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Cultured networks of excitatory projection neuronsand inhibitory interneurons for studying humancortical neurotoxicityJin-Chong Xu,1,2* Jing Fan,1,2* Xueqing Wang,1,2 Stephen M. Eacker,1,2 Tae-In Kam,1,2

Li Chen,1,2 Xiling Yin,1,2 Juehua Zhu,1,2,3 Zhikai Chi,1,2 Haisong Jiang,1,2 Rong Chen,1,2

Ted M. Dawson,1,2,4,5† Valina L. Dawson1,2,4,6†

Translating neuroprotective treatments fromdiscovery in cell and animalmodels to the clinic has proven challenging.To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, wedeveloped a human cortical neuron culture system fromhuman embryonic stem cells or human inducible pluripotentstemcells that generated both excitatory and inhibitory neuronal networks resembling the compositionof thehumancortex. This methodology used timed administration of retinoic acid to FOXG1+ neural precursor cells leading to dif-ferentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neu-ronal networks that were functional and homeostatically stable. In human cortical neuronal cultures, excitotoxicity orischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate(NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatosthat is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell deathwas attenuated by PARPinhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for thestudy of human cortical neurotoxicity and suggests that PARP inhibitorsmay be useful for ameliorating excitotoxic andischemic cell death in human neurons.

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INTRODUCTION

The human cerebral cortex is a complex structure with tightly inter-connected excitatory and inhibitory neuronal networks that are or-ganized in six layers (1, 2). The dynamic interplay between excitatorypyramidal cells and g-aminobutyric acid–releasing (GABAergic) inter-neurons begins at the early stages of neurogenesis (3). Considerableadvances in methods that model human cortical development usinghuman embryonic stem cells (ESCs) or human inducible pluripotentstem cells (iPSCs) have allowed the generation of relatively pure popula-tions of excitatory cortical projection neurons (4–6), forebrain inhibitoryprogenitors (7–11), and inhibitory neurons (12–14) or poorly character-ized mixtures of excitatory and inhibitory neurons (15–17). Because noexisting protocol produces a balanced network of excitatory and in-hibitory neurons observed in the human cerebral cortex (see fig. S1)(4, 16–24), we sought to develop a suitable protocol that would yieldappropriately balanced neuronal networks in vitro that closely resembleneuronal networks in vivo. This is particularly important when modelingglutamate excitotoxicity because neuronal nitric oxide (NO) synthase(nNOS)–expressing interneurons play a major role in the death ofneurons in response to glutamate or ischemia (25–28).

The cerebral cortex is derived from forebrain forkhead box G1(FOXG1)–expressing primordium (29). Here, we describe the produc-

1Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns HopkinsUniversity School ofMedicine, Baltimore,MD21205, USA. 2Department of Neurology, JohnsHopkins University School of Medicine, Baltimore, MD 21205, USA. 3Department of Neurol-ogy, Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road,Nanjing, Jiangsu 210002, China. 4Solomon H. Snyder Department of Neuroscience, JohnsHopkins University School of Medicine, Baltimore, MD 21205, USA. 5Department of Phar-macology andMolecular Sciences, Johns Hopkins University School of Medicine, Baltimore,MD 21205, USA. 6Department of Physiology, Johns Hopkins University School of Medicine,Baltimore, MD 21205, USA.*These authors contributed equally to this work.†Corresponding author. E-mail: vdawson@jhmi.edu (V.L.D.); tdawson@jhmi.edu (T.M.D.)

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tion of a highly enriched population of forebrain region–specific FOXG1neural precursor cells by the isolation of rosette neural aggregates (thatis, neural aggregates derived from rosette type neural stem cells orRONAs) from human ESCs or iPSCs. Spontaneous fate determina-tion of dorsal and ventral telencephalic progenitors coupled with timedretinoic acid administration leads to differentiation of neurons into allclasses of excitatory and inhibitory neurons in a balanced manner, re-flecting the mature human cerebral cortex. These cultures are sensitiveto excitotoxic injury and cell death after oxygen-glucose deprivationmediated by the activation of N-methyl-D-aspartate (NMDA) receptors,nNOS, and poly(ADP-ribose) polymerase (PARP). Clinically availablePARP inhibitors are effective in blocking human cortical neuronaldeath. Thus, this method enables the study of excitatory and inhibitoryfunctional networks and processes that are thought to play importantroles in synaptic physiology and pathophysiology.

RESULTS

Isolation of FOXG1-positive forebrain progenitor from hESCsor hiPSCsHuman ESCs and iPSCs have intrinsic mechanisms enabling differ-entiation into different subtypes of neurons in the setting of appro-priate cues (5, 30–32). We sought to develop a method to inducedifferentiation of human ESCs or iPSCs into FOXG1 forebrain progen-itors to mimic the patterning and neurogenesis necessary to generateexcitatory and inhibitory neurons in the same culture (Fig. 1A). First,human ESCs or iPSCs were detached and grown as embyroid body (EB)suspensions (Fig. 1A) (18). On day 2, dual SMAD inhibition (31) withnoggin and SB431542 was initiated for 4 days until day 7 to allow therobust induction of rosette formation (fig. S2, A to D). On day 7, the EBswere plated on Matrigel- or laminin-precoated plates. At day 9, rosettes

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began to form, which were FOXG1- and Nestin (NES)–positive (Fig. 1B).Rosettes showed profound cellular expansion in a defined central areaof each colony where they formed RONAs (Fig. 1, C and D). The cen-

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ters of these RONAs were FOXG1-positiveand were surrounded by cytokeratin 8(CK8)–positive epithelial-like cells. Thisdemarcation was easily identified byphase-contrast microscopy without im-munostaining, which allowed manualisolation of FOXG1 precursor cells, min-imizing contamination of the nonneuralprecursors without the need for cell sorting(Fig. 1, E to G). On day 17, RONAs weremanually isolated and grown as neuro-spheres in suspension for 1 day. On day18, the neurospheres were disassociatedinto single cells and plated on laminin/poly-D-lysine–coated plates or coverslipswhere they formed neural precursor cell(NPC) clusters (Fig. 1A). To determinewhether this isolation protocol resultedin the full complement of neural precur-sor subtypes [FOXG1, LIM homeobox 2(LHX2), paired box 6 (PAX6), genetic-screened homeobox 2 (GSX2), and NK2homeobox 1 (NKX2.1)], which patternthe human cortical subdivision and specifycortical excitatory and inhibitory neurons(fig. S2E), immunostaining for these mark-ers was performed at day 9 (rosettes),day 16 (RONA), and day 18 (NPC) (Fig.1, H to L). The rosettes on day 9 were pos-itive for the forebrain and dorsal markersFOXG1, PAX6, and LHX2 but not for theventral telencephalic markers NKX2.1 andGSX2. On day 16, RONAs expressed allfive cortical patterning related transcrip-tion factors, with FOXG1, PAX6, andLHX2 predominating and NKX2.1 andGSX2 expressed in patches at the periph-ery of RONAs (fig. S2, F to I). A compa-rable pattern was observed in human ESC(H1) and human iPSC (SC1014 andSC2131) lines, with no significant differ-ences observed among the different lines(Fig. 1, H to L, and fig. S2J). RNA sequen-cing was performed to gain a compre-hensive and quantitative view of thetranscriptome of isolated FOXG1 RONAs.Markers of pluripotency, mesoderm, andendoderm were not detected. Pan-NPCmarkers [SOX2, NES, vimentin (VIM), andcadherin 2 (CDH2)] were highly expressedin isolated RONAs (Fig. 1M). The an-terior forebrain markers FOXG1, LHX2,and OTX2 (orthodenticle homeobox 2)were highly expressed (Fig. 1N). Dorsalexcitatory neuronal lineage markers [PAX6,

empty spiracles homeobox 1 (EMX1), and EMX2] and ventral forebrainGABAergic neuronal markers [ASCL1 (achaete-scute homolog 1) andDLX2 (distal-less homeobox 2)] along with medial ganglionic eminence

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Fig. 1. Generation of forebrain progenitor cells with regional identity fromhuman iPSCs. (A) Schemeof the RONA differentiation protocol showing generation of forebrain progenitors from human ESCs and

iPSCs.Nog, noggin; SB, SB431542. (B) Rosetteswere immunopositive for FOXG1andNESatday9after initiationof neural differentiation. DAPI, 4′,6-diamidino-2-phenylindole. (C andD) With the prolongation of neural differ-entiation, highly proliferative rosettes started to pile up, resulting in three-dimensional (3D) columnar cellularaggregates,whichwerepositive for FOXG1andNES. (D) Phase-contrast imageof RONA. Scalebar, 50mm(B toD).(E toG) FOXG1 immunoreactive RONAs were located in the center (E) and demarcated clearly with surroundingCK8-positive cells. (F) Cells underneath the RONAswere FOXG1−/CK8−. (G) Thewhite inset box in (F) ismagnified(×2.5). (H to L) Expression and quantification of the early forebrain regionalization markers FOXG1 (H), PAX6(I), LHX2 (J), NKX2.1 (K), andGSX2 (L) after 9, 16, and 18 days of differentiation. Data are presented asmeans ±SEM (n = 3). Scale bar, 20 mm. (M and N) RNA sequencing gene expression profiling of RONAs derived fromthe human ESCH1 line showing the enrichment of forebrain progenitors with regional identity. Data show theaverage RPKM (reads per kilobase of transcript per millionmapped reads) value of two biological replicates ofhumanESCH1cell lineRNAsequencingexperiments. ESRRB, estrogen-related receptorb; SMA, smoothmuscleactin; HNF1B, hepatocyte nuclear factor 1 homeobox B; MGE, medial ganglionic eminence; CGE, caudal gan-glionic eminence. (O) Spontaneous neuronal differentiation after isolation of RONAneural progenitorswithoutthe application of exogenous mitogens. Nuclei were counterstained with DAPI (blue). Colors are indicated inthe images. Scale bar, 20 mm. Data are from the human ESC H1 cell line in (B) to (O). DCX, doublecortin.

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markers (NKX2.1 and LHX8) and caudal ganglionic eminence markers[GSX2 and SP8 (specificity protein 8)] were all expressed in isolatedRONAs (Fig. 1N). Diencephalic [RAX (retina and anterior neural foldhomeobox)], midbrain [EN1 (engrailed homeobox 1 and MSX2 Mshhomeobox 2)], hindbrain [homeobox A2 (HOXA2) and HOXB2], andmore posterior markers (HOXA1, HOXB1, HOXC5, HOXB6, andHB9) were expressed at barely detectable levels (Fig. 1N). Completeontology and enrichment analysis for all genes expressed in FOXG1progenitors are shown in data file S1. These results together indicatethat the isolation of the FOXG1 RONAs provided a robust methodfor recapitulating the early induction of the human telencephalon(FOXG1) and its subsequent subdivision into dorsal (PAX6 andLHX2) and ventral (NKX2.1) territories. These NPCs were spontane-ously differentiated into neurons after isolation from the RONAs andplating (Fig. 1O). These data suggested that the FOXG1-positive cellswere being maintained in a precursor state before their isolation, andthat they were capable of spontaneous differentiation into neuronswhen removed from the RONA niche.

Induction of forebrain precursors with regional identity anddifferentiation of inhibitory neuronsIn the absence of exogenous factors, the neural precursors expressed acomplex combination of brain regional markers, including FOXG1,PAX6, LHX2, NKX2.1, and GSX2, directly after isolation of RONAs(Fig. 1), indicating that these neuronal precursors had the potentialcapability of differentiating into both excitatory and inhibitory neurons.After isolation from RONAs, neural precursors spontaneously showeddynamic changes in cell identity. At day 19, close to 100% of the cellswere FOXG1-positive, 50% were PAX6-positive, and less than 5% wereNXK2.1-positive (fig. S3, A to D). At day 24, 80% of the cells werePAX6-positive and 20% were NKX2.1-positive (fig. S3, A to D). Afterprolonged culture, more than 90% of these cells spontaneously differ-entiated into excitatory neurons, whereas less than 5 to 7% showed amature inhibitory neuronal phenotype.

Because the composition of neurons in the human cerebral cortexis about 80% excitatory glutamatergic neurons and 20% GABAergicinterneurons (1, 2), a better representative balance of excitatory andinhibitory neurons was needed. Accordingly, we explored whetherretinoic acid or sonic hedgehog (SHH) signaling could facilitate theoptimal production of excitatory and inhibitory neurons (fig. S3, Ato D). Retinoic acid and either SHH or the SHH pathway agonistpurmorphamine in various combinations were applied at day 19 andmaintained for 6 days or were applied at day 24 and maintained for6 days (fig. S3A). Extensive optimization revealed that administrationof retinoic acid at days 24 to 30 was required for maintaining the iden-tity of both PAX6/FOXG1 and NKX2.1/FOXG1 progenitors and pro-vided an appropriate percentage of excitatory and inhibitory neuronsrepresentative of the human cerebral cortex (Fig. 2A).

Next, the neural precursors were allowed to develop and differentiateafter withdrawal of retinoic acid at day 30 while being maintainedin neuronal differentiation medium. Cultures were assessed at differenttime points for markers of excitatory and inhibitory neurons, as well assynaptogenesis. Two weeks after withdrawal of retinoic acid, the cultureswere composed primarily of b-tubulin III (TUJ1)–positive neuronalcells (>90%) with about 5 to 10% glial fibrillary acidic protein (GFAP)–positive astrocytes (Fig. 1O and fig. S3E). Four weeks after the with-drawal of retinoic acid, the neurons were composed of about 15 to20% inhibitory neurons as assessed by vesicular GABA transporter

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(VGAT)/glutamic acid decarboxylase 67 (GAD67) immunostainingand 80 to 85% excitatory neurons as assessed by vesicular glutamatetransporter (VGLUT)/calmodulin-dependent protein kinase II (CAMKII)immunostaining (Fig. 2B and fig. S3F). Two weeks after the retinoicacid withdrawal, these neurons started to express the presynaptic markersynapsin and the postsynaptic marker postsynaptic density protein 95(PSD95) (Fig. 2C). These cultures expressed a variety of inhibitoryneuronal markers that included calretinin (CR), calbindin (CB), parv-albumin (PV), nNOS, somatostatin (SST), neuropeptide Y (NPY), andvasointestinal polypeptide (VIP). Thirty-two weeks after differentiation,the microtubule-associated protein 2 (MAP2)–positive neurons com-prised about 3.2% CR-, 1% CB-, 3.68% PV-, 2.5% nNOS-, 5.67%SST-, 1.53% NPY-, and 1.39% VIP-positive neurons (Fig. 2, D and E).A comparable composition was observed in cultures of the human iPSCSC1014 cell line (fig. S3G). The composition of interneuron subtypes inadult human cortex and neuronal cells derived from RONAs was com-pared. The RONA culture showed comparable composition of PV, SST,CR, VIP, and nNOS interneurons to human adult cortex (Fig. 2F) andsimilar developmental patterns for PV, SST, CR, nNOS, and CB inter-neurons when compared to human brain tissues (Fig. 2, E and G).Furthermore, these cultures also expressed the NMDA receptor subunitNR1, the AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid) receptor subunit glutamate A1 (GluA1), and nNOS in a maturepunctate pattern (fig. S3, H to J). These data demonstrated that timedexposure to retinoic acid signaling directed the differentiation of FOXG1forebrain progenitor cells derived from human ESCs or iPSCs intoexcitatory neurons and a diverse repertoire of inhibitory neuronal sub-types with high efficiency.

Differentiation of layer-specific cortical excitatory neuronsfrom FOXG1+ forebrain progenitorsTo determine whether the neurons expressed cortical layer–specificmarkers 90 days after differentiation, immunostaining was performedfor a set of neuronal specific transcription factors (Fig. 3A). The neuronalcultures expressed the cortical layer I, V, and VI marker T-box brainprotein 1 (TBR1); the layer II to IV markers brain-2 (BRN2) and specialAT-rich sequence-binding protein 2 (SATB2); and the layer V and VImarker chicken ovalbumin upstream promoter-transcription factorinteracting protein 2 (CTIP2) (Fig. 3A). The proportion of upper super-ficial and deep layer cortical projection neurons was roughly the samein the differentiated cortical system across human ESC H1, H9, andhuman iPSC SC1014 cell lines (Fig. 3B). The human cerebral cortex ischaracterized by an organized six-layered complex structure. In a mod-ified 3D basement membrane culture method (33–35) (Fig. 3C), im-munostaining of cross sections indicated that these neuronal culturesattempted to assemble into cortical layers with the deep layer corticalmarker CTIP2 and the superficial cortical layer marker SATB2 tendingto segregate into different orientations (Fig. 3, D and E).

Functional human cortical excitatory and inhibitorynetworks show homeostasisEight weeks after differentiation, the electrical properties of humanESC–derived cortical neurons were measured. Whole-cell patch-clamprecording of the neurons revealed the presence of voltage-gated sodiumcurrents that were blocked by tetrodotoxin (Fig. 4A and fig. S4A). Inaddition, there were voltage-gated potassium currents that were sen-sitive to the potassium channel blocker 4-aminopyridine (Fig. 4B). Hu-man ESC–derived cortical neurons fired action potentials when they

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were depolarized but not when they were held at low membrane po-tential (Fig. 4C). To test whether the excitatory and inhibitory synapses(see Fig. 2, B and C) were functional under physiological conditions,

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miniature excitatory postsynaptic currents (mEPSCs) and miniatureiPSCs (mIPSCs) were detected by whole-cell patch-clamp recording.To probe the dynamics of mIPSCs during the development of the

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were found associated with synapsin puncta. Data are presented asmeans ± SEM (n = 3). (D) Differentiation of inhibitory neurons expressing

of conditions for differentiation of appropriate balanced excitatory andinhibitory neurons. KoSR, knockout serum replacement; E/I, excitatory/inhibitory. (B) Immunocytochemical analysis of neuronal expression ofthe excitatory marker VGLUT and the inhibitory marker VGAT. Quantifi-cation of the percentages of VGLUT- and VGAT-positive cells with or with-out retinoic acid (RA) exposure. Data are presented as means ± SEM (n = 3).*P < 0.05, analysis of variance (ANOVA) with Tukey-Kramer’s post hoc test.(C) Immunocytochemical analysis of PSD95 (red) and synapsin (SYN; green)after retinoic acid exposure from days 24 to 30 after the initiation of neuraldifferentiation. Quantification of the proportion of PSD95 puncta that

subtype markers. Colors are indicated in the images. Scale bar, 20 mm.(E) Quantification of inhibitory neurons with immunostaining analysesover 32 weeks after differentiation. Data are presented as means ± SEM(n = 3). (F) Composition of interneuron subtypes in adult human cortex(41) and the cultured neurons derived from RONAs treated with retinoicacid. Data from cortical neuronal cultures derived from the human ESCH1 cell line are presented as means ± SEM. (G) Developmental expressionpattern of PV, SST, CR, nNOS, and CB in human cortical interneurons frommid-fetal stage, late fetal stage, to infant (60). Data from human corticalcultures are from the human ESC H1 cell line in (B) to (F).

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neuronal network, the properties of mIPSCs in amplitudes and frequen-cies were recorded and analyzed over time, revealing a kinetic change overthe time measured (fig. S4B). At 8 weeks after induction of neuronal dif-ferentiation, consistent with the presence of GABAergic synaptic inputs,whole-cell patch-clamp analysis demonstrated that cells readily receivedinhibitory postsynaptic currents that could be reversibly blocked by ag-aminobutyric acid type A (GABAA) receptor inhibitor picrotoxin inthe presence of the NMDA receptor antagonist MK801 or the AMPAreceptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)(Fig. 4D). The mEPSCs were frequently detected in the presence of pic-rotoxin with 5 to 15 pA of amplitude. The amplitude and frequency ofmEPSCs of these cortical neurons increased over time (fig. S4C).

Synaptic scaling homeostatically regulates the stability of net-work activity by balancing excitation and inhibition (36). To examinewhether the existing functional network could undergo synapticscaling, homeostatic scaling was assessed in human ESC–derived cor-tical neurons treated for 36 to 48 hours with tetrodotoxin (1 mM),

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which blocked all evoked neuronal activity, or with bicuculline(20 mM), which blocked inhibitory neurotransmission mediated byGABAA receptors and increased neuronal firing (37). There was a robustincrease in the amplitude of mEPSCs after tetrodotoxin treatment(Fig. 4E), which was present 4 weeks after differentiation and furtherincreased after 8 weeks (Fig. 4E and fig. S4, D and E). A rightward shiftof the cumulative probability distributions indicated that the increasein amplitude was distributed across the range of recorded events. Incontrast to the increase in mEPSCs after tetrodotoxin treatment, therewas a robust decrease in the amplitude of mEPSCs after bicucullinetreatment (Fig. 4E), which began at 4 weeks after differentiation andwas more robust after 8 weeks (Fig. 4E and fig. S4, D and E). Therewas a leftward shift of the cumulative probability distributions, indi-cating that the decrease in amplitude was distributed across therange of recorded events (Fig. 4E). These results show that the differ-entiated neurons formed a functionally balanced excitatory and inhib-itory network, and neuronal activity could be homeostaticallyregulated to maintain network stability.

Human cortical neurons are sensitive toNMDA- and oxygen-glucose deprivation–mediated neurotoxicityTo ascertain the susceptibility of human cortical neurons to excito-toxicity, human cortical neurons derived from the human ESC H1 cellline were exposed to 500 mM glutamate for 5, 15, 30, or 60 min. Morethan 60% of neurons died after a 30-min exposure to 500 mM gluta-mate as assessed 24 hours later. Exposure to 500 mM glutamate for 5 or15 min resulted in lower neurotoxicity (Fig. 5A). A dose response toincreasing concentrations of glutamate indicated that human cortical neu-rons were sensitive to a graded concentration of glutamate, with greaterthan 60% toxicity achieved with 500 mM glutamate for 30 min (Fig. 5B).Glutamate neurotoxicity was markedly attenuated by the NMDA re-ceptor antagonist MK801, but only a modest protection was observedwith the AMPA receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline) (Fig. 5C). Because glutamate neuro-toxicity in human neurons occurred predominantly through NMDAreceptors, subsequent studies focused on NMDA receptor stimulationusing NMDA to activate the NMDA glutamate receptor subtype. A timecourse of 500 mMNMDA was performed and revealed a similar patternof neurotoxicity with about 60% neuronal cell death after 30 min ofexposure and no or modest toxicity after 5 or 15 min of exposure to500 mM NMDA assessed 24 hours later (fig. S5A). A dose response toincreasing concentrations of NMDA indicated that human corticalneurons derived from the human ESC H1 cell line were sensitive toa graded concentration of NMDA, with greater than 60% toxicity achievedwith 500 mM NMDA for 30 min (Fig. 5D). Calcium ion influx trig-gered by exposure to NMDA (100 or 500 mM) was almost fully abol-ished by the addition of MK801 (Fig. 5E). Human cortical neuronswere also sensitive to oxygen-glucose deprivation, with cell death observedafter 60 min of oxygen-glucose deprivation and with greater than 60%cell death observed after 120 min of oxygen-glucose deprivation as-sessed 24 hours later (Fig. 5F). Human cortical neuron cultures derivedby a similar protocol but without retinoic acid treatment were chal-lenged with glutamate or NMDA at different doses and exposure timesor with oxygen-glucose deprivation at different exposure times to com-pare their sensitivity to excitotoxicity in the presence or absence of retinoicacid treatment (fig. S5, B to F). The absence of retinoic acid treatment ledto less sensitivity of human neuronal cultures to neurotoxicity mediatedby glutamate, NMDA, or oxygen-glucose deprivation (fig. S5, B to F).

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forebrain progenitor cells. (A) Immunocytochemical analysis of the cortical

layer–specific markers TBR1, CTIP2, BRN2, and SATB2 in human neuronalculture differentiated from FOXG1+ neural progenitors derived from the hu-man ESC H1 cell line. (B) Quantification of the percentages of TBR1, CTIP2,BRN2, and SATB2 in neuronal culture. Data are presented as means ± SEM(n = 3). Data are from the human ESC H1 andH9 cell lines and the humaniPSC line SC1014. (C) Diagram of the 3D human cortical assembly assay. (Dand E) Cross sections from 3D human cortical assemblies were immunos-tained with the cortical neuron layer markers TBR1, CTIP2, BRN2, and SATB2.Nuclei were counterstained with DAPI (blue). The boxed area is magnified(×2.5) in the fifth column of (E). Colors are indicated in the images. Scalebar, 50 mm. Data in (C) to (E) are from the human ESC H1 cell line.

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NMDA-mediated excitotoxicity and oxygen-glucosedeprivation–mediated neurotoxicity depend on NOand the parthanatos pathwayTo evaluate the mechanisms involved in NMDA-mediated or oxygen-glucose deprivation–mediated neurotoxicity in human cortical neuronsderived from the human ESC H1 cell line, cell death was assessed afterexposure to NMDA or oxygen-glucose deprivation in the presence ofantagonists to known cell death signals. Only the selective nNOS in-hibitor Nw-propyl-L-arginine (NPLA) and the PARP inhibitor DPQ

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blocked NMDA- or oxygen-glucose deprivation–mediated neurotoxic-ity (Fig. 6, A and B). Because nNOS neurons play a critical role inexcitotoxicity, we quantified and compared the percentage of nNOSneurons in retinoic acid–induced and non–retinoic acid–induced humanneuronal cultures after 2 months of differentiation. We observed fewernNOS-positive neurons in non–retinoic acid–treated cultures comparedto retinoic acid–treated cultures, which may partly explain the reducedsensitivity to excitotoxicity in non–retinoic acid–treated cultures (fig. S5G).The broad-spectrum caspase inhibitor Z-VAD, the necroptosis inhibitor

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current is inward (n = 11 for each group). (E) Homeostatic scaling of humanESC H1 cell line–derived cortical neurons. Measurement of mEPSCs from

tassium channels (B) present in human ESC H1 line–derived corticalneurons (n = 10 for sodium current and n = 12 for potassium current).4-AP, 4-aminopyridine. (C) Evoked action potentials (whole-cell recording,current clamping) generated by human ESC H1 line–derived cortical neu-rons after 8 weeks of differentiation (n = 9). (D) Measurement of mIPSCs in-dicated the formation of a functional inhibitory network in human corticalneuronal culture.With 40mMchloride ion in the pipette solution, themIPSC

cultured human cortical neurons under control conditions (control),conditions of activity blockade [tetrodotoxin (TTX)], or conditions of ac-tivity enhancement with bicuculline (Bic). Forty-eight hours of activityblockade increased the amplitude of mEPSCs, whereas 48 hours ofenhanced activity decreased the mEPSC amplitude. *P < 0.05, Mann-Whitney test, Control, n = 17; TTX, n = 21; Bic, n = 18. All data shownare from the human ESC H1 cell line.

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the neurons against glutamate excitotoxicity (***P < 0.001), whereas NBQXhadnoeffect onglutamate excitotoxicity. (D) Thirtyminutes of treatmentwith

dide (PI)/Hoechst staining 24 hours after different exposure times to 500 mMglutamate (Glut) andwere quantified and expressed asmeans ± SEM (n= 3).(B) Percentages of neuron death were assessed by PI/Hoechst staining 24hoursafter30minofglutamateexposureatdifferentdosesandwerequantifiedand expressed asmeans ± SEM (n= 3). (C) MK801 (NMDA receptor antagonist)but not NBQX (antagonist of AMPA receptors and kainate receptors) abolished500 mMglutamate–induced human cortical neuron death. Percentages of neu-ron death were quantified and expressed as means ± SEM (n = 3). Glutamatesignificantly killed neurons (***P < 0.001), and MK801 significantly protected

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NMDA at different concentrations. Percentages of neuron death were as-sessed by PI/Hoechst staining, quantified, and expressed as means ± SEM(n = 9). (E) Calcium imaging with Fura-2 of human cortical neuron culturesstimulated with 100 or 500 mM NMDA and 10 mM glycine, followed byMK801. Plots show the means ± SEM of 30 to 31 neurons. (F) Human ESCH1 cell line–derived cortical neurons were subjected to different lengths ofoxygen-glucose deprivation. Percentages of neuron death were assessedby PI/Hoechst staining, quantified, and expressed as means ± SEM (n = 9).**P<0.01 and ***P<0.001, ANOVAwith Bonferroni’s posttest. NT, not treated.

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necrostatin-1 (NEC-1), or the autophagy inhibitor 3-methyladenine (3-MA)was not effective in altering NMDA- or oxygen-glucose deprivation–mediated neurotoxicity (Fig. 6, A to B). NMDA-induced or oxygen-glucosedeprivation–induced NO formation was measured with the Measure-iTHigh-Sensitivity Nitrite Assay Kit (38). The selective nNOS inhibitor

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NPLA attenuated NO production consistent with its neuroprotectiveeffect, whereas the PARP inhibitor had no effect (Fig. 6, C and D).NMDA-induced or oxygen-glucose deprivation–induced PARP activitywas assessed by a PAR immunoblot assay. The PARP inhibitor DPQand the NOS inhibitor L-NAME or the selective nNOS inhibitor

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of treatment with 500 mMNMDA or 2 hours of oxygen-glucose deprivation.Data are shown as means ± SEM of NO units (fluorescence measured at

inhibitor) and DPQ (PARP inhibitor), but not Z-VAD (caspase inhibitor), NEC-1 (necroptosis inhibitor), or 3-MA (autophagy inhibitor), protected humanESC H1 cell line–derived cortical neuronal cultures from cell death due to(A) 30 min of exposure to 500 mM NMDA or (B) 2 hours of oxygen-glucosedeprivation.Quantitative data from thePI/Hoechst-positive cell number ratioare shown as means ± SEM. For NMDA-treated neurons (A), n = 8 for thedimethyl sulfoxide (DMSO) control group and the NMDA plus DMSOgroup; for oxygen-glucose deprivation–treated neurons (B), n = 3 forthe DMSO control group and the DMSO + oxygen-glucose deprivation(OGD) group. For the NMDA and oxygen-glucose deprivation neuron groups,n = 3 for Z-VAD, NEC-1, 3-MA, NPLA, and DPQ treatments. (C andD) An nNOSinhibitor (NPLA), but not a PARP-1 inhibitor (DPQ), inhibited NO produc-tion in humanESCH1 cell line–derived cortical neuronal cultures after 30min

excitation/emission wavelengths of 365 nm/450 nm) normalized to medium-onlywells (n=4). (E and F) Representative blots showing that treatmentwitha NOS inhibitor (NPLA or L-NAME) or a PARP inhibitor (DPQ) blocked PARpolymer formation (theproduct generatedbyPARP) after 30minof treatmentwith500mMNMDAor2hoursofoxygen-glucosedeprivation inhumanESCH1cell line–derived cortical neuronal cultures;means ± SEMof PAR polymer/actinband density ratio (n = 3). (G and H) Lentiviral vector–based PARP-1 CRISPR/Cas9 sgRNAs protect human ESC H1 line–derived cortical cultures fromNMDA- or oxygen-glucose deprivation–induced cell death. Data are shownasmeans± SEM (n=3). (I and J) PARP-1 inhibitors protect humanESCH1 line–derivedcortical neuronal cultures fromNMDA-oroxygen-glucosedeprivation–induced cell death. Data are shown as means ± SEM (n = 4). *P < 0.05, **P <0.01, ***P < 0.001, and ****P < 0.0001 (ANOVA with Bonferroni’s posttest).

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NPLA attenuated PAR formation (Fig. 6, E and F). To confirm that ac-tivation of PARP was critical to NMDA- or oxygen-glucose deprivation–mediated neurotoxicity, PARP-1 was knocked out by CRISPR/Cas9 using two different single-guide RNAs (sgRNAs) (fig. S6A).Knockout of PARP-1 by either sgRNA substantially attenuatedNMDA- or oxygen-glucose deprivation–mediated neurotoxicity(Fig. 6, G and H). Furthermore, four PARP inhibitors that are current-ly being evaluated in clinical trials for cancer were evaluated forneuroprotective actions against NMDA- or oxygen-glucose depriva-tion–mediated neurotoxicity. AG-014699, ABT-888, olaparib, andBMN 673 all markedly attenuated NMDA- or oxygen-glucose depri-vation–mediated neurotoxicity in human cortical neurons (Fig. 6, I to J).In rodent models, questions have been raised regarding the role of PARPactivation in male versus female mice (39). Thus, human cortical neuronswere derived from the human ESC H9 cell line, which is female, for com-parison to male human ESC H1 cell line–derived cortical cultures. Un-der identical conditions, we observed that NMDA or oxygen-glucosedeprivation elicited identical neurotoxicity in the H1 cell line– versusthe H9 cell line–derived cortical cultures, which was attenuated by PARPinhibitors equally in both cortical cultures (fig. S6, B to G).

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DISCUSSION

This study presents an efficient method for differentiation of humanESCs or iPSCs into excitatory and inhibitory neurons by isolation ofhighly enriched FOXG1 neural precursors from human ESC or iPSCRONAs. In particular, timed administration of retinoic acid led to balancedexcitatory and inhibitory neuronal networks that exhibited all six layers ofcortical projection neurons and a rich diversity of GABAergic inter-neurons. This method enabled the formation of functional excitatoryand inhibitory networks that exhibited homeostatic scaling and suscep-tibility to excitotoxicity and oxygen-glucose deprivation in a NO- andPARP-dependent manner.

FOXG1 is one of the earliest expressed transcription factors thatdirect the development of the telencephalon (29). In the mouse brain,FOXG1 is required for the development of both the ventral telencephalonand the anterior neocortex, thus providing a source of both excitatoryand inhibitory neurons (29, 40). After efficient neuroectoderm conversionof human ESCs or iPSCs expressing high levels of FOXG1, these FOXG1precursors expressed markers of the dorsal forebrain (PAX6 and LHX2)and ventral forebrain (NKX2.1 and GSX2). The RONA culture methoddescribed here does not require cell sorting, is cost-effective using min-imal numbers of expensive growth factors, and should be a widely ap-plicable method. It supports the formation of cortical dorsal andventral subdivisions, reminiscent of forebrain patterning along the dor-soventral axis. This method results in about 20% inhibitory neurons,which is reflective of the known abundance of inhibitory neurons in bothmouse and human brain tissue (1, 2). The expression of nNOS neuronsin the human brain and RONA-derived neuron culture is equivalent tothat observed in mouse and human brain (41, 42). The composition ofPV, SST, CR, VIP, and nNOS in the interneuronal culture derived fromRONAs is similar to those in human adult cortex (41), which indicatesthat the percentage of these interneuronal populations is more re-flective of human than of mouse cortex. A review of other human corticalneuron differentiation and culture methods indicates that the methoddescribed here provides a more balanced representation of both excit-atory and inhibitory neurons than previously reported methods (fig.

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S1). There is a modest underrepresentation of nNOS, CR, and CBinterneurons (Fig. 2 and fig. S1), and future studies and optimizationwill be required to provide a more accurate representation of the in-terneuron composition of the human cortex. The composition ofPV, SST, and VIP interneurons shared by RONA-derived neuronsand the human cortex differs from the mouse brain; thus, RONA-derived human neurons may be more suitable for studying neuro-logical diseases involving human excitatory and inhibitory neuronalnetworks. In addition to the highly enriched neuron culture 8 to 12weeks after induction of neural differentiation, we observed that about5 to 10% of the cells were GFAP-positive astrocytes, which is consistentwith the astrogenesis pattern shown in 3D cultures of laminated cerebralcortex–like structures (43).

Proper brain function requires a balance of excitatory and inhibi-tory neurotransmission (36). In neurological and neuropsychiatricdiseases such as autism, Alzheimer’s disease, and schizophrenia, thereis evidence that the balance of excitatory and inhibitory neurotrans-mission is disturbed (44). There has been progress in establishing invitro systems for studying human disease using populations of maturecortical interneurons, excitatory pyramidal neurons, or mixed neuralcultures with limited characterization of the interplay between excit-atory and inhibitory networks. The protocol described here provides amore accurate representation of the human cortex, allowing the studyof the interplay between excitatory and inhibitory networks in bothnormal and pathological conditions. This method generates functionalneurons with relatively mature electrical properties including excitatoryand inhibitory postsynaptic currents, mEPSCs and mIPSCs, which areindicative of the copresence of excitatory and inhibitory networks. Inresponse to neural activity, neuronal networks undergo compensatorysynaptic scaling to maintain a proper balance of excitation and inhibi-tion (45). Our cortical cultures exhibit homeostatic scaling becausepharmacological perturbation by tetrodotoxin and bicuculline resultsin adaptive synaptic function. Homeostatic scaling has been widelystudied in rodent models, and our data indicate that homeostaticscaling can occur in human cortical neurons as well.

Although cultured human fetal cortical neurons are sensitive to glu-tamate neurotoxicity (46), the underlying mechanisms of glutamate ex-citotoxicity have not been explored in human neuronal cultures (47, 48).The dynamic interplay between inhibitory neurons and pyramidal ex-citatory neurons is particularly important in studying neurotoxicity orneuronal injury. In particular, modeling excitotoxicity in primary neu-ronal cultures from mice and rats requires the presence of nNOS inhib-itory neurons (27, 28, 49, 50). A recent study of NMDA excitotoxicity inhuman embryonic stem cell–derived excitatory neurons required a 24-hourexposure to NMDA to elicit neurotoxicity (47). The requirement ofprolonged exposure to NMDA to elicit excitotoxicity is markedly dif-ferent from the rapidly triggered delayed death that is characteristicof acute neuronal injury that occurs in stroke or trauma (51). On theother hand, the human neuronal culture method reported here exhibitedrapidly triggered delayed death in response to glutamate-mediated andNMDA-mediated excitotoxicity and oxygen-glucose deprivation. Theenhanced susceptibility is most likely due to an appropriate balance ofexcitatory and inhibitory neurons (52), including nNOS neurons. Ourmethod revealed that human neurons are sensitive to NO and partha-natos, similar to rodent neuronal cultures, cortical brain slices, and in vivoanimal studies (26, 49, 53–58). Because neuronal cell death is dependenton the length and strength of the stimulus, other cell death pathwaysmay be recruited with longer exposure to excitotoxins or oxygen-glucose

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deprivation. However, in acute injury paradigms, NO and parthanatospredominate much in the same manner as has been reported in rodentmodel systems (26, 53, 57, 59). These results are consistent with thenotion that interfering with parthanatos is a particularly attractive ap-proach to treat acute neuronal injury in humans, particularly becausethere are clinically available PARP inhibitors (54).

In summary, we report a robust and easily reproducible system forgeneration of excitatory projection neurons and inhibitory interneurons.These human cortical neurons can be used to study the mechanisms ofexcitotoxicity and screen for clinically relevant neuroprotective com-pounds. Furthermore, in future studies, this methodology can be usedto generate cultures that will be useful in exploring the genetic basis ofneuronal connectivity in neuropsychiatric diseases such as autism andschizophrenia, which are thought to involve imbalances in excitatoryand inhibitory neural transmission. Moreover, these cultures may beuseful in the study of neurodegenerative diseases relevant to the cortex,including Alzheimer’s disease.

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MATERIALS AND METHODS

Study designTo ensure adequate power to detect an effect size, the effect size wasfirst calculated on the basis of pilot experiments and then used inpower analysis by the software G*Power to determine sample sizeby given error probability (a = 0.05) and power (1 − b > 0.95). Allsamples were included for data analysis. Imaging and cell count forneural differentiation and neurotoxicity were performed by eitherautomated software or a blinded observer. National Institutes of Health(NIH) registry human ESC lines H1 and H9 (which are widely distrib-uted and researched) and two characterized human iPSC lines, SC1014and SC2131 (reprogrammed by non-integrating Sendai virus and lenti-virus), were used for the neural differentiation and neurotoxicity assays.The human ESC/iPSC lines used and quantified in all the experimentsare described in the individual figures and figure legends.

Culture of human ESCs or iPSCsHuman ESC lines H1 and H9 (WiCell) and human iPSC lines SC1014and SC2131 (Johns Hopkins Medical Institutions) were maintainedon inactivated mouse embryonic fibroblasts (MEFs) according to stan-dard protocols. Briefly, human ESCs or iPSCs were maintained in humanESC medium containing Dulbecco’s modified Eagle’s medium/nutrientmixture F-12 (DMEM/F12; Invitrogen), 20% knockout serum replace-ment (Invitrogen), fibroblast growth factor 2 (FGF2; 4 ng/ml; PeproTech),1 mM GlutaMAX (Invitrogen), 100 mM nonessential amino acids(Invitrogen), and 100 mM 2-mercaptoethanol (Invitrogen). Medium waschanged daily. Cells were passaged using collagenase (1 mg/ml inDMEM/F12) at a ratio 1:6 to 1:12. All experiments using human ESCs/iPSCs were conducted in accordance with the policy of the Johns Hop-kins University (JHU) School of Medicine that research involving humanESCs or iPSCs being conducted by the JHU faculty, staff, or students orinvolving the use of JHU facilities or resources shall be subject to oversightby the JHU Institutional Stem Cell Research Oversight Committee.

Neural differentiation of human ESCs or iPSCsTo initiate differentiation, human ESC or iPSC colonies were treatedwith collagenase (1 mg/ml in DMEM/F12) in an incubator for about5 to 10 min. The colony borders began to peel away from the plate, and

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collagenase was washed off the plate with growth medium. While thecolony center remained attached, the colonies were selectively detachedwith the MEFs undisturbed. Detached ESC or iPSC colonies were thengrown in suspension in human ESC medium without FGF2 for 2 daysin low-attachment six-well plates (Corning). From days 2 to 6, noggin(50 ng/ml; R&D system) or dorsomorphin (1 mM; Tocris) and SB431542(10 mM; Tocris) were supplied in human ESC medium (without FGF2,defined as KoSR medium). On day 7, free-floating EBs were transferredto Matrigel- or laminin-precoated culture plates to allow the completeattachment of EB aggregates with the supplementation of N2 inductionmedium containing DMEM/F12 (Invitrogen), 1% N2 supplement(Invitrogen), 100 mMMEMnonessential amino acid solution (Invitrogen),1 mM GlutaMAX (Invitrogen), and heparin (2 mg/ml; Sigma). Cul-tures were fed with N2 medium every other day from days 7 to 12.From day 12 onward, N2 induction medium was changed everyday.Attached aggregates broke down to form a monolayer colony on days8 to 9 with typical neural-specific rosette formation. With the ex-tension of neural induction, highly compact 3D column-like neuralaggregates (termed RONAs) formed in the center of attached colonies.RONAs were manually microisolated, taking special care to minimizethe contaminating peripheral monolayer of flat cells and the cells under-neath RONAs. RONA clusters were collected and maintained as neuro-spheres in Neurobasal medium (Invitrogen) containing B27 minus VitA(Invitrogen) and 1 mM GlutaMAX (Invitrogen) for 1 day; the next day,the neurospheres were dissociated into single cells and plated onlaminin/poly-D-lysine–coated plates for further experiments. For neuronaldifferentiation, retinoic acid (2 mM), SHH (50 ng/ml), purmorphamine(2 mM), or the combination of retinoic acid, SHH, and purmorphaminewas supplemented in neural differentiation medium containingNeurobasal/B27 (NB/B27; Invitrogen), brain-derived neurotrophicfactor (BDNF; 20 ng/ml; PeproTech), glial cell line–derived neuro-trophic factor (GDNF; 20 ng/ml; PeproTech), ascorbic acid (0.2 mM;Sigma), dibutyryl adenosine 3′,5′-monophosphate (cAMP; 0.5 mM;Sigma) at the indicated time points after neurospheres were dis-sociated into single cells. For long-term neuronal culture, neural dif-ferentiation medium containing rat astrocyte–conditioned Neurobasalmedium/B27, BDNF, GDNF, ascorbic acid, and dibutyryl cAMP wasused for maintenance.

RNA sequencingTwo biological replicates of RONA colonies were dissected away fromthe supporting cells, disassociated, and plated. Twenty-four hours afterplating, total RNA was harvested from the culture using TRIzol(Invitrogen) following the manufacturer’s protocol. Ribosomal RNAswere subtracted from total RNA using Ribo-Zero magnetic kit (Epi-centre). Libraries from ribosomal RNA–subtracted RNA were generatedusing ScriptSeq 2 (Epicentre) and sequenced on an Illumina HiSeq 2500.Reads were mapped to the human genome (release 19) using TopHat2.Reads were annotated using a custom R script using the UCSC KnownGenes table as a reference. Gene ontology (GO) shows output from aenrichment analysis on DAVID (Database for Annotation, Visualization,and Integrated Discovery; https://david.abcc.ncifcrf.gov/), searching thefollowing categories: GO Biological Process, GO Molecular Function,PANTHER (Protein Analysis Through Evolutionary Relationships)Biological Process, PANTHER Molecular Function, and KEGG (KyotoEncyclopedia of Genes and Genomes) and PANTHER pathway. En-richment terms and associated genes and statistics presented with ad-ditional statistical analyses are also provided. P values presented in the

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text are corrected for multiple comparisons using the Benjamini method(column L).

Immunocytochemistry, immunohistochemistry, imaging,and quantificationCultured cells were washed in phosphate-buffered saline (PBS) andfixed in 4% paraformaldehyde for 15 min. Attached RONAs and humancortical assemblies were sectioned (25 mm) using a cryostat (CM3050;Leica) and collected on SuperFrost Plus glass slides (Roth). Cells andsections were blocked with blocking buffer containing 10% (v/v) donkeyserum and 0.2% (v/v) Triton X-100 in PBS. Then, they were incubatedovernight at 4°C with primary antibodies diluted in blocking buffer,washed three times in blocking buffer, treated with secondary antibody(Invitrogen) for 1 hour, and washed three times in blocking buffer. Afterstaining, coverslips were mounted on glass slides, and sections werecoverslipped using ProLong Gold Antifade Reagent (Invitrogen). Theprimary antibodies used were human-specific NES (Millipore, MAB5326andMAB5922), PAX6 (Covance, PRB-278P), FOXG1 (Abcam, ab18259),CTIP2 (Abcam, ab18465), SATB2 (Abcam, ab51502), BRN2 (Santa CruzBiotechnology, sc-6029), TBR1 (Abcam, ab31940), DCX (Santa CruzBiotechnology, sc-8066), TUJ1 (Millipore, AB1637), MAP2 (Sigma,M2320; Millipore, AB5622), nNOS (Sigma, N2880), VGLUT1 (SYSY,135311), synapsin I (Millipore, AB1543), PSD95 (Abcam, ab2723), VGAT(SYSY, 131003), parvalbumin (Sigma, P3088), CB (Sigma, C9848), CR(BD, 610908), Somatostatin (Millipore, MAB354), GSX2 (Abcam,ab26255), NKX2.1 (TTF1, Epitomics, 2044-1), VIP (Sigma, V0390), NPY(Abcam, ab30914), prospero homeobox 1 (Abcam, ab37128), CK8(Santa Cruz Biotechnology, sc-101459), LHX2 (Millipore, AB10557),GluN1 (SYSY, 114011), GluA1 (Epitomics, 1308-1), CAMKII (Cell Sig-naling Technology, 3362S), and GAD67 (Millipore, MAB5406).Thefollowing cyanine 2 (Cy2)–, Cy3-, and Cy5-conjugated secondary anti-bodies were used to detect primary antibodies: donkey antibody againstmouse, donkey antibody against rat, donkey antibody against goat, anddonkey antibody against rabbit (Invitrogen).

3D human cortical assembly assayThe cell culture insert was initially coated with 2%Matrigel, forming agelled bed of basement membrane. Isolated NPCs were seeded ontothis bed as a single-cell suspension at a high density of 0.75 × 106 to 1 ×106/cm2 in a neural differentiation medium containing NB/B27(Invitrogen), 1%Matrigel, BDNF (20 ng/ml), GDNF (20 ng/ml), ascorbicacid (0.2 mM), and dibutyryl cAMP (0.5 mM). The neural differenti-ation medium was replaced every 3 days.

Oxygen-glucose deprivationDissolved O2 was first removed from glucose-free medium by bubblingwith oxygen-glucose deprivation gas (5% CO2, 9.8% hydrogen, and85.2% N2; Airgas Ltd.) for 30 min. Neurons were then washed withglucose-free medium. Oxygen-glucose deprivation was started by addi-tion of glucose-free medium prebubbled with oxygen-glucose depriva-tion gas after incubation in a hypoxia chamber attached with an O2

sensor/monitor (Biospherix Ltd.) for the indicated times. Neuronalcultures were then refed with normal medium and maintained undernormal culture conditions (5% CO2 and 20% O2 at 37°C).

Cell death assessmentTwo- to three-month-old human cortical neurons were treated withNMDAor oxygen-glucose deprivation at various indicated timeperiods

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and doses. Percent of cell death was determined by staining with 5 mMHoechst 33342 and 2 mMPI (Invitrogen). Images were taken and countedby a Zeiss microscope equipped with automated computer-assistedsoftware (Axiovision 4.6, Zeiss). Z-VAD (20 mM; Sigma, V116), 20 mMNEC-1 (Sigma, N9037), 500 mM 3-MA (Calbiochem, 189490), 20 mMNPLA (Tocris, 1200), 30 mM DPQ (Enzo, ALX-270-21-M005), 500 nMAG-014699 (Selleckchem, S1098), 10 mM ABT888 (Active Biochem,A-1003), 2 mM olaparib (LC Laboratories, O-9201), and 20 nM BMN673 (Selleckchem, S7048) were applied to evaluate the effect of differ-ent antagonists to known cell death signals.

Calcium imagingHuman cortical neurons were administrated with Fura-2 (Molecular Probes)for 30 min at room temperature. After washing, calcium imaging wasconducted at 340- and 380-nm excitation in 2-month-old human cor-tical neuron cultures. Neurons were stimulated with 100 or 500 mMNMDA followed by MK801 to detect intracellular free calcium.

Measurement of NO production in culture mediumThe NO production of 2-month-old humanH1 cortical cultures after a30-min treatmentwithNMDAor2hours of oxygen-glucose deprivationwas assessed using the Measure-iT High-Sensitivity Nitrite Assay Kit(Life Technologies).

PARP-1 knockout in human cortical neuronal cultureusing CRISPR/Cas9PARP-1 sgRNAs (#2,GTGGCCCACCTTCCAGAAGC;#3,ATACCAAA-GAAGGGAGT-AGC) were synthesized and subcloned into lentiCRISPRvector (Addgene, pXPR_001, plasmid 49535). Lentiviral vectors werecotransfected into HEK293FT cells with the lentivirus packaging plas-mids pVSVg andpsPAX2using FuGENEHD. Supernatants containingvirus were collected 48 and 72 hours after transfection, passedthrough a nitrocellulose filter (0.45 mm), and applied on cells inculture. At 8 to 12 weeks after differentiation, human cortical neuronswere transduced with lentiCRISPR lentivirus carrying control sgRNAor PARP-1 sgRNAs. Cells were then challenged with NMDA or oxy-gen-glucose deprivation at 5 days after transduction, and cell deathwas assessed by PI/Hoechst staining 24 hours later. Another batch ofcells was collected at 5 days after transduction and analyzed by Westernblotting for PARP-1 expression.

Western blotSDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferwere performed according to laboratory protocol with slight modifica-tions. In brief, cultured cells were lysed in radioimmunoprecipitation as-say buffer containing 1% Triton, 0.5% Na-deoxycholate, 0.1% SDS, 50 mMNaF, 10 mM Na4P2O7, 2 mM Na3VO4, and EDTA-free protease in-hibitor mixture (Roche Diagnostics) in PBS (pH 7.4). Protein extractswere separated by 4 to 12% SDS-PAGE. Proteins were transferred atconstant voltage of 80 V for 150 min at 4°C from the SDS to poly-vinylidene difluoride membranes. The membranes were then blockedwith 5% nonfat milk and incubated with primary antibodies overnightat 4°C. The antibodies used were anti-PAR (Trevigen, 4336-APC-050), actin (Cell Signaling, 5125), PARP-1 (BD, 611039), and b-actin(Cell Signaling Technology, #5125S). After washes with TBST (tris-buffered saline with 0.1% Tween 20), the membranes were incubatedwith horseradish peroxidase–conjugated secondary antibodies for 1 hourat room temperature. Immunoreactive bands were visualized by the

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enhanced chemiluminescent substrate (Pierce) on x-ray film and quan-tified using the image software TINA.

Electrophysiological recordingsWhole-cell voltage-clamp recordings were obtained with whole-cellconfiguration using an EPC-10 amplifier (HEKA Elektronik). Patch-clamp recordings from human ESC-derived neurons were made usingrecording electrodes of 8 to 10 megohms pulled from Corning KovarSealing #7052 glass pipettes (PG52151-4, WPI) by a Flaming-Brownmicropipette puller (Sutter Instrument Co.). The extracellular solutioncontained 125 mM NaCl, 2.5 mM KCl, 25 mM D-glucose, 25 mMNaHCO3, 1.25 mM NaH2PO4, 1 mM CaCl2, and 1 mM MgCl2, and 5%CO2 and 95% O2 bubbled (pH 7.4). Intracellular solutions contained135 mM KMeSO4, 8 mM NaCl, 2 mM ATP-Mg, 0.1 mM Na3GTP,0.3 mM EGTA, and 10 mMHepes (pH 7.2). Action potential recordingswere obtained with current clamp configuration. Spontaneous mEPSCswere obtained with voltage-clamp configuration, the membranepotential was held at −70 mV, and 100 mM picrotoxin and 0.5 mM te-trodoxin were in the extracellular solution; for iPSC recording, 10 mMCNQX and 10 mM MK801 were in the extracellular solution. Sponta-neous mEPSC and mIPSC recordings were acquired and stored digi-tally using the data acquisition/analysis package PULSE (HEKAElectronik). Potential recordings were acquired at 6 kHz and filteredat 3 kHz. We used Mini Analysis 6 (Synaptosoft) to detect mEPSCand mIPSC events and create cumulative amplitude histograms ofthe pooled data.

Statistical analysisData are presented as means ± SEM. Statistical analysis was performedusing GraphPad Prism and noted in the text and figure legends. In-formation on sample size (n, given as a number) for each experimentalgroup/condition, statistical methods, and measures are available in allrelevant figure legends. Unless otherwise noted, significance was as-sessed as P < 0.05.

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www.sciencetranslationalmedicine.org/cgi/content/full/8/333/333ra48/DC1Fig. S1. Comparison of human forebrain neuron differentiation method.Fig. S2. Neural induction of human iPSCs by dual inhibition of SMAD signaling enhances theefficiency of neural conversion via the RONA method.Fig. S3. Isolated FOXG1+ forebrain progenitors preserve excitatory and inhibitory neurogenicpotential.Fig. S4. Development regulates the excitatory/inhibitory network formation and synapticscaling.Fig. S5. NMDA induces human cortical neuron cell death.Fig. S6. PARP activation in male versus female human ESCs.Data file S1 (Excel file)

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Funding: This workwas supported by grantsMSCRFII-0429 andMSCRFII-0125 to V.L.D., grant 2013-MSCRF-0054 to J.-C.X., grant 2013-MSCRF-0028 to J.F., grant 2014-MSCRFE-0758 to S.M.E, NIH/National Institute of Neurological Disorders and Stroke grant NS67525 and NIH/National Insti-tute on Drug Abuse grant DA00266 to T.M.D. and V.L.D. T.M.D. is the Leonard andMadlynAbramsonProfessor in Neurodegenerative Diseases. Author contributions: J.-C.X., J.F., T.M.D., and V.L.D. de-signed the experiments. J.-C.X. and J.F. differentiated cortical neurons from human ESCs andiPSCs. J.-C.X. and J.F. performed immunohistochemistry and confocalmicroscopy studies and carriedout neurotoxicity studies on the neurons derived from human ESCs or iPSCs. X.W. carried outelectrophysiology studies. X.W. and X.Y. analyzed electrophysiology data. S.M.E carried out theRNA sequencing experiment and analyzed the RNA sequencing data. T.-I.K., L.C., J.Z., Z.C., H.J., andR.C. analyzed thedata. J.-C.X., J.F., T.M.D., and V.L.D. wrote themanuscript.Competing interests: Theauthors declare that they have no competing interests.

Submitted 20 July 2015Accepted 18 March 2016Published 6 April 201610.1126/scitranslmed.aad0623

Citation: J.-C. Xu, J. Fan, X. Wang, S. M. Eacker, T.-I. Kam, L. Chen, X. Yin, J. Zhu, Z. Chi, H. Jiang,R. Chen, T. M. Dawson, V. L. Dawson, Cultured networks of excitatory projection neurons andinhibitory interneurons for studying human cortical neurotoxicity. Sci. Transl. Med. 8, 333ra48(2016).

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studying human cortical neurotoxicityCultured networks of excitatory projection neurons and inhibitory interneurons for

Haisong Jiang, Rong Chen, Ted M. Dawson and Valina L. DawsonJin-Chong Xu, Jing Fan, Xueqing Wang, Stephen M. Eacker, Tae-In Kam, Li Chen, Xiling Yin, Juehua Zhu, Zhikai Chi,

DOI: 10.1126/scitranslmed.aad0623, 333ra48333ra48.8Sci Transl Med

of cortical neurons.These cultures can be used to study the mechanisms of neurotoxicity in human disorders that involve the demise

dependent manner.− and poly(ADP-ribose) polymerase-1−show that human cortical neurons die in a nitric oxide derived from human embryonic stem cells or human inducible pluripotent stem cells. Using this new method, they

describe a method to culture human neurons with a representative ratio of both excitatory and inhibitory neurons . nowet alhuman neuronal cultures that exhibit a balanced network of excitatory and inhibitory synapses. Xu

The study of the mechanisms of cell death in human cortical neurons has been hampered by the lack ofInsights into neuronal cell death

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