Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.

To address these problems, we here developed approaches that allow rapid and reproducible production of human iN cells from ESCs or iPSCs. The experiments we describe utilize a renewable resource and are scalable and result in iN cells with reproducible properties that are independent of the starting ESC or iPSC line. Strikingly, our approach requires only a single transcription factor and generates large amounts of human iN cells with robust synapse formation capabilities. Moreover, we demonstrate that the resulting iN cells can be used for analysis of human neuronal short-term plasticity, large-scale Ca 2+ -imaging, or analysis of loss-of-function states mimicking a human genetic disorder. Thus, the approach we describe may be generally useful not only to explore the cellular phenotype associated with neuropsychiatric disorders, but also for drug screening endeavors and for mechanistic studies.

The two major limitations of current technologies for generating human neurons outlined above motivated us and others to develop methods for direct conversion of human fibroblasts into induced neurons, referred to as iN cells (). Although these efforts were successful and allow rapid production of human iN cells, all of the currently available protocols for generating human iN cells (as opposed to mouse iN cells) suffer from relatively low yields and low efficiency and are further hampered by the limited availability and renewability of fibroblasts as starting materials. Moreover, the resulting iN cells often exhibited decreased competence for synapse formation. Specifically, we () and others () found that the same three transcription factors that convert mouse fibroblasts into iN cells (Brn2, Ascl1, and MytL1;) also transdifferentiate human fibroblasts into iN cells when combined with a fourth transcription factor (NeuroD1), a process that may be additionally facilitated by coexpression of microRNAs (). However, apart from the limited capabilities of iN cells produced by these procedures, these experiments did not clarify the minimal requirement of defined factors for transdifferentiating human nonneuronal cells into neurons and suggested that ancillary factors, such as specific culture conditions, may introduce further variability into these transdifferentiation protocols. Together, these features make analysis of disease-related phenotypes using human iN cells difficult, especially since these protocols do not generate large amounts of iN cells that are fully competent to form synapses.

The second limitation is related to the cumbersome, variable, and slow procedures needed for deriving neurons with functional properties from ESCs or iPSCs. Generating neurons by differentiation of ESCs or iPSCs requires months of tissue culture procedures and renders large-scale studies difficult (). Moreover, differentiation of ESCs and iPSCs into neurons depends on specific environmental factors such as pharmacological agents and bioactive proteins that may be difficult to obtain with a consistent composition, injecting a further element of variation ().

The first limitation is based on characteristic differences between particular pluripotent cell lines (). These differences influence the properties of the neurons that are derived from these lines. For example, neurons derived by the same protocol from two different ESC lines exhibited quite distinct properties (). Moreover, ESC and iPSC lines may change as a function of time in culture (). A comparison of the neural differentiation potential of different ESC and iPSC lines revealed a large variation in differentiation efficiency, and it is likely that maturation stages and functional properties of the resulting neurons are also variable ().

The generation of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and their in vitro differentiation into potentially any desired cell type hold great promise and may revolutionize the study of human disease (). Given the lack of alternative sources, a major effort has been directed toward the development of differentiation protocols that convert pluripotent stem cells into neurons to allow examination of healthy human neurons and of neurons derived from patients with a variety of neurological diseases. In this approach, fibroblasts from patients with poorly understood diseases—such as schizophrenia or Alzheimer’s disease—are converted into iPSCs that are then differentiated into neurons to study the pathogenesis of these diseases (reviewed in). Moreover, elegant studies have described differentiation protocols that produce distinct types of neurons in vitro, although the number and properties of different types of human neurons in situ are largely unknown and are only now beginning to be defined. Overall, these studies suggest that derivation of neurons from human stem cells may allow scientists to examine specific subtypes of neurons, to generate human neurons for regenerative medicine, and to investigate changes in human neurons in neuropsychiatric disorders (e.g., see). However, this approach of studying human neurons at present suffers from two major limitations.

Immunofluorescence staining revealed that the injected iN cells had dispersed throughout the striatum and formed extensive dendritic arborizations ( Figure 6 A). Numerous EGFP-positive processes were found throughout the striatum and extending through the corpus callosum into the nontransplanted hemisphere. The human iN cells were selectively labeled by antibodies to human nuclei ( Figure 6 B), human NCAM ( Figure 6 C), and NeuN ( Figure 6 D). Electrophysiological recordings from acute slices in current-clamp mode showed that the transplanted iN cells exhibited a resting potential of ∼−60 mV, fired trains of action potentials when injected with current, and displayed a near physiological action potential firing threshold and action potential amplitude ( Figures 6 E and 6F). Moreover, recordings in voltage-clamp mode demonstrated that the transplanted neurons received highly active spontaneous inhibitory synaptic inputs as documented by the blockade of the synaptic events by picrotoxin ( Figure 6 G). Inhibitory synaptic events would be expected given the preponderance of inhibitory medium spiny neurons in the striatum. Accordingly, inhibitory postsynaptic currents could also be elicited by extracellular stimulation, confirming that the transplanted neurons received abundant inhibitory synaptic inputs from the surrounding neurons in the striatum ( Figure 6 H).

(E) Example traces showing action potential generation (upper traces) in response to current pulses (lower traces) applied with 10 pA step sizes. For the recording configuration from transplanted iN cells, see Figure S6

To probe the competence of Ngn2-induced iN cells to form synapses in vivo and not only in vitro, we injected EGFP-labeled iN cells on day 6 into the striatum of newborn mice (postnatal day 2) and analyzed the mouse brains 6 weeks later.

Finally, we examined whether iN cells can potentially be used to monitor a disease state. We produced a knockdown (KD) of Munc18-1, resulting in a ∼75% decrease in Munc18-1 mRNA levels ( Figure 5 G). Heterozygous loss-of-function mutations of Munc18-1 (gene symbol STXBP1) have been associated not only with severe infantile epileptic encephalopathies (Ohtahara and West syndromes), but also with moderate to severe cognitive impairment and nonsyndromic epilepsy, suggesting that the functions of human neurons are very sensitive to Munc18-1 levels (). Strikingly, KD of Munc18-1 in human iN cells, such that Munc18-1 levels are decreased but not abolished, led to a major decrease in the frequency but not the amplitude of spontaneous EPSCs, which based on their size probably represent mEPSCs ( Figure 5 H). Moreover, KD of Munc18-1 caused a > 50% decrease in evoked EPSCs in iN cells ( Figure 5 I). Thus, decreasing the Munc18-1 levels in human iN cells produces a major phenotype consistent with the deleterious phenotype observed in heterozygous loss-of-function mutations observed in Ohtahara syndrome.

In another experiment, we tested whether synapses in Ngn2 produced iN cells can be modulated. Recent studies revealed that retinoic acid rapidly upregulates postsynaptic AMPA-type glutamate receptors via a direct synaptic action and that this signaling pathway is activated by activity blockade (). We thus explored the ability of exogenously applied retinoic acid to upregulate postsynaptic glutamate receptors ( Figure 5 F). Indeed, acute treatment (∼45–90 min) of iN cells with retinoic acid significantly enhanced the amplitude of postsynaptic mEPSCs that are mediated by AMPA-type glutamate receptors without changing the frequency of mEPSCs, demonstrating that the retinoic acid-dependent synaptic signaling pathway is operational in iN cells and thus also applies to humans. The effect was equally observed with iN cells derived from H1 ESCs and with iN cells derived from two different iPSC lines ( Figures 5 F and S5 ).

We then examined the possibility of monitoring activity-dependent Catransients in entire populations of iN cells using the genetically expressed Casensor gCamp6M, which is an advanced version of gCamp5 (). We found that Catransients induced even by single isolated action potentials could be detected in our iN cells ( Figure 5 D). The amplitude of the Casignal correlated well with the number of action potentials elicited. Conversely, when we cocultured iN cells with mouse neurons, we observed typical network activity in iN cells that was induced by addition of the GABA-receptor blocker picrotoxin ( Figure 5 E). These Caimaging examples demonstrate that it is possible to use iN cells for monitoring network activity of iN cells over larger populations of cells, for example during drug screening projects.

To explore the potential use of ESC- or iPSC-derived iN cells for monitoring drug activities, studying human synaptic plasticity, or modeling human disease states, we examined Ngn2 iN cells in a variety of paradigms. We first tested the use of optogenetics to directly probe the formation of presynaptic specializations of iN cells onto cocultured mouse neurons ( Figures 5 A–5C). When we selectively expressed the channelrhodopsin variant oChiEF in iN cells and cocultured the iN cells with mouse neurons, we found that this approach led to an accurate definition of presynaptic function in the human iN cells that allows measurement of synaptic transmission between two connected neurons without the need to separately patch these neurons.

(G–I) Effect of a Munc18-1 loss-of-function on synaptic transmission in human iN cells. (G) Quantification of the Munc18-1 knockdown (KD) efficiency in iN cells. Munc18-1 mRNA levels were measured by quantitative RT-PCR in conrol iN cells and iN cells infected with Munc18-1 KD lentivirus, and normalized to MAP2 as an endogenous control (n = 3 independent experiments). (H) Representative traces of spontaneous EPSCs monitored in control and Munc18-1 KD iN cells from H1 ESCs (left), and quantifications of the frequency (center) and amplitudes of spontaneous EPSCs (right). Numbers of cells/ independent experiments performed are indicated. (I) Representative traces of evoked EPSCs of control and Munc18-1 KD iN cells derived from H1 cells (left), and quantification of spontaneous EPSCs amplitudes (right). Numbers of cells/independent experiments performed are shown in the bars. Data are means ± SEMs; statistical significance was assessed by Student’s t test ( * p < 0.05; *** p < 0.001).

(F) iN cells derived from H1 ESCs or two different iPSC lines exhibit retinoic acid (RA)-dependent increases in synaptic strength as a model of homeostatic plasticity. iN cells were incubated with 2 μM RA for 45–90 min, and the amplitude of spontaneous miniature EPSCs (mEPSCs) was recorded in TTX. Data shown are means ± SEMs. Statistical significance was assessed by Student’s t test ( ** p < 0.01; *** p < 0.001).

(D and E) Ca 2+ imaging of human iN cells. (D) Representative images of GCaMP6M-expressing iN cells cultured alone (top) or (E) of cocultured with cortical primary neurons (top) in the absence (left panels) or presence of a Ca 2+ signal (right panels). Traces of Ca 2+ signals induced by field stimulation (D; indicated action potential (AP) inducing pulses were delivered at 50 Hz) or by network activity triggered by 0.1 mM picrotoxin (E) monitored in these iN cells are shown on the bottom.

(A–C) Optogenetic mapping of functional presynaptic specializations formed by iN cells onto cocultured cortical mouse neurons. (A) Strategy for the generation of channelrhodopsin-expressing iN cells (top) and combined tdTomato fluorescence and DIC images (overview) or DIC images only (images of patched mouse neurons and iN cells) to illustrate the selectively patching human iN cells or mouse neurons (bottom). ESCs were coinfected with lentivirus expressing the channelrhodopsin variant oChiEF as a td-Tomato fusion protein at the time of Ngn2 transduction. (B) Synaptic responses triggered by presynaptic optogenetic stimulation of iN cells and monitored in postsynaptic mouse neurons (top, representative traces; bottom, summary graph of the evoked EPSC amplitudes). Responses were triggered by 3 ms blue light pulses without or with 0.5 μM TTX (to block presynaptic action potentials induced by channelrhodopsin). (C) Channelrhodopsin-mediated presynaptic depolarizations monitored in human iN cells (top, representative traces; bottom, summary graph of the light-evoked EPSC). As in (B), responses were triggered by light pulses in the absence or presence of 0.5 μM TTX, but TTX has no effect because the recorded current is directly induced by channelrhodopsin activation which is not inhibited by TTX.

Stimulus trains of 10 Hz revealed fast synaptic depression, showing that iN cell synapses exhibit short-term plasticity ( Figure 4 H). No inhibitory synaptic events were observed when Ngn2-induced human iN cells were cocultured with glia cells, but strong inhibitory synaptic inputs onto the iN cells were detected when we cocultured iN cells with mouse cortical neurons ( Figures S4 C–S4E). This experiment demonstrated that iN cells integrate into a synaptic network with the mouse cortical neurons and that they are fully capable of forming inhibitory postsynaptic specializations. Quantifications showed that the vast majority of all iN cells, when cocultured with mouse glia cells or cortical neurons, contained voltage-gated Naand Kcurrents, exhibited spontaneous synaptic activity, and displayed evoked EPSCs ( Figure 4 I).

We next probed the ability of Ngn2-induced iN cells to differentiate into electrophysiologically active neurons and to form synapses. To promote synapse formation, we cocultured iN cells with mouse glial cells (). The iN cells reliably produced robust action potentials, and exhibited voltage-gated Naand Kcurrents that were indistinguishable between iN cells derived from H1 ESC and different iPSC lines ( Figures 4 A–4C and S4 A). iN cells exhibited massive spontaneous synaptic activity that was blocked by the AMPA-receptor antagonist CNQX ( Figure 4 D). Extracellular stimulation evoked EPSCs of large amplitudes, documenting abundant synapse formation ( Figures 4 E and 4F). The kinetics of evoked EPSCs were identical at −70 mV and +40 mV holding potentials, and EPSCs were blocked by CNQX at both holding potentials. Thus, consistent with the gene expression profile described in Figure 3 A, EPSCs are entirely due to activation of AMPA-type and not of NMDA-type glutamate receptors, although we did observe small NMDA-receptor mediated synaptic currents in iN cells after more than 3 weeks of culture ( Figure S4 B). When we quantified the frequency and amplitude of spontaneous EPSCs and the amplitude of evoked EPSCs, we found that they were indistinguishable between iN cells derived from H1 ESCs and two different iPSC lines and were reproducible between experiments ( Figure 4 G).

(I) Quantification of the rate of successful observations of voltage-gated Na + currents, spontaneous EPSCs (sEPSCs), and evoked EPSCs (eEPSCs) in iN cells. Numbers in top bars indicate the number of cells/independent experiments performed.

(G) Quantification of the frequency (left panel) and amplitude of spontaneous EPSCs (middle panel) and of the amplitude of evoked EPSCs (right panel). Data are means ± SEM; numbers in the left bars indicate the number of cells/independent experiments performed, and apply to all panels. Note that iN cells derived from different ESC/iPSC lines exhibit quantitatively similar synaptic properties.

(E and F) Representative traces of evoked EPSCs monitored at −70 mV (E) and at +40 mV (F); lower panels show that CNQX completely blocks all EPSCs. Two superimposed traces are shown.

(C) Quantification of I/V curves of Na + and K + currents in iN cells derived from H1 ESCs and from two different iPSC lines. Data are means ± SEMs; numbers of cells/cultures analyzed are shown in the lower-right corner.

(B) Representative traces of whole-cell voltage-clamp Na + and K + currents recorded in iN cells. iN cells were subjected to 10 mV step depolarizations from −90 mV to +50 mV at a −70 mV holding potential (pipette solution (in mM): 123 K-gluconate, 10 KCl, 1 MgCl 2 , 10 HEPES-KOH pH 7.2, 1 EGTA, 0.1 CaCl 2 , 1 K 2 ATP, 0.2 Na 4 GTP, and 4 glucose).

Arguably the most important question in the production of iN cells—in fact, in the in vitro production of all human neurons—is reproducibility between lines. We therefore assessed this question for the Ngn2-based protocol in great detail. Comparison of the gene expression profiles between iN cells produced by forced differentiation of H1 ESCs and of two independent lines of iPSCs revealed a striking concordance in expression patterns ( Figures 3 B and S3 D). There was no major difference between stem cells in the expression of the genes tested. The highly similar transcriptional effects of Ngn2 indicate that forced expression of Ngn2 can override presumptive epigenetic differences between various pluripotent stem cell lines to induce differentiation of a single homogenous population of excitatory forebrain neurons.

We next aimed to gain insight into the nature of the neurons generated and, more importantly, to assess the reproducibility of Ngn2-induced production of iN cells from different ESC and iPSC lines. Toward this end, we quantitatively analyzed expression of 73 genes at the single-cell level using fluidigm-dependent mRNA measurements ( Table S1 ). All fluidigm-mediated quantitative RT-PCR assays were validated using standard curves ( Table S1 ). Analysis of more than 100 individual iN cells revealed a uniform but discrete pattern of gene expression in iN cells derived from H1 ESC and two different iPSC lines ( Figures 3 and S3 ). Specifically, Ngn2-iN cells expressed at high levels the telencephalic markers Brn-2, Cux1, and FoxG1, which are characteristic of layer 2/3 excitatory cortical neurons, but lacked other prominent forebrain transcription factors (e.g., Tbr1 and Fog2). iN cells consistently expressed AMPA-type glutamate receptors GluA1, A2, and A4, but lacked NMDA-type glutamate receptors 3 weeks after induction ( Figure 3 A). Moreover, nearly all iN cells expressed vGlut2, and approximately 20% of iN cells expressed vGlut1. iN cells highly expressed GABAreceptors but lacked the vesicular GABA transporter vGAT or the GABA-synthetic enzyme glutamate decarboxylase (GAD). Ngn2 iN cells expressed all panneuronal markers tested, but lacked expression of markers for various glia cell types or for stem cells ( Figures 3 A and S3 B). These measurements show that Ngn2 iN cells are relatively homogeneous and that they constitute excitatory neurons that express telencephalic markers suggestive of cortical layers 2/3.

(B) Comparison of gene expression profiles in iN cells differentiated from H1 ESC and two different lines of iPSCs. The plot depicts average Ct values of the genes indicated at the bottom, with a cutoff of 27 cycles (on top of the 18 cycle preamplification).

(A) Single-cell quantitative RT-PCR analysis (Fluidigm) of the expression levels of the genes indicated on the right. Expression levels (expressed as Ct values) are color coded as shown on the bottom. mRNA levels were quantified in cytoplasm aspirated from individual iN cells using patch pipettes after 3 weeks of induction.

Measurements of the yield of iN cell conversion in three stem cell lines, H1 ESCs and two different iPSC lines, showed that nearly 100% of surviving lentivirally infected ESCs and iPSCs were converted into neurons, revealing an unprecedented efficiency of conversion ( Figure 2 D). When we calculated the number of iN cells generated as a function of starting ESCs or iPSCs, we observed an apparent increase with H1 ESC-derived iN cells but not with the two iPSC-line-derived iN cells ( Figure 2 D). The increase in cell numbers in H1 ESC-derived iN cells is due to the continuing division of H1 cells after plating; iPS-cell derived iN cells do not show such increased cell numbers because they exhibit some cell death in response to culture splitting and lentiviral infection, resulting in a partial loss of the iPSCs as iN cells are being generated. Overall, these data demonstrate that forced expression of a single transcription factor—Ngn2—induces neuronal differentiation with high yield.

Immunoblotting experiments showed that the neuronal precursor cell (NPC) markers nestin and Sox2 were only detectable in the ESCs and iPSCs, whereas a series of well-established synaptic genes were only expressed in 3-week-old Ngn2 iN cells ( Figures 2 C and S2 A). Quantitative RT-PCR measurements of the expression of the NPC markers Sox2 and nestin in the first 2 weeks after Ngn2 induction revealed a transient brief increase in these markers immediately after induction, with a rapid decline in expression ( Figure S2 B). Furthermore, upon coculture with mouse astrocytes, H1-cell-derived iN cells formed synapses with each other and with cocultured COS cells expressing neuroligin-1 ( Figures S2 C–S2E). Thus, iN cell generation involves a switch from a stem cell to a neuronal gene expression phenotype with stimulation of endogenous Ngn2 expression.

We stained H1 ESCs and H1-derived iN cells at 21 days after induction for EGFP (to identify cells with viral transduction), the stem cell markers Sox2, Oct4, and Nanog, and the glial cell marker GFAP (to mark mouse astrocytes added to the iN cell culture for promotion of synapse formation). At the level of immunolabeling, expression of stem cell markers was abolished in iN cells, consistent with a conversion of H1 ESCs into iN cells ( Figure 2 A). Quantitative RT-PCR analyses revealed that iN cells expressed increased levels of endogenous Ngn2 as well as of two neuronal markers, NeuN and MAP2, whose levels were elevated ∼100-fold ( Figure 2 B). In addition, we observed an even larger induction of the expression of the transcription factors Brn2 and FoxG1, which are markers for excitatory cortical neurons ( Figure 2 B).

(E) Yield of iN cell conversion of H1 ESCs and two different iPSC lines. The percentage of EGFP-positive cells that also express the neuronal marker MAP2 after 2 or 3 weeks of conversion is shown on the left, and the yield of NeuN-positive cells at the right. NeuN-positive cell yields are calculated both in terms of the percentage of EGFP-positive cells (dark bars) or in terms of starting cell numbers (light bars). For the latter, the yield exceeds 100% for H1-derived but not iPS-derived iN cells because H1 cells still proliferate after lentiviral infection, while iPSCs do not because they are more sensitive to lentivirally induced cell death. Data are means ± SEMs (n = 3 independent experiments). For additional data, see Figure S2

(D) Representative images of H1 ESC-derived iN cells visualized via their EGFP fluorescence and immunolabeled for MAP2 or NeuN as indicated.

(C) Immunoblot analyses of proteins extracted from human postmortem cortex (hu Brain), mouse brain (m Brain), and cultured mouse glia cells (m Glia) and of proteins solubilized from iN cells that were derived from H1 ESC and two different iPSC lines (3 weeks after induction) and from the starting ESCs and iPSCs as indicated. Proteins are identified on the left (Cpx1/2, complexin-1 and −2; Syb2, synaptobrevin-2; Syt1, synaptotagmin-1; Syn1, synapsin-1; Synt1A/B, syntaxin-1A and -1B).

(B) Quantification of selected mRNA levels in H1 ESCs and in H1 ESC-derived iN cells after 2 and 3 weeks of lentiviral infection. Levels are normalized for GAPDH mRNA levels as an internal control and are shown on a logarithmic scale. Note that endogenous Ngn2 is induced ∼20-fold, and endogenous Brain-2 and FOXG1 expression is induced > 1,000-fold. Data are means ± SEM (n = 3 independent experiments).

(A) Immunofluorescence images of H1 ESCs and H1 ESC-derived iN cells (3 weeks after induction). H1 ESCs but not iN cells are positive for the ESC markers Nanog, Sox2, and Oct4, while GFAP is only present in cocultured astrocytes but not iN or ESCs.

Because the effects of NeuroD1 and Ngn2 were similar, we decided to focus only on one factor and chose Ngn2. To selectively culture only cells expressing the transcription factor, we coexpressed a puromycin resistance gene with Ngn2 (allowing us to select for cells expressing Ngn2), and we additionally coexpressed EGFP (allowing us to identify lentivirally transduced cells). In the standard protocol ( Figure 1 A), ESCs or iPSCs were plated on day −2, the cells were infected with lentiviruses on day −1, and Ngn2 expression was induced with doxycyclin on day 0. A 24 hr puromycin selection period was started on day 1, and mouse glia (primarily astrocytes) were added on day 2 to enhance synapse formation ( Figure 1 B;). Strikingly, forced Ngn2 expression converted ESCs and iPSCs into neuron-like cells in less than 1 week and produced an apparently mature neuronal morphology in less than 2 weeks ( Figures 1 C and 1D). This is faster than any currently available method for generating neurons from human ESCs or iPSCs ( Table 1 ).

* Based on 1 marker expression, ** based on multiple markers, *** based on functional data. All time periods listed are from the undifferentiated ESC state; ND: not determined.

Following our initial observation that the combined expression of Brn2, Ascl1, and MytL1 induces functional neurons from human ESCs (), we examined whether forced expression of a series of single transcription factors in ESCs and iPSCs might initiate iN cell differentiation. As in previous studies (), we used lentiviral delivery for constitutive expression of rtTA () and tetracycline-inducible expression of exogenous proteins driven by a tetO promoter. Surprisingly, we found that overexpressing either neurogenin-2 (Ngn2) or NeuroD1 alone rapidly converted ESCs and iPSCs into neuronal cells ( Figure 1 and Figure S1 , available online). Since this conversion was based on forced expression of a lineage-specific transcription factor and appears to be a direct lineage conversion similar to lineage conversion between somatic cells, we refer to the resulting neurons as iN cells as previously ().

(D) Representative images of converted iN cells from two different iPSC lines at day 6 and day 14. Note that iN cells are clearly identifiable already on day 6. For iN cells generated with NeuroD1 expression, see Figure S1

(C) Representative images illustrating the time course of the conversion of H1 ESCs into iN cells. Corresponding differential interference contrast (DIC) and GFP fluorescence pictures are shown on top and bottom.

(A) Design of lentiviral vectors for Ngn2-mediated conversion of ESCs and iPSCs to iN cells. Cells are transduced with (1) a virus expressing rtTA and (2) either a single additional virus expressing an Ngn2/EGFP/puromycin resistance gene as a fusion protein linked by P2A and T2A sequences, or with two viruses that separately express Ngn2/puromycin resistance gene and EGFP.

Discussion

2+imaging (e.g., for drug screening purposes), and for disease modeling as exemplified in our Munc18-1 KD experiments. Thus, we believe that the approach described here has the potential to enable mechanistic and translational studies on human neurons that exceed currently existing capabilities and hope that the simplicity of the approach will allow its wide dissemination. In the present study, we describe a new, highly effective method that generates a homogeneous population of iN cells by forced expression of a single transcription factor in ESCs or iPSCs. We demonstrate that the new method results in the reproducible generation of the same type of neuron with quantitatively the same properties independent of the ESC or iPSC line used. The entire procedure generates iN cells in only a few weeks, allowing a rapid turnaround of experiments, and the resulting iN cells exhibit short-term plasticity, are modulated at the level of their synapses, and integrate into neuronal networks when transplanted into the mouse brain. Moreover, the new iN cells can be used for studying synaptic properties including plasticity, for large-scale Caimaging (e.g., for drug screening purposes), and for disease modeling as exemplified in our Munc18-1 KD experiments. Thus, we believe that the approach described here has the potential to enable mechanistic and translational studies on human neurons that exceed currently existing capabilities and hope that the simplicity of the approach will allow its wide dissemination. Table 1 shows a comparison of the properties of the method described here with selected other widely used methods to illustrate the advantages and disadvantages of the various approaches that have been described.

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Wernig M. Induced neuronal cells: how to make and define a neuron. We do not at present understand what determines the ability of a neuron to form synapses. Most neuron-like cells can produce action potentials and elaborate postsynaptic specializations—even nonneuronal cells can be made to generate postsynaptic specializations by simple expression of postsynaptic cell-adhesion molecules ()—but the ability to form presynaptic specializations seems to be specific for a “real” neuron (). Ngn2-induced iN cells form robust synapses among themselves when cultured in the presence of mouse astrocytes (which supply unknown synaptogenic factors) in a manner that was not previously observed for any human iN cells and occurs much faster than during conventional guided ESC/iPSC differentiation approaches. The synapses that are thus generated exhibit full function and are capable of short-term plasticity and direct modulation by retinoic acid, suggesting that Ngn2 iN cells provide a useful system for studies in which the effects of mutations or pharmacological agents on synaptic transmission in human neurons is investigated.