Hamburg, Germany) in a lentivirus backbone. The shrna against a target sequence on the human MeCP2 gene was cloned in the LentiLox3.7 lentivirus vector. Retrovirus vectors containing the Oct4, c-myc, Klf4 and Sox2 human cdnas from Yamanaka s group (Takahashi et al., 2007) were obtained from Addgene. Two days after infection, fibroblasts were plated on mitotically inactivated mouse embryonic fibroblasts (Chemicon) with hesc medium. After 2 weeks, ipsc colonies were directly transferred to feeder-free conditions on matrigelcoated dishes (BD) using mtesr1 (StemCell Technologies, and passed manually. The detailed protocols to obtain NPCs and mature neurons are described in the supplemental material. For the rescue experiments, 10 hg/ml of IGF1 (Peprotech) or Gentamicin (Invitrogen; at 100 or 400 mg/ml) was added to neuronal cultures for 1 week. Protocols were previously approved by the University of California San Diego and Salk Institute Institutional Review Board and the Embryonic Stem Cell Research Oversight Committee. Immunocytochemistry and Neuronal Morphology Quantification Cells were briefly fixed in 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 in PBS. Cells were then blocked in PBS containing 0.5% Triton X-100 and 5% donkey serum for 1 hr before incubation with primary antibody overnight at 4 C. After three washes with PBS, cells were incubated Cell 143, , November 12, 2010 ª2010 Elsevier Inc. 535

16 Figure 6. Decreased Frequency of Spontaneous Postsynaptic Currents in RTT Neurons (A) Fluorescence micrographs of representative WT and RTT neurons. The scale bar represents 10 mm. (B) Electrophysiological properties of WT and RTT neurons. From top to bottom: Transient Na + currents and sustained K + currents in response to voltage step depolarizations (command voltage varied from 20 to +30 mv in 5 mv increments when cells were voltage-clamped at 70 mv, Bars = 400 pa and 50 ms). Action potentials evoked by somatic current injections (cells current-clamped at around 60 mv, injected currents from 10 to 40 pa, Bars = 20 mv and 100 ms), sepscs (Bars = right, 20 pa, 100 ms; left: 10 pa, 500 ms), and sipscs (Bars = right, 20 pa, 500 ms; left: 20 pa, 400 ms). (C) Sample 4 min recordings of spontaneous currents when the cells were voltage-clamped at 70 mv (Bars = 20 pa and 25 s). (D) Cumulative probability plot of amplitudes (left panel, 1 pa bins; p < 0.001) and inter-event intervals (right panel, 20 ms bins; p < 0.05) of sepscs from groups of WT (black) and RTT (red) cells, respectively. (E) Cumulative probability plot of amplitudes (left panel, 1 pa bins; p < 0.05) and inter-event intervals (right panel, 20 ms bins; p < 0.05) of sipscs from each group (WT, black; RTT, green). with secondary antibodies (Jackson ImmunoResearch) for 1 hr at room temperature. Fluorescent signals were detected using a Zeiss inverted microscope and images were processed with Photoshop CS3 (Adobe Systems). Primary antibodies used in this study are described in the supplemental information. Cell soma size was measure in bright field using ImageJ software after identification of neurons using the Syn::EGFP. The morphologies of neuronal dendrites and spines were studied from an individual projection of z-stacks optical sections and scanned at 0.5-mm increments that correlated with the resolution valued at z-plane. Each optical section was the result of 3 scans at 500 lps followed by Kalman filtering. For synapse quantification, images were taken by a z-step of 1 mm using Biorad radiance 2100 confocal microscope. Synapse quantification was done blinded to genotype. Only VGLUT1 puncta along Map2-positive processes were counted. Statistical significances were tested using Two-way ANOVA test and Bonferroni post-test. Cell Cycle Analysis One million NPCs were fixed in 70% EtOH for at least 2 hr at 4 C. After PBS washing, cells were stained with 1 ml of propidium iodide (PI) solution (50 mg/ml PI in 3.8 Mm sodium citrate) and treated with 20 ml/ml of RNaseA. Cells were analyzed by fluorescence-activated cell sorting (FACS) on a Becton Dickinson LSRI and cell cycle gating was examined using FLOWJO - Flow Cytometry Analysis Software. RNA Extraction and RT-PCR Total cellular RNA was extracted from 5x10 6 cells using the RNeasy Protect Mini kit (QIAGEN, Valencia, CA), according to the manufacturer s instructions, and reverse transcribed using the SuperScript III First-Strand Synthesis System RT-PCR from Invitrogen. The cdna was amplified by PCR using Accuprime Taq DNA polymerase system (Invitrogen). Primer sequences used are described in Supplemental information. Teratoma Formation in Nude Mice Around 1-3 x10 6 fibroblasts or ipscs were injected subcutaneously into the dorsal flanks of nude mice (CByJ.Cg-Foxn1nu/J) anesthetized with isoflurane. Five to six weeks after injection, teratomas were dissected, fixed overnight in 10% buffered formalin phosphate and embedded in paraffin. Sections were stained with hematoxylin and eosin for further analysis. Control mice injected with RTT fibroblasts failed to form teratomas. Protocols were previously approved by the University of California San Diego Institutional Animal Care and Use Committee. 536 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

17 Karyotyping and DNA Fingerprinting Standard G-banding chromosome and DNA fingerprinting analysis was performed by Cell Line Genetics (Madison, WI). DNA and RNA FISH Xist RNA exon 6 probes (GenBank U80460: a gift from Dr. Jeannie T. Lee, Massachusetts General Hospital, Harvard Medical School) were transcribed by using T7 RNA polymerase (Roche) with AlexaFluor UTP. X chromosome probe and Xist slide hybridization were performed by Molecular Diagnostic Services, Inc. (San Diego, CA). Protein Isolation and Western Blot Analysis Cells were isolated, suspended in 13 RIPA lyses buffer (Upstate) supplemented with 1% protease inhibitor cocktail (Sigma), triturated and centrifuged at 10,000 3 g for 10 min at 4 C. Twenty micrograms of total protein was separated on 12% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and probed with a primary antibody against MeCP2 (1:5,000; Diagenode), followed by horseradish-peroxidase-conjugated secondary antibody (1:5,000; Promega), and then visualized using ECL chemiluminescence (Amersham). As a control, membranes were stripped and re-probed for b-actin (1:10,000; Ambion) or a tubulin (1:5,000, Ambion). For semiquantitative analysis, MeCP2 signal intensity was analyzed and corrected with respect to b-actin. Microarray Analysis The Affymetrix Power Tools (APT) suite of programs and Affymetrix Human Gene 1.0 ST Arrays library files and annotation were obtained from and details of the analysis are available in Supplemental information. Calcium Imaging Neuronal networks derived from human ipscs were previously infected with the lentiviral vector carrying the Syn:DsRed reporter construct. Cell cultures were washed twice with sterile Krebs HEPES Buffer (KHB) and incubated with 2 5 mm Fluo-4AM (Molecular Probes/Invitrogen, Carlsbad, CA) in KHB for 40 min at room temperature. Excess dye was removed by washing twice with KHB and an additional 20 min incubation was done to equilibrate intracellular dye concentration and allow de-esterification. Time-lapse image sequences (1003 magnification) of 5000 frames were acquired at 28 Hz with a region of pixels, using a Hamamatsu ORCA-ER digital camera (Hamamatsu Photonics K.K., Japan) with a 488 nm (FITC) filter on an Olympus IX81 inverted fluorescence confocal microscope (Olympus Optical, Japan). Images were acquired with MetaMorph 7.7 (MDS Analytical Technologies, Sunnyvale, CA). Images were subsequently processed using ImageJ ( rsbweb.nih.gov/ij/) and custom written routines in Matlab 7.2 (Mathworks, Natick, MA). Detailed quantitative analysis of calcium transients is available in the Supplemental material. Electrophysiology Whole-cell patch clamp recordings were performed from cells co-cultured with astrocytes after 6 weeks of differentiation. The bath was constantly perfused with fresh HEPES-buffered saline (see supplemental methods for recipe). The recording micropipettes (tip resistance 3 6 MU) were filled with internal solution described in the Supplemental materials. Recordings were made using Axopatch 200B amplifier (Axon Instruments). Signals were filtered at 2 khz and sampled at 5 khz. The whole-cell capacitance was fully compensated. The series resistance was uncompensated but monitored during the experiment by the amplitude of the capacitive current in response to a 10 mv pulse. All recordings were performed at room temperature and chemicals were purchased from Sigma. Frequency and amplitude of spontaneous postsynaptic currents were measured with the Mini Analysis Program software (Synaptosoft, Leonia, NJ). Statistical comparisons of WT and RTT groups were made using the nonparametric Kolmogorov-Smirnov two-tailed test, with a significance criterion of p = EPSCs were blocked by CNQX or DNQX (10 20 mm) and IPSPs were inhibited by bicuculine (20 mm). SUPPLEMENTAL INFORMATION Supplemental Information includes Extended Experimental Procedures, five figures, and two tables and can be found with this article online at doi: /j.cell ACKNOWLEDGMENTS The work was supported by the Emerald Foundation and by the National Institutes of Health through the NIH Director s New Innovator Award Program, 1-DP2-OD F.H.G. is supported by California Institute for Regenerative Medicine RL and RC , The Lookout Fund and the Mathers Foundation. C.C. is a fellow from the International Rett Syndrome Foundation. M.C.N.M. is a Christopher and Danna Reeve Foundation fellow. G.C. was supported by the Glenn Foundation. We would like to thank Monica Coenraads for critical discussion; the Greenwood Genetic Center clinical diagnostic laboratory for X-inactivation analysis; Dr. Jeannie T. Lee for the Xist probe; and M.L. Gage for editorial comments. Received: February 9, 2010 Revised: August 4, 2010 Accepted: October 8, 2010 Published: November 11, 2010 REFERENCES Allen, R.C., Zoghbi, H.Y., Moseley, A.B., Rosenblatt, H.M., and Belmont, J.W. (1992). Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am. J. Hum. Genet. 51, Amir, R.E., Van den Veyver, I.B., Wan, M., Tran, C.Q., Francke, U., and Zoghbi, H.Y. (1999). Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-cpg-binding protein 2. Nat. Genet. 23, Autism and Developmental Disabilities Monitoring Network Surveillance Year 2000 Prinicipal Investigators; Centers for Disease Control and Prevention. (2007). 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20 Supplemental Information EXTENDED EXPERIMENTAL PROCEDURES Cell Culture and Retrovirus Infection Female RTT (1155del32, GM11272; Q244X, GM16548; T158M, GM17880 and R306C, GM11270; from Coriell Institute) and WT fibroblasts (AG09319; from Coriell Institute, CRL2529; from ATCC). WT-126, ADRC40 and WT-33 were cultured in Minimum Essential Medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone Laboratories). The hesc Cyth25 (CyThera Inc., San Diego) and HUES6 (Harvard) cell lines were cultured as previously described (Muotri et al., 2005). Recombinant viruses were produced by transient transfection in 293T cells, as previously described (Muotri et al., 2005). To obtain NPCs, EBs were formed by mechanical dissociation of cell clusters and plating onto low-adherence dishes in hesc medium without FGF2 for 5-7 days. After that, EBs were plated onto poly-ornithine/laminin (Sigma)-coated dishes in DMEM/F12 (Invitrogen) plus N2. Rosettes were visible to collect after 7 days. Rosettes were then dissociated with accutase (Chemicon) and plated again onto coated dishes with NPC media (DMEM/F12; 0.5X N2; 0.5X B27 and FGF2). Homogeneous populations of NPCs were achieved after 1-2 passages with accutase in the same condition. To obtain mature neurons, floating EBs were treated with 1 mm of retinoic acid for 3 more weeks (total time of differentiation 4 weeks). Mature EBs were then dissociated with Papain and DNase (Worthington) for 1 hr at 37 C and plated in poly-ornithine/laminin-coated dishes in NPC media without FGF2. Primary Antibodies Used for Immunofluorescence in This Study Primary antibodies used in this study were TRA-1-60, TRA-1-81 (1:100, Chemicon); Nanog and Lin28 (1:500, R&D Systems); human Nestin (1:100, Chemicon); Tuj-1 (1:500, Covance); Map2 (1:100; Sigma); MeCP2 (1:1000, Sigma); VGLUT1 (1:200, Synaptic Systems); Psd95 (1:500, Synaptic Systems), GFP (1:200, Molecular Probes-Invitrogen); Sox1 (1:250, BD Biosciences), Musashi1 (1:200, Abcam) and me3h3k27 (1:500, Millipore). Oligonucleotide Sequences Used in This Study ShRNA against the human MeCP2 gene (5 0 -GGAGTCTTCTATCCGATCTGT-3 0 ) was cloned in the LentiLox3.7 lentivirus vector, which forms a part of hairpin loop 5 0 -GGAGTCTTCTATCCGATCTGTTCAAGAGACAGATCGGATAGAAGACCTCC-3 0. The primer sequences were: hoct4-f: gggaggggaggagctagg and hoct4-r: tccaaccagttgccccaaac; hsox2-f: tgggaggggtgcaaaagagg and hsox2-r: gagtgtggatgggattggtg; hnanog-f: cctatgcctgtgatttgtgg and hnanog-r: ctgggaccttgtcttccttt; hmsx1-f: 5 0 aggaccccgtggatgcagag and hmsx1-r: 5 0 ggccatcttcagcttctccag; hgapdh-fw: 5 0 accacagtccatgccatcac 3 0, hgapdh-rv: 5 0 tcca ccaccctgttgctgta 3 0. PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide and visualized by UV illumination. Microarray Analysis Gene-level signal estimates were derived from the CEL files by RMA-sketch normalization as a method in the apt-probeset-summarize program. Hierarchical clustering of the full dataset by probeset values was performed by complete linkage using Euclidean distance as a similarity metric in Matlab. Quantification of Calcium Transients The recipe for Krebs HEPES Buffer (KHB) used for calcium Imaging was: 10 mm HEPES, 4.2 mm NaHCO 3, 10 mm dextrose, 1.18 mm MgSO 4 $2H 2 O, 1.18 mm KH 2 PO 4, 4.69 mm KCl, 118 mm NaCl, 1.29 mm CaCl 2 ; ph 7.3). Neurons were selected after the confirmation that calcium transients were blocked with 1 mm of tetrodotoxin (TTX) or the glutamate receptor antagonists CNQX/APV (6-cyano-7- nitroquinoxaline-2,3-dione at 10 mm / (2R)-amino-5-phosphonovaleric acid; (2R)-amino-5-phosphonopentanoate at 20 mm, respectively) treatments. Calcium transients increased after 30 mm Gabazine treatment. For quantification of calcium transients, ImageJ, an NIH-funded open source, JAVA-based morphometric application, was used to allow manual selection of individual neurons on the Syn::DsRed image that correspond to each calcium movie using circular regions of interest (ROI) of 4 pixels (5mm) in diameter. Each cell was considered as an individual ROI and the average fluorescence intensity was calculated for each ROI through the entire acquired image sequence. Quantitative signal analysis and processing were done with custom-written Matlab routines. Individual temporal fluorescence intensity signals indicative of intracellular calcium fluctuations were filtered using power spectrum calculated from Fourier transforms to reduce noise. Amplitude of signals was presented as relative fluorescence changes (DF/F) after background subtraction. A first-derivative filter was used to identify regions of increase in calcium signal and a calcium event was identified by a positive derivative value of 2 SD or more above background with a rise phase that persisted a minimum of 5 consecutive frames (70ms). Electrophysiology Recipe for HEPES-buffered saline: 115 mm NaCl, 2 mm KCl, 10 mm HEPES, 3 mm CaCl2, 10 mm glucose and 1.5 mm MgCl2 (ph 7.4). Recipe for solution inside the recording micropipettes (tip resistance 3 6 MU): 140 mm K-gluconate, 5 mm KCl, 2 mm MgCl 2,10 mm HEPES and 0.2 mm EGTA, 2.5 mm Na-ATP, 0.5 mm Na-GTP, 10 mm Na 2 -phosphocreatine (ph 7.4). Cell 143, , November 12, 2010 ª2010 Elsevier Inc. S1

21 SUPPLEMENTAL REFERENCES Mnatzakanian, G.N., Lohi, H., Munteanu, I., Alfred, S.E., Yamada, T., MacLeod, P.J., Jones, J.R., Scherer, S.W., Schanen, N.C., Friez, M.J., et al. (2004). A previously unidentified MECP2 open reading frame defines a new protein isoform relevant to Rett syndrome. Nat. Genet. 36, Muotri, A.R., Nakashima, K., Toni, N., Sandler, V.M., and Gage, F.H. (2005). Development of functional human embryonic stem cell-derived neurons in mouse brain. Proc. Natl. Acad. Sci. USA 102, S2 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

22 Cell 143, , November 12, 2010 ª2010 Elsevier Inc. S3

23 Figure S1. Generation of ipscs Derived from RTT Patients Fibroblasts Carrying Distinct Mutations in the MeCP2 Gene, Related to Figure 1 (A) Morphology of fibroblasts before retroviral infection. (B) Aspect of ipscs colonies growing in the absence of feeder layer. Colonies are compact and have welldefined borders. Cells display high nucleus-to-cytoplasm ratio and are morphologically similar to hescs. (C) Representative immunofluorescence analysis of RTT-iPSC clones. Expression of pluripotent markers such as Nanog and Tra-1-81 is observed. The scale bar represents 100 mm. (D) Hierarchical clustering and correlation coefficients of microarray profiles of triplicate WT Fibroblasts, RTT Fibroblasts, WT-iPSC clone 1, WT-iPSC clone 2, RTT-iPSC clones 15 and 18 (1155del32), RTT-iPSC clones 1 and 2 (Q244X) and the hesc line HUES6. Color bar indicates the level of correlation (from 0 to 1), with color bar reporting log2 normalized expression values (green/red indicates high/low relative expression). (E), Reprogrammed ipscs showed expressions similar to hesc-enriched genes (Lin28, CXADR, Nanog and PTRZ1; black bars) and showed distinct differences from fibroblast-enriched genes (GREM1, MMP1, DKK1 and PTX3; white bars). (F) RT-PCR from reprogrammed ipscs showed endogenous expressions of hesc-enriched genes (Oct4, Sox2 and Nanog) but not from a fibroblast-enriched gene (Msx1). S4 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

24 Figure S2. Neuronal Differentiation from Individual WT and RTT-iPSC Clones, Related to Figure 2 Clones from WT and RTT-iPSCs were differentiated into neurons for approximately 1 month. (A) Neurons were stained with the Map2 neuronal marker. (B) Neurons were infected with a lentiviral vector expressing the DsRed reporter under the control of the Synapsin promoter region. (C) Inhibitory neurons were revealed in the cultures after staining with anti-gaba antibody. Each bar represents 3 independent experiments for each individual clone. Data shown as mean ± s.d.m. Cell 143, , November 12, 2010 ª2010 Elsevier Inc. S5

25 Figure S3. Androgen Receptor Analysis, Related to Figure 3 Example of X-inactivation analysis using the X-linked androgen receptor locus for the RTT-1155del32 C15 genomic DNA. After the PCR, 2 different-sized amplicons were detected (different peaks) and digested with a methylation-sensitive restriction enzyme (HpaII). The PCR using undigested DNA shows if two distinct alleles are present and also allows a correction factor due to the advantage on the amplification of the smaller allele. When the template DNA is digested, amplification occurs if the restriction sites are methylated. If the site is unmethylated, digestion will occur between the flanking oligonucleotides and amplification will not be possible. The peak areas after HpaII restriction digestion of genomic DNA are used to distinguish each parental X chromosome. (A) When random inactivation is present, the maternal and paternal alleles are represented at similar proportions. (B) In contrast, in a condition where nonrandom inactivation is present, the more commonly inactive allele will be preferentially amplified and this will be detected by a stronger peak. (C) A male control is displayed showing a single peak before HpaII digestion. (D) A PCR was run without DNA template as a control. (E) Fibroblasts carrying the 1155del32 MeCP2 mutant (GM11272) displayed random X-inactivation. (F) RTT-1155del32-derived neurons showed highly skewed X-inactivation. S6 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

26 Figure S4. Phenotypic Analysis of ipsc-derived Neurons from Several Clones, Related to Figure 4 (A) Representative images showing co-localization between VGLUT1 and Psd95 (arrows). The scale bar represents 5 mm. (B) Experimental and clonal variation of VGLUT1 puncta quantification in different individuals. (C) Efficient expression and knockdown of both MeCP2 isoforms by a specific shrna against MeCP2. The scale bar represents 50 mm. Two alternatively spliced MeCP2 transcripts have been characterized, isoforms A and B, which differ only in their most 5 0 regions. The MeCP2 isoform B is more prevalent in the brain and during neuronal differentiation (Mnatzakanian et al., 2004). (D) Graph shows cell soma radius for several RTT and WT clones. (E) WT MeCP2 protein levels detected in control and RTT neurons (Q244X). Gentamicin treatment in RTT neurons increased protein levels after 2 weeks. Numbers of neurons analyzed (n) are shown within the bars in graphs (B) and (D). Data shown as mean ± SEM. Cell 143, , November 12, 2010 ª2010 Elsevier Inc. S7

27 Figure S5. Calcium Transient Analysis in ipsc-derived Neurons, Related to Figure 5 Neurons were selected after the confirmation that calcium transients were blocked with 1 mm of TTX or the glutamate receptor antagonists CNQX/APV treatments. (A) Blocking glutamatergic signaling in the neuronal network using CNQX and APV resulted in significant reduction in intracellular calcium transients. (B) Blocking voltage-gated sodium channels using TTX prohibited the generation of action potentials and resulted in complete elimination of neuronal intracellular calcium transients. (C) Gabazine increased the number of calcium transients in the ipsc-derived neuronal networks. Red traces correspond to the calcium rise phase detected by the algorithm used. (D) Bar graph shows the normalized frequency of neurons with calcium transients after drug treatments. (E) Bar graph shows the event frequency decrease in RTT and shmecp2-treated WT neurons compared to WT control neurons. (F) Bar graph shows the percentage of signaling neurons in RTT and shmecp2-treated WT neurons compared to WT control neurons. S8 Cell 143, , November 12, 2010 ª2010 Elsevier Inc.

28 Cell Stem Cell Article Non-Cell-Autonomous Effect of Human SOD1 G37R Astrocytes on Motor Neurons Derived from Human Embryonic Stem Cells Maria C.N. Marchetto, 1 Alysson R. Muotri, 1 Yangling Mu, 1 Alan M. Smith, 2 Gabriela G. Cezar, 2 and Fred H. Gage 1, * 1 Laboratory of Genetics, The Salk Institute for Biological Studies, North Torrey Pines Road, La Jolla, CA 92037, USA 2 Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA *Correspondence: gage@salk.edu DOI /j.stem SUMMARY Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. ALS can be induced by mutations in the superoxide dismutase 1 gene (SOD1). Evidence for the noncell-autonomous nature of ALS emerged from the observation that wild-type glial cells extended the survival of SOD1 mutant motor neurons in chimeric mice. To uncover the contribution of astrocytes to human motor neuron degeneration, we cocultured hesc-derived motor neurons with human primary astrocytes expressing mutated SOD1. We detected a selective motor neuron toxicity that was correlated with increased inflammatory response in SOD1-mutated astrocytes. Furthermore, we present evidence that astrocytes can activate NOX2 to produce superoxide and that effect can be reversed by antioxidants. We show that NOX2 inhibitor, apocynin, can prevent the loss of motor neurons caused by SOD1- mutated astrocytes. These results provide an assay for drug screening using a human ALS in vitro astrocyte-based cell model. INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative adult disease characterized by fatal paralysis in both the brain and spinal cord motor neurons. ALS can be induced by inherited mutations in the gene encoding the ubiquitously expressed enzyme superoxide dismutase 1 (SOD1) (Boillee et al., 2006; Deng et al., 1993; Lobsiger and Cleveland, 2007; Rosen et al., 1993). Initial evidence for the non-cell-autonomous nature of ALS became apparent when chimeric mice containing a mixture of mutated and normal human SOD1 were developed (Clement et al., 2003). Further confirmation came to light through the observation that diminishing mutant SOD1 levels within either microglia or astrocytes sharply slowed disease progression (Boillee et al., 2006; Yamanaka et al., 2008). In agreement with in vivo observations, cocultures of mouse-derived, mutant SOD1-expressing astrocytes and mouse motor neurons have demonstrated that mutant astrocytes reduced motor neuron survival over a 2 week period (Di Giorgio et al., 2007; Nagai et al., 2007). The symptomatic phase of ALS is characterized by a massive activation of microglia and astrocytes (Boillee et al., 2006). Misfolded SOD1 can increase oxidative stress and secretion of proinflammatory toxic factors by glial cells such as nitric oxide (NO), amplifying the disease symptoms (Barbeito et al., 2004). Recent data suggest that the increase in the production of reactive species of oxygen (ROS) in an ALS mouse model is partially caused by elevated levels of NADPH oxidase (NOX) (Harraz et al., 2008; Marden et al., 2007; Wu et al., 2006). Moreover, deletion of one of the catalytic subunits of the NOX gene (NOX2/ gp91 phox ), as well as treatment with a specific NOX inhibitor, significantly increased life span and improved survival in SOD1- mutated transgenic mouse models (Harraz et al., 2008; Marden et al., 2007; Wu et al., 2006). We consistently generated a population of human neurons in vitro that expressed postmitotic motor neuron markers, made neuromuscular junctions, and fired action potentials. Subsequently, we cocultured the human embryonic stem cell (hesc)- derived motor neurons with human primary astrocytes expressing either the wild-type or the mutated form of SOD1 protein (SOD1 WT or SOD1 G37R, respectively). In our cocultures, we detected a specific decrease in the number of motor neuron markers in the presence of SOD1-mutated astrocytes, with no detectable effect on other subtypes of neurons. Furthermore, we showed that the toxicity conferred by the SOD1-mutated astrocytes was generated in part by an increase in astrocyte activation and production of ROS. The physiological changes observed in SOD1 G37R human astrocytes were well correlated with intensification of the proinflammatory activity of the induced nitric oxide synthase enzyme (inos or NOS2A), neurosecretory protein Chromogranin A (CHGA), secretory cofactor cystatin C (CC or CST3), and NADPH oxidase (NOX2/gp91 phox or CYBB) overexpression. Activation of NOX2 and production of oxygen radicals had already been demonstrated to be mediators of microglial toxicity in familial ALS mouse models (Barbeito et al., 2004; Wu et al., 2006). Our data suggest that human astrocytes overexpressing mutated SOD1 can activate NOX2 to produce oxygen radicals, and the addition of antioxidants can reverse this process. Moreover, by treating the cells with one of the prescreened antioxidant compounds, we were able to prevent the loss of motor neurons caused by coculture with SOD1-mutated astrocytes. Key elements of our findings are (1) the development of a human model Cell Stem Cell 3, , December 4, 2008 ª2008 Elsevier Inc. 649

29 Cell Stem Cell Human SOD1 G37R -Expressing Astrocytes Kill Motor Neurons of disease using hescs, providing an important proof of principle toward developing high throughput drug discovery assays for ALS; and (2) the identification of a class of compounds to consider for future clinical investigation. RESULTS hescs Generate Functional Motor Neurons In Vitro hesc-derived rosettes expressed motor neuron progenitor markers such as Pax6, Nestin, Olig2, and Islet1 after 2 3 weeks of differentiation (Figures 1A 1D). After 4 weeks under differentiation conditions, the cells started to express panneuronal markers such as TuJ1, and after 6 8 weeks, the cells exhibited motor neuron postmitotic lineage-specific markers, such as homeobox gene Hb9, HoxC8, and choline acetyltransferase neurotransmitter, ChAT (Figures 1E 1G). Motor neuron identity was also confirmed at the transcription level by RT-PCR. Accordingly, we detected downregulation of the hesc undifferentiated marker Nanog and upregulation of the postmitotic motor neuron markers Hb9 and ChAT (Figure 1H). At the 8 week differentiation stage, cells were also positive for synapsin and could incorporate a-bungarotoxin when cocultured with C2C12 myoblasts, indicating that the cells could form functional neuromuscular junctions (Figures 1I and 1J). Live postmitotic human motor neurons could be visualized after transduction with a lentivirus expressing the green fluorescent protein gene (GFP) under the control of the Hb9 promoter (Lee et al., 2004) (Lenti Hb9::GFP). We confirmed the promoter specificity by costaining the Hb9:: GFP-positive cells with the endogenous Hb9 protein in hescderived neurons as well as in rat purified spinal cord motor neurons (Figure 1K and see Figures S2A S2C available online). We preformed RT-PCR for the endogenous human Hb9 transcript in sorted Hb9::GFP-positive versus Hb9::GFP-negative cells and only detected endogenous Hb9 expression in Hb9::GFP-positive cells (Figure S2D). The Hb9::GFP-positive neurons also colocalized with ChAT marker (Figure 1L). We determined the functional maturation of the hesc-derived neurons using electrophysiology. Whole-cell perforated patch recordings were performed from cultured HB9-expressing cells that had differentiated for at least 8 weeks in culture (Figures 1M 1R). Expression of Mutated SOD1 G37R Protein in Astrocytes Affects Motor Neuron Survival We then examined the effects of astrocytes expressing either a wild-type (SOD1 WT ) or mutated (SOD1 G37R ) form of the human SOD1 protein on the survival of hesc-derived motor neurons upon coculture. Primary human astrocytes were transduced with a lentivirus vector expressing either SOD1 WT or SOD1 G37R (Figures S1A and S1B). We then cocultured the Hb9::GFP motor neurons with SOD WT - or SOD1 G37R -expressing astrocytes (Figure 2A). After coculture for 4 weeks, cells were subjected to fluorescence-activated cell sorting (FACS) for Hb9::GFP quantification (Figure 2B). We detected a decrease of 49% of Hb9:: GFP-positive cells when cocultured with SOD1 G37R astrocytes. For comparison, we included noninfected human astrocytes and did not detect significant differences in the number of Hb9::GFP-positive cells when compared to SOD1 WT cocultures (see graph in Figure 2B). To further confirm our findings, we counted the number of cholinergic neurons in cocultures with SOD WT or SOD1 G37R astrocytes (Figure 2C). We detected a similar decrease (52%) in ChAT-positive cells when cocultured with SOD1 G37R astrocytes. Moreover, the toxic or detrimental effect was specific to the motor neuron population, since other subtypes of neurons concomitantly present in the differentiated cultures, such as GABAergic neurons, were not affected (Figure 2D). We also determined that the toxic effect of mutated astrocytes was specific for glial cell type and was not present in human primary fibroblasts overexpressing SOD1 WT or SOD1 G37R that were cocultured with hesc-derived motor neurons (Figures S3A S3C). Astrocytes Activate an Inflammatory Response in the Presence of SOD1 G37R Next we investigated the possible causes of the astrocytic toxicity conferred by the mutated SOD1 to hesc-derived motor neurons by analyzing the behavior of the mutated astrocytes in culture. Primary astrocytes usually respond to inflammation by activation. Activated astrocytes increase the assembly of their intermediate filaments (produced by glial fibrillary acidic protein, GFAP) and the number and size of the processes extended from the cell body. Furthermore, activated astrocytes intensify their oxidation levels and the production of proinflammatory factors (Barbeito et al., 2004). We detected a significant increase in the number of activated (GFAP-positive) astrocytes when SOD1 G37R was present in comparison to control astrocytes (Figure 3A). We also confirmed that the population of astrocytes was still homogeneous after SOD1 overexpression by staining the cells with A2B5, a general astrocyte marker (Figure 3A). Moreover, we did a cell-death analysis for both SOD1 G37R and SOD1 WT astrocytes, and both had similar amounts of propidium iodide (PI) staining (Figure S1C), so we concluded that the viability of astrocyte SOD G37R is similar to SOD WT. In parallel, we measured an increase in the number of cells producing ROS by the astrocytes expressing the mutated SOD1 (Figure 3B), a hallmark of ALS pathology (Barber et al., 2006). We also calculated the intensity of fluorescence present in the oxidation experiments but did not detect significant changes between groups (Figure 3B). In addition, we observed an increase in the expression of proinflammatory factors such as inos, an overexpression of the neurosecretory protein known to interact specifically with mutated SOD1, CHGA (Urushitani et al., 2006), induction of a superoxide producer enzyme NOX2 (gp91 phox subunit), and an increase of cystein protease inhibitor CC expression (Figure 3C). Curiously, we did not observe a clear decrease in EAAT2 glutamate transporter protein or mrna in the mutated astrocytes (data not shown). The increment in inos enzyme was accompanied by a rise in the NO levels in the SOD1 G37R astrocyte-conditioned media, indirectly measured by nitrite concentration (Figure 3D). Astrocyte ROS Production Is Reversed by Antioxidants: A Model for Drug Screening A total of five compounds and their respective vehicles (EtOH or DMSO) were tested in SOD G37R -mutated astrocyte cultures to address their antioxidant potential (Figure 4A). Treatment with both NOX2 inhibitor apocynin and antioxidant a-lipoic acid for 48 hr decreased the percentage of cells that were able to produce ROS (percentage of oxidation) in comparison to treatment with vehicle only (EtOH) (Figure 4B). Likewise, treatment with the 650 Cell Stem Cell 3, , December 4, 2008 ª2008 Elsevier Inc.

30 Cell Stem Cell Human SOD1 G37R -Expressing Astrocytes Kill Motor Neurons Figure 1. Differentiation and Functional Characterization of hesc-derived Motor Neurons (A D) Neuroectodemal rosettes expressing motor neuron-progenitor markers, Pax6, Nestin, Olig2, and Islet1, after 2 3 weeks of differentiation. (E G) Expression of motor neuron postmitotic markers Hb9, HoxC8, and ChAT was detected after 4 weeks of differentiation. (H) RT-PCR of hesc-derived motor neurons showing downregulation of the hesc marker Nanog and confirming the expression of motor neuron subtype markers such as Hb9 and ChAT. (I and J) (I) Synapsin-expressing neurites and (J) a-bungarotoxin incorporation at neuromuscular junctions following coculture with C2C12 myoblasts observed after 7 8 weeks of differentiation. (K) Expression of endogenous Hb9 colocalizing with Hb9::GFP-positive cells in human motor neurons. (L) Colocalization between ChAT-positive (inset) and Hb9::GFP motor neuron. (M) Fluorescence micrograph of the Hb9-positive cell from which data shown in (N) (R) were obtained. (N) Transient Na + and sustained K + currents (upper panel; the asterisk and arrow indicate Na + and K + currents, respectively) in response to step depolarizations (lower panel; cell voltage clamped at 70 mv, command voltage from 90 to +100 mv, 10 mv step). (O and P) I-V relations corresponding to peak Na + currents (O) and steady-state K + currents. (Q) Sub- and suprathreshold responses (upper panel) to somatic current injections (lower panel; cell current clamped at around 80 mv, currents from 10 to 30 pa, 10 pa step). (R) Spontaneous action potentials when the cell was current clamped at 60 mv. Scale bars, (A) (F), 100 mm; (G), 80 mm; (I), 20 mm; and (J) (M), 40 mm. Cell Stem Cell 3, , December 4, 2008 ª2008 Elsevier Inc. 651

31 Cell Stem Cell Human SOD1 G37R -Expressing Astrocytes Kill Motor Neurons Figure 2. hesc-derived Neuronal Cocultures with Human Astrocytes (A) Experimental design: human primary astrocytes were infected with LentiSOD1 WT or LentiSOD1 G37R for SOD1 (wild-type or mutated) overexpression. hescs were differentiated into motor neuron precursors (rosettes), gently dissociated, and plated on two different glial monolayers. The cocultures were then infected with LentiHb9::GFP and carried out for three more weeks. The motor neurons were detected by GFP fluorescent sorting (FACS) or ChAT immunofluorescence. (B) Hb9::GFP-positive neurons cocultured with Astro SOD1 WT or Astro SOD1 G37R and corresponding GFP fluorescence quantification by FACS. Mean ± SD; n = 3. (C) Astrocyte cocultures overexpressing either SOD1 WT or SOD1 G37R and quantification of cholinergic motor neurons. Mean ± SD; n = 3. (D) Representative fields of GABAergic neurons detected by glutamic acid decarboxylase 65 (GAD65) immunoreactivity present in the cocultures concomitantly with the motor neurons and corresponding quantification. Scale bars, 80 mm. Mean ± SD; n = 3. antioxidant flavonoid epicatechin decreased the oxidation levels of SOD1 G37R astrocytes when compared to vehicle (DMSO). The drugs resveratrol and luteolin, on the other hand, did not seem to have a detectable effect on the number of SOD1 G37R astrocytes that are producing ROS. We then chose the compound apocynin for further verification in a coculture assay using hesc-derived motor neurons and either SDO1 WT or SOD G37R astrocytes. Apocynin treatment rescued the motor neuron survival in the presence of SOD1 G37R (Figure 5), confirming previous observation in SOD1-mutated transgenic mice treated with the same drug (Harraz et al., 2008; Marden et al., 2007; Wu et al., 2006). DISCUSSION We successfully differentiated hescs in electrophysiologically active Hb9-expressing human motor neurons to establish a system for modeling ALS using human cells. Our model consists of coculturing healthy human motor neurons with human astro- cytes carrying either the wild-type or mutated SOD1 cdna. Under these conditions, we could confirm the role of astrocytes in ALS disease, as motor neuron numbers decreased about 50% in the presence of mutant SOD1-expressing astrocytes. Moreover, the toxicity seemed to be restricted to the motor neuron subpopulation, with no effects on other neuronal subtypes. Other groups have proposed a similar astrocyte-dependent damage in mouse systems using in vitro assays (Di Giorgio et al., 2007; Nagai et al., 2007). Notably, Di Giorgio et al. (2008) (in this issue of Cell Stem Cell) observed a comparable reduction in the human motor neuron population using mouse astrocytes expressing a different SOD1 mutant (SOD1 G93A ), supporting the observation that the mechanism of motor neuron toxicity is likely conserved in familial ALS. We have evidence that the mechanism of astrocyte-specific motor neuron toxicity involves both secretory and inflammatory pathways. CC, a secretory cofactor involved with inhibition of cystein proteinases and neurogenesis, has been identified in cerebral spinal fluid (CSF) proteomic profiles as a potential biomarker for ALS (Pasinetti et al., 2006; Taupin et al., 2000). The role of CC in ALS pathology has yet to be elucidated, and research has demonstrated no obvious mutation in the CC gene in either familial or sporadic patients (Watanabe et al., 2006). However, CC is one of the two proteins that immunostain the so-called Bunina bodies, small intraneuronal inclusions that are the only specific pathological ALS hallmark (Okamoto et al., 1993). Our findings are consistent with a previous report that activated astrocytes increase expression of the neurosecretory protein CHGA, and suggest that the secretion of mutant SOD1 may 652 Cell Stem Cell 3, , December 4, 2008 ª2008 Elsevier Inc.

32 Cell Stem Cell Human SOD1 G37R -Expressing Astrocytes Kill Motor Neurons Figure 3. Inflammatory Response in Astrocytes Expressing Mutated SOD1 (A) Astrocytes reactivity, measured by expression of GFAP in control (Astro SOD1 WT ) versus mutated (AstroSOD1 G37R ) astrocytes. Note that the expression of A2B5, a marker that is not related to astrocytic immune response, is the same in both conditions. Mean ± SD; n = 3. (B) Quantification of the production of ROS in control versus SOD1 G37R astrocytes. Graphs show percentage of cells producing ROS and fluorescence intensity. Mean ± SD; n = 3. (C) Western blot showing differential expression of inducible NO synthase (inos), the gp91phox (NOX2) subunit from NOX, secretory proteins CHGA, and CC in control versus mutated astrocytes. Mean ± SD; n = 3. (D) Indirect measure of NO by Griess method in astrocytes conditioned media. Scale bar, 80 mm. represent one of the neurotoxic pathways for the non-cellautonomous nature of ALS (Urushitani et al., 2006). Interestingly, the CHGA induction in SOD1 G37R -mutated astrocytes is accompanied by an increase in ROS production, likely mediated by NOX2, which is also upregulated in human ALS astrocytes. The induction of proinflammatory inos has been shown to be caused by release of ROS via the NOX pathway in rat astrocytes activated by lipopolysaccharides (LPS) (Pawate et al., 2004). Consistently, attenuated induction of inos was observed in primary astrocytes derived from NOX2-deficient mouse (Pawate et al., 2004). In ALS patients, damaged motor neurons are typically surrounded by a strong activation of inos-expressing glia in the ventral horn of both familial and sporadic forms of the disease (Almer et al., 1999; Anneser, 2000). Intriguingly, we did not detect a significant decrease in glutamate transporter (EAAT2) expression in astrocytes containing the mutated SOD1, so we conclude that, in our system, the motor neuron toxicity is independent of glutamate excitotoxicity. We cannot rule out the possibility that EAAT2 protein levels would decrease after longer periods of culture (in our system, we cultured the SOD1 G37R astrocytes for no more than 6 weeks). In accordance with our observations, recent data have also shown that EAAT2 expression in astrocytes is not responsible for the recovery of motor function in the loxsod1 G37R /GFAP-Cre+ mouse model (Yamanaka et al., 2008). Importantly, our data show that antioxidant apocynin decreased the ROS production in SOD1 mutant-expressing astrocytes, likely by inhibition of NADPH oxidase (NOX2), and in turn restored motor neuron survival. We believe that we can use SOD1 mutant astrocytes as a rapid drug screening test for oxidative damage to identify the best candidates for a following long-term coculture experiment (Figure 6). Our findings support the idea that astroglia can contribute to ALS symptoms, promoting extrinsic toxicity to motor neurons. Further investigation should elucidate whether the motor neuron toxicity is a result of a specific toxic factor produced by mutated astrocytes or via the lack of a surviving factor (maybe a combination of both events). Nonetheless, the data we present here show for the first time that an antioxidant agent can improve the survival of motor neurons in a completely humanized model. The use of human coculture models will have an increasing impact on developing drug discovery and screening assays for both familial and sporadic ALS. Mouse and rat ALS models still have a critical impact in unveiling the complexity of the metabolic pathways involved in the disease. Nevertheless, a variety of drugs that had demonstrated significant efficacy in murine models showed inefficacy in both preclinical and clinical human trials (DiBernardo and Cudkowicz, 2006; Scott et al., 2008). Currently, there is only one FDA-approved treatment for ALS, namely riluzole (Doble, 1996), and it only extends the course of the disease for 2 months (Miller and Moore, 2004). There is an urgent need for new ALS models that have the potential to be translated into clinical trials and could, at a minimum, be used in conjunction with the murine models to verify targets and drugs. We propose that a human ALS model based on hesc-derived motor neurons in coculture with human astrocytes is a robust and invaluable model to study ALS disease and to initiate species-specific drug development assays. Cell Stem Cell 3, , December 4, 2008 ª2008 Elsevier Inc. 653

33 Cell Stem Cell Human SOD1 G37R -Expressing Astrocytes Kill Motor Neurons Coculture of Motor Neurons and Myocytes C2C12 myoblasts were purchased from American Type Culture Collection (ATCC) and cultured according to the specifications of the manufacturer. After reaching a specific confluence, the myoblasts formed myotubes. The manually dissected rosettes (motor neuron progenitors) were plated on top of the myotubes, and the medium was replaced with the differentiation medium (described previously). After 4 6 weeks in coculture, the cells were fixed, and the formation of neuromuscular junctions was observed by incorporation of a-bungarotoxin conjugated with Alexa 568 (1:200, Molecular Probes, Invitrogen, Carlsbad, CA). Purification and Culture of Rat Primary Motor Neurons Primary rat motor neurons were purified following previously published procedures (Arce et al., 1999; Henderson et al., 1993), with some modifications. Briefly, spinal cords were dissected from E14 rat embryos, treated with trypsin (2.5% w/v; final concentration 0.05%) for 10 min at 37 C, and then dissociated. The largest cells were isolated by centrifugation for 15 min at g over a 5.2% Optiprep cushion (Sigma, St. Louis, MO), followed by centrifugation for 10 min at g through a 4% BSA cushion. Purified motor neurons were plated inside 35 mm Petri dishes on 12 mm coverslips previously coated with polyornithine/laminin and grown 7 10 days in L15 medium with sodium bicarbonate (625 mg/ml), glucose (20 mm), progesterone ( m), sodium selenite, putricine (10 4 m), insulin (5 mg ml 1 ), and penicillin-streptomycin. BDNF (1 ng ml 1 ), and 2% horse serum were also added to the medium. Figure 4. Screening of Compounds and Their Ability to Decrease Oxidation in SOD1 G37R Astrocytes (A) Detection of ROS production in SOD G37R astrocytes. Green fluorescence marks cells that undergo oxidation. (B) Quantification of the number of cells producing ROS. Mean ± SD; n = 3. (C) Relative intensity of fluorescence. Scale bar, 80 mm. Mean ± SD; n = 3. EXPERIMENTAL PROCEDURES Culture Conditions and Differentiation of hescs The cells lines used in this study were HUES9 (Douglas Melton-WiCell) and Cythera 203 (Novocell Inc., San Diego, CA). The hescs were differentiated in vitro in motor neurons, adapting the protocol previously described elsewhere (Li et al., 2005). Briefly, the cells were manually dissociated to form embryoid bodies (EBs) and cultured in suspension for 5 6 days. The EBs were then plated in laminin/poliornithin-coated plates in the presence of a neural induction medium consisting of F12/DMEM (Invitrogene, Carlsbad, CA), N2 supplement, and 1 mm retinoic acid (RA). The cells started to organize into neural tube-like rosettes and, after 7 8 days in culture, sonic hedgehog (SHH, 500 ng/ml, R&D Systems) and camp (1 mm) were added to the culture media for one more week. The rosettes were then manually selected using a 103 magnifier (Zeiss) and gently dissociated (by pipetting up and down in a Hanks enzyme-free cell dissociation buffer, Invitrogene). After dissociation, rosettes were pelleted at 1000 rpm and replated either on laminin/poliornithine-coated coverslips (for direct differentiation) or on top of astrocyte feeder layers for the coculture experiments. The media was changed for a differentiation medium that consisted of neurobasal medium (Invitrogene), N2 supplement, RA (1 mm), SHH (50 ng/ml), camp (1 mm), BDNF, GDNF, and IGF (all at 10 ng/ml, Peprotech Inc.). The neurons were cultured in the differentiation media for three to five more weeks with or without the astrocyte feeder. Immunofluorescence Astrocyte monolayers or astrocyte and motor neuron cocultures were fixed for 15 min with 4% paraformaldehyde in PBS, and immunofluorescence was performed as described previously (Muotri et al., 2005). Briefly, slides were washed with PBS and permeabilized with 0.1% Triton X-100 for 30 min and incubated for 2 hr at room temperature in blocking solution (0.1% Triton X-100, 5% donkey serum in PBS). The samples were incubated overnight at 4 C with primary antibodies diluted in blocking solution, washed in PBS, and further incubated for 1 hr at room temperature with secondary antibodies (rabbit, mouse, or goat Alexa Fluor-conjugated antibodies, Molecular Probes- Invitrogen, Carlsbad, CA) diluted in blocking solution. The slides were then washed with PBS and mounted. The primary antibodies used were anti- Pax6, anti-islet 1, and anti-hb9 (all used at 1:100 and acquired from Developmental Studies Hybridoma Bank, DSHB Iowa City, IA), anti-human Nestin (1:200), anti-olig2 (1:200), anti-chat (1:100) and anti-a2b5 (1:500) (all from Chemicon, Temecula, CA), anti-tuj1 and anti-hoxc8 (both 1:200 from Covance Research Products, CA), anti-gfp (Molecular Probes-Invitrogen, CA), anti-gfap (1:500 from DAKO Carpinteria, CA), and anti GAD65 (1:200 from Sigma-Aldrich, MO). Lentiviral Vectors The viral vectors used in this research were Lenti-SOD1 WT, Lenti-SOD1 G37R, Lenti-Hb9::GFP, and Lenti-Hb9::RFP (for electrophysiological recordings). Concentrated lentiviral stocks were produced as described (Consiglio et al., 2004). Assessment of virus titering of Lenti-SOD1 WT and Lenti-SOD1 G37R was performed in rat neural stem cells (NSCs) using an antibody that specifically recognizes human SOD1 protein (1:500, Sigma-Aldrich, St Louis, MO; see Figure S1A) and was estimated as units/ml. Electrophysiology Whole-cell perforated patch recordings were performed from cultured Hb9:: RFP-expressing cells that had differentiated for at least 8 weeks. The recording micropipettes (tip resistance 4 8 MU) were tip filled with internal solution (115 mm K-gluconate, 4 mm NaCl, 1.5 mm MgCl 2, 20 mm HEPES, and 0.5 mm EGTA [ph 7.3]) and then back filled with internal solution containing amphotericin B (200 mg/ml). Recordings were made using an Axopatch 200B amplifier (Axon Instruments). Signals were filtered at 2 khz and sampled at 10 khz. The whole-cell capacitance was fully compensated, whereas the series resistance was uncompensated but monitored during the experiment by the amplitude of the capacitive current in response to a 5 mv pulse. The bath was constantly perfused with fresh HEPES-buffered saline (115 mm NaCl, 2 mm KCl, 10 mm HEPES, 3 mm CaCl 2, 10 mm glucose, and 1.5 mm MgCl 2 [ph 7.4]). For current-clamp recordings, cells were clamped at a range 654 Cell Stem Cell 3, , December 4, 2008 ª2008 Elsevier Inc.

34 Cell Stem Cell Human SOD1 G37R -Expressing Astrocytes Kill Motor Neurons Figure 5. Recovery of Motor Neuron Survival after Treatment with Apocynin (A) Immunofluorescence of representative images from cocultures of hesc-derived motor neurons and SOD WT or SOD G37R astrocytes that were treated with apocynin or vehicle. (B) Quantification of ChAT-positive cells in the different conditions. Scale bar, 80 mm. Mean ± SD; n = 3. Primary Astrocyte Culture Human primary astrocytes (HA1800) were obtained from ScienCell Research Laboratories (Carlsbad, CA) and were cultured according to the providers guidelines. Briefly, the astrocytes were isolated from fetal human brain (cerebral cortex) and cultured for no more than 15 passages in astrocyte media (AM 1801). The infections were performed in 80% confluent T75 flasks followed by incubation with the lentivirus expressing either the wild-type of SOD1 (LV-SOD1 WT ) or the mutated form of SOD1 (LV-SOD1 G37R ). For the coculture experiments, the astrocytes were plated on laminin/polyornithine (Invitrogen and Sigma-Aldrich, St. Louis, MO, respectively)-coated coverslips 1 day prior to the coculture. The rosettes were then cultured on top of the astrocytes feeder layer (see Culture Conditions and Differentiation of hescs, above). Cocultures were held for 3 weeks. Cell Death Detection Cell death was quantified by flow cytometry using 5 mg/ml PI in astrocyte cultures that had been previously infected with LentiSOD1 WT or LentiSOD1 G37R. of 60 to 80 mv. For voltage-clamp recordings, cells were clamped at 70 mv. All recordings were performed at room temperature. Amphotericin B was purchased from Calbiochem. All other chemicals were from Sigma. RNA Isolation and RT-PCR Total cellular RNA was extracted from cells using the RNeasy Protect Mini kit (QIAGEN, Valencia, CA) according to the manufacturer s instructions and reverse transcribed using the SuperScript III First-Strand Synthesis System RT-PCR from Invitrogen. The cdna was amplified by PCR using Taq polymerase (Promega, San Luis Obispo, CA), and the primer sequences were as follows: hnanog-fw, 5 0 cctatgcctgtgatttgtgg 3 0 ; hnanog-rv, 5 0 ctggga ccttgtcttccttt 3 0 ; hhb9-fw, 5 0 cctaagatgcccgacttcaa 3 0 ; hhb9-rv, 5 0 ttctgtt tctccgcttcctg 3 0 ; hchat-fw, 5 0 actccattcccactgactgtgc 3 0 ; hchat-rv, 5 0 tccaggcatacaaggcagatg 3 0 ; hgapdh-fw, 5 0 accacagtccatgccatcac 3 0 ; and hgapdh-rv, 5 0 tccaccaccctgttgctgta 3 0. PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and visualized by UV illumination. Product specificity was determined by sequencing the amplified fragments excised from the gel. Detection of ROS Production Detection of total cellular ROS was performed using the Image-iT LIVE Green Reactive Oxygen Species Detection Kit, according to the manufacturer s directions (Molecular Probes, Invitrogen). Briefly, this assay is based on the principle that the live cell permeable compound, carboxy- H 2 DCFDA, emits a bright green fluorescence when it is oxidized in the presence of ROS. The quantification of the ROS production was addressed in two ways: (1) counting the number of fluorescent cells and (2) measuring the intensity of the fluorescence emitted by the cells. The relative fluorescence intensity (arbitrary units ranging from 0 to 255, or black to white) was measured in randomly selected fields for each treatment and was analyzed and quantified using ImageProPlus software. Antioxidants Treatment Antioxidant stock solutions were diluted in astrocyte media and directly applied to astrocyte monolayers. The cultures were treated for 48 hr prior to ROS detection. The compounds used in the experiment were epicatechin (E4018 Sigma Aldrich, 10 mm), luteolin (L9283 Sigma-Aldrich, 5 mm), resveratrol (R5010 Sigma-Aldrich, 5 mm), apocynin ( Calbiochem, 300 mm), and a-lipoic acid (T5625, Sigma-Aldrich, 50 mg/ml). For neuronal cocultures, the astrocytes were pretreated for 48 hr with apocynin, and the rosettes were plated on top of them. The cocultures were carried for three more weeks and the medium containing apocynin was replaced three more times during the coculture period. Cell Stem Cell 3, , December 4, 2008 ª2008 Elsevier Inc. 655

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