Investigating synapse formation and function using human pluripotent stem cell-derived

Ji-Eun Kima, Matthew L. O’Sullivana, Christopher A. Sancheza, Minju Hwanga, Mason A. Israelb, Kristen Brennandc, Thomas J. Deerinckd, Lawrence S. B. Goldsteinb, Fred H. Gagec, Mark H. Ellismand, and Anirvan Ghosha,1

aNeurobiology Section, Division of Biological Sciences, bDepartment of Cellular and Molecular Medicine, dNational Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California at San Diego, La Jolla, CA 92093; and cLaboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037

Edited by Charles F. Stevens, Salk Institute for Biological Studies, La Jolla, CA, and approved December 2, 2010 (received for review June 1, 2010)

A major goal of stem-cell research is to identify conditions that them to signals that are known to drive neural tube cells to ac- reliably regulate their differentiation into specific cell types. quire forebrain fates, and then to induce terminal differentiation This goal is particularly important for human stem cells if they to study synaptic function. We cultured HUES9 hES cells that are to be used for in vivo transplantation or as a platform for drug were generated by Douglas Melton and colleagues (14). We also development. Here we describe the establishment of procedures used in parallel a polyclonal subline that was generated with to direct the differentiation of human embryonic stem cells and a CMV promoter-driven EGFP (HUES9-CMV-EGFP) lentivirus human induced pluripotent stem cells into forebrain neurons that (15). Undifferentiated HUES9-CMV-EGFP cells are karyotypi- are capable of forming synaptic connections. In addition, HEK293T cally stable up to passage 50 (Fig. S1 A and B) and express GFP, cells expressing Neuroligin (NLGN) 3 and NLGN4, but not those OCT4, and Alkaline phosphatase (Fig. S1C). We adapted a forced containing -associated mutations, are able to induce pre- aggregation protocol to efficiently generate embryoid bodies (EBs) synaptic differentiation in human induced pluripotent stem cell- of more uniform sizes (16, 17). We preincubated undifferentiated derived neurons. We show that a mutant NLGN4 containing an in- feeder-free hES cells with the ρ-kinase inhibitor Y-27632 (5 μM), frame deletion is unable to localize correctly to the cell surface and then gently triturated to single cells in a serum-free medium when overexpressed and fails to enhance synapse formation in containing both Y-27632 and recombinant Noggin (200 ng/mL).

human induced pluripotent stem cell-derived neurons. These find- Antagonism of Bone morphogenetic protein (BMP) signaling by ings establish human pluripotent stem cell-derived neurons as a Noggin has been shown to be effective in the neural induction of – viable model for the study of synaptic differentiation and func- hES cells (18 20). In our experiments, mouse recombinant Noggin tion under normal and disorder-associated conditions. produced the most consistent results; however, its prolonged ap- plication in culture was cost-prohibitive. The small molecule dor- neural differentiation | disorders | somorphin was also reported to be an effective BMP antagonist for neural induction (21, 22) and we were able to partially substitute it for Noggin in later experiments (SI Materials and Methods). revious reports have described the differentiation of human – Cells were force-aggregated in either V-bottom 96-well plates PES (hES) cells into neurons (1 6), but they have not directly or AggreWell plates by centrifugation in serum-free conditions explored the kinds of in vitro experiments that may be carried (Fig. 1 A, C–E). The next day, the aggregates formed brownish out using such neurons. In addition, whether human ES and cellular clumps, which were then transferred to a bacterial-grade induced pluripotent stem (hiPS) cell-derived neurons follow the Petri dish for growth in suspension culture for 1 to 2 d. Cystic EBs same differentiation programs has not been extensively exam- were not observed at this time. After attachment to a Matrigel ined. We sought to devise a method to direct the differentiation substrate, the EBs spread out (Fig. 1 F and G), and as early as 24 h of hES and hiPS cells to anterior forebrain fates, as many neu- after plating, the cells in the EB self-organized into neuro- rodevelopmental and cognitive disorders, such as autism and epithelial structures known as rosettes. For HUES9 cells, greater schizophrenia, affect forebrain function. Current methodologies than 95% of EBs generated this way formed rosettes, regardless are often adapted from mouse ES cell cultures and range from of the presence of Noggin. Although Noggin was not required for cocultures with a stromal cell line (7, 8) to application of – – the initial formation of the rosettes in the EBs, withdrawal of recombinant factors and small molecules (3 5, 9 12). We show Noggin resulted in dissolution of rosette structures and flattening in the present study that we were able to derive forebrain neural of cells, and therefore, Noggin was continuously added until the progenitor cells (NPCs) from both hES and hiPS cell lines, and rosettes were clearly visible for manual dissection (Fig. 1J). that these NPCs can subsequently differentiate into electro- We used RT-PCR analysis to analyze the influence of culture physiologically functional neurons. Furthermore, we applied conditions on the expression of region- and cell type-specific such hiPS cell-derived forebrain neurons in a bioassay to test the genes. PCR primers were validated using 7- or 12-wk-old human synapse-inducing ability of Neuroligins. Neuroligins comprise whole-embryo RNA extracts as positive controls. Many EB dif- a small family of transmembrane synaptic-cell adhesion mole- ferentiation protocols use medium containing FBS or knockout fi cules that are known for their ability to induce arti cial synapses serum replacement (KSR). The presence of KSR up-regulated in heterologous nonneuronal cells and to modulate synapse mesodermal Brachyury expression and significantly hampered numbers when overexpressed in rodent neurons (13). We used expression of the neural markers SOX1 and PAX6 (Fig. 1B). this artificial synapse formation assay to compare the synapto- genic potential of two X-linked Neuroligin mutants that have fi been identi ed in rare autistic families. In sum, human neurons Author contributions: J.-E.K. and A.G. designed research; J.-E.K., M.L.O., C.A.S., M.H., differentiated in vitro from human pluripotent stem cells may M.A.I., K.B., and T.J.D. performed research; L.S.B.G., F.H.G., and M.H.E. contributed new serve as a useful culture system to study the functions of synaptic reagents/analytic tools; J.-E.K., M.L.O., C.A.S., M.A.I., and K.B. analyzed data; and J.-E.K. molecules implicated in neurodevelopmental disorders. and A.G. wrote the paper. The authors declare no conflict of interest. Results This article is a PNAS Direct Submission. Derivation of Neural Progenitor Cells Using a Spin Embryoid Body 1To whom correspondence should be addressed. E-mail: [email protected]. Protocol. Our general strategy to direct the differentiation of hES This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cells into forebrain neural progenitors (Fig.1A) was to expose 1073/pnas.1007753108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1007753108 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 up-regulated in Noggin-treated EBs compared with same-stage controls (Fig. 1B).

Monolayer Cultures of Neural Progenitor Cells. To obtain neural progenitor cells from the EB-derived rosettes, we dissociated the rosettes into single cells with accutase, and replated them on a polyornithine and substrate in the presence of FGF2 and EGF, which are known to promote the proliferation of neural progenitor cells (Fig. 1H). The presence of Noggin in these cul- tures increased the proportion of cells expressing FORSE-1 (Fig. 1K), an antigen associated with radial glial progenitors that give rise to neurons and in the forebrain (24). The onset of neural differentiation in these cultures was assessed by expression of the -specific class III β-tubulin antibody TuJ1. Noggin-treated EBs contained greater proportions of TuJ1+ neurons differenti- ated after withdrawal of FGF2 (Fig. 1L). To assess the transition of EBs to NPCs in terms of gene ex- pression, we carried out RT-PCR on RNA samples isolated at three distinct stages of differentiation: undifferentiated hES cells, EBs cultured in the presence of Noggin for 7 d, and NPCs derived from rosettes (Fig. 1M). NANOG expression decreased dramat- ically with differentiation and was almost undetectable in NPCs. OCT4 expression was markedly reduced but still present at the NPC stage. SOX2 was expressed at the EB stage in neural pro- genitors as they are actively dividing, but was down-regulated in the dissociated NPCs. Expression of the neuroectodermal marker NCAM increased with differentiation, whereas the early meso- dermal marker BRACHYURY and endodermal marker AFP were not expressed. SOX1 is the earliest neuroectodermal marker detected in the early neural tube and it was highly up-regulated in EBs and NPCs. Dorsal forebrain markers, such as PAX6, EMX2, and DACH1 showed high expression in both EBs and NPCs. Expression of SIX3, one of the most anterior markers found in the neural tube, was high at the EB stage but absent in NPCs. Fig. 1. Generation of forebrain neural precursors from hES cells. (A) Schematic HOXB4, a hindbrain marker, was not detected. outline of the spin EB method. (B) RT-PCR panel showing the effect of serum removal and addition of Noggin to the differentiation medium. Spin EBs Dorso-Ventral Patterning of NPCs. Strong expression of dorsal but generated in the absence of serum and in the presence of 200 ng/mL Noggin not ventral telencephalic markers led us to question whether – show enhanced expression of anterior forebrain markers. (C H) Generation ventral cell types could be further patterned in the these NPCs. and plating of spin EBs: (C) hES cells were centrifuged in an AggreWell; (D) ∼24 h later, spherical hES cell aggregates formed; (E) hES cell aggregates were Addition of cyclopamine, which was shown to enhance expression cultured as free-floating EBs for a day; (F) spin EBs were replated onto growth of dorsal markers in mouse ES cell-derived neurons (11), did not factor-reduced Matrigel-coated plates (1 d later, the spin EB flattened out); (H) significantly alter gene-expression patterns, indicating that the 2 d after plating, the EB spread out even more; (H) attached EBs were dissoci- NPCs obtained through our protocol had already acquired ated with accutase and cells were replated on poly-L-ornithine/laminin–coated a dorsal forebrain identity (Fig. S2). To explore the possibility of plates. (I) Relative expression of OCT4 and SOX1 (measured by quantitative ventral patterning in our NPCs, we treated EBs starting from the RT-PCR) in spin EBs compared with undifferentiated hES cells, grown in the day of EB attachment with purmorphamine, an activator of the absence or presence of Noggin. Noggin-treated EBs showed a threefold in- < Sonic hedgehog (SHH) signaling pathway (25). The forebrain crease in SOX1 expression compared with nontreated EBs (*P 0.05). Data were marker BF1(FOXG1) was expressed at the NPC stage. In pur- averaged from four independent experiments. (J) Attached EBs treated with Noggin maintain compact rosette morphology, whereas control EBs show areas morphamine-treated samples, the dorsal markers PAX6, EMX2, of flattened cells. (K) Nestin-positive NPCs generated from Noggin-treated spin and GLI3 were suppressed, but the ventral markers SHH and EBs had up-regulated expression of forebrain progenitor marker FORSE-1. (L) NKX2.1 were induced at both the EB and NPC stages. This Spin EBs differentiated with Noggin contain greater numbers of neuronal class finding shows that in our protocol, the cells acquired an anterior III β-tubulin–expressing neurons after 4 wk in culture. (M) RT-PCR panel dorsal forebrain character by a default pathway, and that ventral showing differential expression of various markers at the undifferentiated ES, cell types could be induced simply by adding a SHH agonist at the rosette-bearing EB, and monolayer NPC stages of differentiation. BRACHY, EB stage. Brachyury. Terminal Differentiation of NPCs. To determine if hES cell-derived forebrain NPCs can differentiate into neurons, we cultured the In early embryogenesis, BMP4 stimulates differentiation of the NPCs at low density on a rat primary glial feeder layer (Fig. S3). overlying ectodermal tissue. Inhibition of the BMP4 signal by The hES cell-derived cells could be distinguished from rat cells molecules, such as Noggin, Chordin, and Follistatin, causes the based on GFP expression. GFP+ cells changed morphology over ectoderm to differentiate into the neural plate (23). Although time and by 3 wk many of them had extended and den- BMP4 expression remained high in differentiating hES cells, ad- drites, although some cells remained in clusters with a flattened dition of Noggin promoted the conversion of undifferentiated morphology (Fig. S3A). Cells expressing the neuronal progenitor HUES9 cells into SOX1-expressing neuroectoderm-like cells and marker nestin, as well as cells expressing the immature neuronal helped maintain compact rosette morphology compared with marker TuJ1 could be detected in these clusters (Fig. S3 B and nontreated EBs (Fig. 1 I and J). The neural progenitor marker C). Some weak human-specific GFAP expression was observed PAX6, as well as the forebrain markers DACH1 and EMX2, were in the clusters, suggestive of glial differentiation.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1007753108 Kim et al. Downloaded by guest on September 26, 2021 To test whether hES cell-derived forebrain neurons (hES- Generation of hiPS Cell Lines. We next investigated whether the neurons) formed synaptic connections, we cocultured them with procedures to generate forebrain neurons from hES cells could be E18 rat cortical neurons. After 5 wk in the coculture, the elec- applied to hiPS cells, as they may be used in studies investigating trophysiological characteristics of hES-neurons (identified by molecular mechanisms of human neurological disease (26, 27). We generated two independently derived hiPS cell lines. The fluorescence) were assessed by patch clamp recordings (Fig. S4A). “8011” line was generated from skin-biopsy fibroblasts from Voltage clamp recordings showed voltage-sensitive inward and an 86-y-old healthy donor using retroviral expression of four outward currents with activation and inactivation kinetics char- reprogramming factors discovered by the groups of Yamanaka and + + acteristic of neuronal Na and K currents (Fig. S4 B and B′). colleagues (26). These cells are karyotypically stable and plurip- Current clamp recordings showed a train of adapting action otent (Fig. S5). The “BJiPS#1” line was generated using human potentials in response to depolarizing current injection (Fig. S4C). foreskin BJ fibroblasts using doxycycline-inducible lentiviruses Spontaneous spiking was observed as biphasic inward and out- expressing five reprogramming factors (Fig. S6). Their pluri- E ward transmembrane currents in cell-attached configuration (Fig. potency was assessed by their ability to form teratomas (Fig. S6 ). S4 D and D′). Finally, voltage clamp recordings demonstrated Neural Differentiation of hiPS Cell Lines Using the Spin EB Protocol. spontaneous inward (excitatory) and outward (inhibitory) synap- + − fi Using our spin EB protocol, we were able to generate SOX1 tic currents, at Vhold of 80 and 0 mV, respectively, con rming NPCs from both hiPS cell lines. However, rosette formation in the integration into synaptic networks (Fig. S4E). Thus, hES-neurons hiPS cells appeared to be delayed by 2 to 3 d compared with the are able to fire action potentials and receive synaptic inputs from HUES9 cells, and the addition of 200 ng/mL Dkk1, an inhibitor of the host network. Wnt signaling that has been shown to induce neural differentia- NEUROSCIENCE

Fig. 2. Characterization and comparison of hES- and hiPS cell-derived neurons. Human iPS cell- derived NPCs were infected with human promoter-driven GFP lentivirus 2 d before replating on rat glia. (A) GFP (green) staining colocalized with MAP2 (red) expression. (B) Scanning electron micrographs showing detailed external morpho- logical features of the 8011 hiPS-neurons. Compar- ison of voltage-dependent Na+ and K+ currents of hES-neurons (C–H′) and hiPS-neurons (I–N′). (C and I) (i) Rapidly activating and inactivating inward cur- rents flowing in response to 500-ms depolarizing square voltage steps from −80 mV to −100, −60, −40, −20, 0, 20, 40, and 60 mV. (ii). Local perfusion with the selective voltage-gated Na+ channel an- tagonist TTX (1 μM) abolishes fast inward currents. (iii). Subtraction of ii from i gives TTX-sensitive cur- rent. (D and J)(i) Recording in K+ internal solution reveals large, sustained outward depolarization- activated currents. (ii) Local perfusion with the broad-spectrum K+ channel antagonist TEA (10 mM) blocks the majority of the depolarization-activated outward currents. (iii) Subtraction of ii from i gives the TEA-sensitive current. (E, F, K, and L) I-V plot of TTX- (E and K) and TEA- (F and L) sensitive currents. The calculated reversal potential of Na+ is shown by the gray point in E and K.(G) Whole-cell current clamp recording from an hES-neuron with 500-ms current steps of −20, 0, +30, and +50 pA. (M) Whole- cell current clamp recording from an hiPS-neuron with 500-ms current steps of −10, 0, +10, and +20 pA. (H and N) Cell-attached recordings from an hES- neuron (H) and hiPS-neuron (N) demonstrating spontaneous firing. The boxed regions in H and N are shown on a different scale in H′ and N′, respectively.

Kim et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 tion (9), appeared to enhance the formation of rosettes. Differ- ences were also noted in the extent of marker expression between the two lines (Fig. S7). For both lines, addition of Noggin en- hanced expression of SOX1, PAX6, and GLI3; however, expres- sion of these genes in the BJiPS#1 line was weaker at the NPC stage compared with the 8011 line. This finding could indicate that there may be an intrinsic preprogramming of the 8011 cells to generate more dorsal cell types. Intrinsic differentiation potential of individual lines has been documented among various hES and hiPS cells (28, 29). We observed that the majority of neurons generated in 8011 iPS cell cultures were VGlut1+/MAP2+ ex- citatory neurons, but the proportion of GABA+/MAP2+ neurons were higher in the BJiPS#1 cultures suggesting that iPS cell- derived neurons might have differentiation biases (Fig. S8).

Electrophysiological Comparison of hES Cell-Derived Neurons and hiPS Cell-Derived Neurons. Human iPS cell-derived NPCs were infected with a lentivirus expressing a human Synapsin promoter- driven GFP (pCSC-hSyn-GFP), and could be further differen- tiated into MAP2+ neurons when cultured on rat glial feeder layers for 3 to 5 wk (Fig. 2A). Human iPS cell-derived neurons (hiPS-neurons) had elaborate dendritic arbors and many swel- lings in the axons suggestive of boutons, as observed by scanning electron microscopy (Fig. 2B). To compare the electrophysiological properties of hES- and hiPS-neurons, we recorded voltage-sensitive currents in 3-wk-old hES- and hiPS-neurons (Fig. 2 C–L). TTX-sensitive Na+ and TEA- sensitive K+ currents had similar current-voltage (I-V) relation- ships between hES-neurons (Fig. 2 C–F) and hiPS-neurons (Fig. 2 I–L). In hES-neurons, the maximal TTX-sensitive current was −1,575 ± 417.3 pA and the maximal TEA-sensitive current was 1,218 ± 382.9 pA, but for the hiPS-neurons, it was -1,329 ± 267.7 pA and 1,579 ± 304.6 pA, respectively. Both hES-neurons (Fig. 2G) and hiPS-neurons (Fig. 2M) were able to fire trains of action potentials in response to depolarizing Fig. 3. Synapse formation in hiPS-neurons. (A) An example of a MAP2+ somatic current injection. Ten of 13 (76.9%) hES-neurons under neuron derived from the 8011 hiPS cell line. (B) Enlarged view of current clamp fired one or more overshooting action potentials, from three different cells stained with Synapsin antibodies. (C) Transmission and six of them (46.2%) fired a train of two or more action electron micrograph of an terminal (axo) in close apposition to a den- potentials. Similarly, 14 of 16 (87.5%) hiPS-neurons fired one or drite (den) in hiPS-neuron cultures at 6 wk. Note the presence of numerous more overshooting action potentials and eight of them (50.0%) synaptic vesicles (SV) in the . (D) RT-PCR analysis of neuronal and glial markers in NPCs (lane 1) and in 3-wk-old (lane 2) and 5-wk-old (lane fired a train of two or more action potentials. Starting at around 5 fi fi 3) hiPS-neurons. Expression of MAP2, Synaptotagmin1 (SYT1), and PSD95 is wk in culture, spontaneous ring in the cell-attached con gura- increased in hiPS-neuron cultures. GFAP expression is detected only in 5-wk- tion could be detected in a subset of neurons in the hES-neuron old cultures. (E) Human iPS-neurons can show spontaneous DNQX-sensitive (Fig. 2 H and H′) and hiPS-neuron (Fig. 2 N and N′) cultures. Two synaptic currents. (E)(i) Raster plot of sEPSC occurrence in a hiPS-neuron. of six (33.3%) hES-neurons recorded were spontaneously spiking DNQX was bath-applied to block AMPARs at the time indicated by the black at a frequency of 0.25 ± 0.14 Hz. Five of 11 (45.5%) hiPS-neurons bar, followed by a wash period. (ii) Average sEPSCs before application were spontaneously spiking at 0.95 ± 0.35 Hz. Thus, both hES can (black) and after wash (gray) of DNQX from the same experiment. (iii) μ hiPS cells can be comparably differentiated into electrophysio- Summary of sEPSC frequency before and in the presence of 20 M DNQX. Baseline frequency 0.11 ± 0.032 Hz; DNQX frequency 0 ± 0Hz;n =3;P = logically functional forebrain neurons using the protocol de- 0.027. (F–L) Induction of presynaptic differentiation in 4- to 5-wk-old hiPS- scribed here and are able to form functional connections with very neurons cultured with HEK293T cells expressing GFP together with empty similar efficiencies. vector (G), FLAG-tagged WT NLGN4 (H), or FLAG-tagged NLGN4 carrying an autism-associated deletion in exon 4 (I). Side panels show resliced z-projec- Modeling Synaptic Disorders Using hiPS-Neurons. Investigation of tions of confocal stacks through the HEK293T cells. (J) Percent area occupied synaptic maturation in hiPS-neuron cultures revealed that hiPS- by Synapsin staining in HEK293T cells. (K) Mean intensity of Synapsin neurons begin to receive more numerous presynaptic inputs at 4 staining. (L) Average cell sizes across conditions. to 5 wk in culture, as evidenced by Synapsin staining (Fig. 3 A and B). We also confirmed the presence of synaptic vesicles at sites of contact between axons and dendrites by transmission The ability of hiPS-neurons to form electrophysiologically and fi electron microscopy (Fig. 3C). As the hiPSC-derived NPCs immunologically identi able synapses suggested that they could matured into neurons in the absence of rat glia, increased ex- be used to develop in vitro models of synaptic dysfunction. In pression of synapse-associated markers, such as Synaptotagmin1 particular, we were interested in human neurological and cog- and PSD95, was detectable by RT-PCR, and glial GFAP ex- nitive diseases that are characterized by mutations in synapse- pression appeared at 5 wk, which corresponds to the time when associated genes. For example, mutations in two synapse- spontaneous activity is detectable in these cultures (Fig. 3D). associated genes, NLGN3 and NLGN4, are linked to autism Furthermore, 6,7-dinitroquinoxaline-2,3-dione (DNQX)-sensitive spectrum disorders, but the effects of these mutations in synapse spontaneous excitatory synaptic currents were detected by 6 wk, development or function are not known (13). confirming the presence of glutamatergic synapses in hiPS-neu- To determine if NLGNs regulate synapse formation in human rons (Fig. 3E). neurons, we used hiPS-neurons in an artificial synapse formation

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1007753108 Kim et al. Downloaded by guest on September 26, 2021 assay, wherein the cultured hiPS-neurons are cocultured with HEK293T cells that have been transfected with postsynaptic synapse-inducing molecules (30). We found that hiPS-neurons are indeed able to form presynaptic specializations onto HEK293T cells expressing WT NLGN4 (Fig. 3 F–L). Because the hiPS- neurons were cocultured with rat glia for most experiments, we wanted to address the possibility of some postnatal rat neurons or neuronal progenitors surviving the glial cell preparation. We maintained exclusively glial cultures in NPC neuronal differenti- ation medium and used them as controls in the assay. No hemi- synapse-inducing activity was detected in the rat glia-only cultures (Fig. 3F). Thus, we are confident that the synapsin-positive pre- synaptic contacts are made by the hiPS-neurons. Furthermore, expression of an autism-linked splice variant of NLGN4 with a skipped exon 4, which we refer hereupon as NLGN4 ΔE4 (31), was unable to induce synapses in the same assay. This finding was quantified in terms of percent-area of synapsin staining and mean intensity, which are significantly in- creased in the NLGN4-expressing cells but not in control or ΔE4 cells (Fig. 3 G–L). Skipping of exon 4 results in a 62 amino acid in-frame deletion within the esterase-like domain of NLGN4, likely causing misfolding of the protein. Confocal analysis of FLAG immunofluorescence showed a lack of surface trafficking of the FLAG-tagged mutant protein in the HEK293T cells. We also compared the effects of another autism-linked muta- tion, an R451C mutation in NLGN3 (32), which appears to cause Fig. 4. Artificial synapse formation in hiPS-neurons and rat cortical neuron aberrant protein processing and retention in the endoplasmic cultures. Human iPS-neuron cultures were differentiated 2 to 3 wk on rat glial reticulum (33). Comparison of the NLGN3 R451C and NLGN4 feeders, and E18 rat cortical cultures were cultured for 10 to 11 d before the NEUROSCIENCE ΔE4 mutants in the artificial synapse formation assay shows that assay. Then, HEK293T cells transfected with various constructs were added, there is residual synaptogenic activity in the R451C mutant in and the coculture was fixed after 24 h. Comparison of NLGN3 and NLGN4 both hiPS-neuron cultures and rat neuron cultures (Fig. 4 A and with autism-associated mutant versions in hiPS-neurons (A) and rat neurons B). Indeed, the R451C has been previously reported to be in- (B). Comparison of NLGN1, 2, 3, and 4 in hiPS-neurons (C) and rat neurons (D). correctly processed and retained in the endoplasmic reticulum, Data are averages of two experiments. Sixty to 100 cells were analyzed per with only a small fraction that reaches the cell surface and displays condition. (E–G) NLGN4 overexpression can enhance presynaptic differenti- Δ partial synaptogenic function (33–35). Finally, we compared the ation in hiPS-neurons. Overexpression of WT NLGN4 (E) but not E4 mutant synapse-inducing activity of four NLGNs. NLGN1, -3, and -4 had (F) increases Synapsin puncta density along dendrites in hiPS-neurons (G). comparable synapse-inducing abilities, whereas NLGN2, which is found selectively on inhibitory synapses (36), was the least syn- C Functional neurons with spontaneous network activity can be aptogenic in our hiPS-neuron cultures (Fig. 4 ). We performed differentiated from these NPCs in the presence of rat glial feed- experiments in parallel with rat dissociated cortical neurons, ers. Moreover, these neurons can be applied in a coculture assay which yielded similar results, but NLGN4 was not as strong to test the efficacy of synaptogenic molecules expressed on a a hemisynapse inducer as was NLGN1 or -3 (Fig. 4 B and D). nonneuronal cell type. We examined cellular localization of NLGN4 in hiPS-neurons Even though the same forebrain markers are induced in both by overexpressing the FLAG-tagged NLGN4 WT and ΔE4 pro- lines, dorsal markers are enriched in the 8011 line. This finding teins in hiPS-neurons. Three weeks following delivery of pCSC- hSyn-NLGN4 constructs, the hiPS-neurons were stained for suggests that the two iPS cell lines display a bias toward the cell types generated and that particular cell lines will be more useful MAP2, FLAG, and calreticulin, a calcium-binding protein abun- fi dant in the endoplasmic reticulum. WT NLGN4 localized to in exploring applications that require investigating a speci c cell − fi MAP2+ dendrites and also to small MAP-2 secondary branchlets type. For example, we show that carrying out the arti cial synapse in the iPS-neurons. On the other hand, the ΔE4 mutant was ex- assay in the 8011 hiPS-neurons showed that NLGN2, which has pressed at very low levels and the signal overlapped significantly been shown to primarily induce presynaptic differentiation of with calreticulin-positive intracellular compartments (Fig. S9). GABAergic axons, had very little synapse-inducing activity, pre- Finally, we quantified synaptic differentiation after overexpressing sumably because of lower numbers of inhibitory neurons in such NLGN4 WT and ΔE4 directly in hiPS-neurons. In 3-wk-old cul- cultures (Fig. S8). These observations indicate that it will be im- tures, where the synapsin density is quite low, we observed a portant to characterize the differentiation properties of hiPS cell- threefold increase in the density of Synapsin puncta made onto derived neurons before generalizing across different lines. the dendrites of hiPS-neurons overexpressing NLGN4 WT but not Rare missense mutations in NLGN4 associated with autism the ΔE4 mutant (Fig. 4 E–G). Thus, the autism-associated ΔE4 have been identified in a small number of families (38). We report mutation in NLGN4 compromises the ability of NLGN4 to induce a sensitive assay for exploring the effects of these mutations using synaptic differentiation. the hiPS-neuron coculture assay. In light of the observation that neuroligin variants associated with autism tend to misfold and fail Discussion to reach the cell surface, it will be interesting to test whether With the advent of novel reprogramming technologies, it is now endoplasmic reticulum stress leading to an unfolded protein re- possible to generate hiPS cells from individual patients carrying sponse may underlie some of the neuronal dysfunction in affected a disease mutation in their genome (27, 37). To study the effects cells. In line with the hypothesis that overload in protein pro- of these mutations, it will be necessary to differentiate the hiPS duction and clearance can underlie neuronal dysfunction in au- cells to the appropriate cell types affected in a given disease. In tism, mutations or copy number variations of genes involved in this study, we show that hES and hiPS cells can be differentiated either the mammalian Target of rapamycin (mTor) pathway (39) to neural progenitor cells of a dorsal anterior forebrain identity. or the ubiquitin-proteasome pathway (40) have been shown to be

Kim et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 strongly associated with autism. By modeling disease in patient- Materials and Methods derived hiPS cells differentiated to hiPS-neurons, we may be able Human ES/hiPS Cell Line Maintenance, Spin EB generation, NPC Cultures, and to gain insight into how individual mutant forms affect neuronal Neuronal Differentiation. For routine maintenance of HUES9 cultures, hES cells function at the synaptic level. were passaged on mouse embryonic fibroblasts and cultured according to Although hiPS-neurons behave similarly to rat cortical neu- standard guidelines (found on http://www.mcb.harvard.edu/melton/hues). fi More detailed methods on the generation and characterization of the two rons in the arti cial synapse assay, there are some noteworthy hiPS cell lines can be found in SI Materials and Methods. differences that suggest that it may be important to use hiPS- neurons in assessing synaptic defects associated with human mu- RT-PCR Analysis, Immunofluorescence, Electron Microscopy, and Electro- tations. Specifically, NLGN4 appeared to be less synaptogenic physiology. Detailed protocols can be found in SI Materials and Methods. compared with NLGN1 and -3 in the rat cortical neuron assay, Information about primers used in the RT-PCR analysis are listed in Table S1. fi although it appeared to be just as ef cient as the others in the fi C D Arti cial Synapse Formation Assay. One day before the assay, HEK293T cells hiPS-neuron assay (Fig. 4 and ). It may be possible that rat were transfected with a pcDNA3.1 vector FLAG-NLGN4 WT or ΔE4 neurons lack the complement of specific receptors that allow the mutant, together with pBOS-EGFP reporter plasmid using FuGENE 6 trans- full synaptogenic potential of NLGN4 to manifest in the assay fection reagent (Roche). The next day, HEK293T cells were dissociated to and that working with human neurons may be able to uncover a single cell suspension using accutase and replated over hiPS-neurons fi growing on coverslips that were 2 to 5 wk old. The cocultures were in- additional molecular interactions speci c for NLGN4. fi In conclusion, the procedures described here show that forced cubated 24 to 28 h before xation. Coverslips were stained with antibodies, and then imaged under confocal microscopy (Leica SP5). Image J software aggregation of human ES and iPS cell lines in serum-free me- was used to analyze confocal z-stacks. Both image acquisition and analyses dium with defined factors can restrict cells to an anterior fore- were performed blinded to the transfection conditions. neural progenitor cell fate. Uniform induction of anterior neural fate provides a useful system to study the molecular basis ACKNOWLEDGMENTS. We thank Zoë Vomberg for helpful advice on hES cell fi cultures; Peyton Paulick and Rebecca Brewster for technical assistance; and of region-speci c differentiation of human neurons. In addition, Karl Willert, who kindly provided us with the HUES9-CMV-EGFP subline. The the robustness of the artificial synapse formation assay using the NLGN4 ΔE4 mutant was cloned by Meghan T. Miller, and all NLGN constructs human iPS cell-derived neurons underscores their potential to be were a generous gift from Davide Comoletti and Palmer Taylor. This work was supported in part by the Scientific Excellence through Exploration and applied in exploring both basic biological processes and human Development (SEED) Grant (to A.G.) and a Postdoctoral Fellowship (to J.-E.K) disease mechanisms. from the California Institute for Regenerative Medicine.

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