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Generation of pure GABAergic neurons by transcription factor programming

Nature Methods volume 14, pages 621–628 (2017)Cite this article

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Abstract

Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges—(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.

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Figure 1: Rapid and efficient generation of human inhibitory iN cells with Ascl1, Dlx2 and Myt1l.
Figure 2: Transient expression of Ascl1 and Dlx2 (AD) generates GABAergic iN cells from human ES cells.
Figure 3: Functional maturation and synaptic integration of AD-iN cells in vitro and long-term stability of GABAergic fate in vivo.
Figure 4: Induced GABAergic neurons for human neurological disease modeling.

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Acknowledgements

This work was supported by the New York Stem Cell Foundation-Robertson Award (to M.W.), the NIH grants U19MH104172-01 and R01MH092931 (to M.W. and T.C.S.), HD032116 (to A.A.B.), MH049428 (to J.L.R.), P50-HG007735 (to H.Y.C.) and the DoD grant W81XWH-14-1-0307 (to M.W.). M.W. is a New York Stem Cell Foundation-Robertson Investigator, a Howard Hughes Medical Institute Faculty Scholar, and the Tashia and John Morgridge Faculty Scholar from the Child Health Research Institute at Stanford. T.C.S. is a Howard Hughes Medical Institute Investigator. N.Y. is supported by a 2014 NARSAD Young Investigator Award and a Siebel Foundation Scholarship. S.C. was supported by a postdoctoral grant (Stanford ChEM-H112878). H.A. was supported by postdoctoral fellowships from Swedish Research Council and Swedish Society for Medical Research.

Author information

Author notes

  1. Henrik Ahlenius & John L Rubenstein

    Present address: Present address: Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Division of Neurology and Lund Stem Cell Center, Lund, Sweden.,

  2. Nan Yang and Soham Chanda: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine and Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

    Nan Yang, Soham Chanda, Samuele Marro, Yi-Han Ng, Justyna A Janas, Daniel Haag, Cheen Euong Ang, Moritz Mall, Henrik Ahlenius & Marius Wernig

  2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA

    Soham Chanda & Thomas C Südhof

  3. Department of Neurological Surgery and the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, USA.,

    Yunshuo Tang, Quetzal Flores & Arturo Alvarez Buylla

  4. Program in Epithelial Biology and Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, California, USA

    Orly Wapinski & Howard Y Chang

  5. Institute for Computational and Mathematical Engineering, Stanford University School of Medicine, Stanford, California, USA

    Mavis Li

  6. Department of Psychiatry, University of California, San Francisco, San Francisco, California, USA

    John L Rubenstein

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Contributions

N.Y., S.C., T.C.S. and M.W. designed and performed the experiments and analyzed the data. S.M. helped with single-cell experiments and data analyses; Y.H.N. and J.A.J. provided experimental material and helped with biochemistry experiments. D.H. and C.E.A. helped with the disease modeling experiment. M.M. provided Myt1l antibody. H.A., Y.T., Q.F., A.A.B. and J.L.R. provided experimental material and helped with in vivo study and data analyses. O.W., M.L. and H.Y.C. helped with RNA-sequencing experiment and data analyses. N.Y., S.C. and M.W. wrote the paper.

Corresponding author

Correspondence to Marius Wernig.

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Competing interests

A.A.B. and J.L.R. are cofounders, stockholders, and currently on the scientific board of Neurona, a company studying the potential therapeutic use of interneuron transplantation.

Integrated supplementary information

Supplementary Figure 1 Identification of candidate factors for the generation of inhibitory neurons.

(a) Tuj1+ cells derived from human ES cells using different transcription factors 3 days after induction. Scale bars, 50 μm. (b) Schematic representation of the strategy to test candidate GABAergic neuron-inducing factors. (c) Example traces recorded from iN cells generated in different conditions. Left box: AMPAR-mediated spontaneous excitatory postsynaptic currents (sEPSC) recorded from an example cell (Ngn2-iN cell). Expanded view of events reveals fast kinetics (middle). Center box: GABAR-mediated spontaneous inhibitory postsynaptic currents (IPSCs, top) with slower kinetics (middle) recorded from an example cell (AM+Dlx2-iN cell). Right box: Spontaneous EPSCs with fast kinetics and spontaneous IPSCs with slower kinetics recorded from an example cell (AM+Dlx5-iN cell). (d) Representative traces of spontaneous postsynaptic currents (sPSCs) with fast kinetics as recorded from an Ngn2+EGFP-expressing neuron (red, i), or sPSCs with slow kinetics as recorded from an AM+Dlx2-expressing neuron (blue, ii), that were blocked by NBQX (50 μM, AMPAR-antagonist) or picrotoxin (50 μM, GABAAR-antagonist), respectively. Insets = expanded views of boxed areas (dotted lines). (e) Bar-graphs show the average amplitude (top panels) and τdecay (bottom panels) of sEPSCs (red) and sIPSCs (blue) in cells infected with indicated transcription-factor combinations. Bar-graphs represent mean values ± SEM (n = 5-7 cells). Filled circles represent values measured from individual cells. Asterisks (red) indicate absence of EPSC events in AM+Dlx2-expressing neurons.

Supplementary Figure 2 Transcription factor combination AMD induces the differentiation of human pluripotent stem cells into a homogenous population of GABAergic neurons with high yield and reproducibility.

(a) Representative traces of sIPSCs recorded from AMD-iN cells generated from three different human pluripotent stem cell lines. (b) AMD-iN cells generated from different cell lines show similar intrinsic properties in terms of membrane capacitance (Cm, bi), input-resistance (Rm, bii), and the average amplitude and frequency of the sIPSCs (biii and biv, respectively). Data are presented as mean ± SEM (one-way ANOVA, p > 0.05, n = 18 cells/condition), and filled circles represent values measured from individual cells.

Supplementary Figure 3 RNA-seq analysis of AMD-iN cells reveals expression of genes characteristic of different forebrain inhibitory neuron subtypes.

(a-d) Shown are human-specific RPKM expression values for the indicated genes in AMD-iN cells 32 days after transgene induction and co-cultured with mouse glia for 4 weeks. Total RNA was collected and sequenced, but only human-specific reads were considered. DA, dopaminergic; Glu, glutamatergic; ACh, cholinergic; cIN, cortical interneuron. Data are represented as mean of two biological replicates and the filled circles represent values measured from the 2 experiments.

Supplementary Figure 4 Detection of GABAergic neuron subtype markers in 5 WPI AD-iN cells.

(a) The robust generation of GABAergic neurons expressing CR, CB and SST from different human ES and iPS cells using Ascl1 and Dlx2. (b) ISL1 was observed in ~ 20% of the AD-iN cells and specific staining of NR2F2 in a small fraction of AD-iN cells (<2%) was detected (c). The bar graph shows the percentage of MAP2 positive cells that also express ISL1. Scale bar: 50 μm.

Supplementary Figure 5 Notch inhibitor enhanced the morphologic complexity of AD-iN cells.

(a) Schematic diagram represents experimental strategy for treating and characterizing cellular phenotype resulting from DAPT treatment in human neurons. (b-c) Brief DAPT treatment (3 or 4 days) at early stage during neuronal induction did not exhibit any effect on the neuronal morphology of AD induced neuronal cells revealed by MAP2 immunofluorescence. However, the mean process length of iN cells significantly increased from day 4 to day 5 after transgene induction independent of DAPT treatment. (d-g) Whereas brief DAPT treatment during day 2-7 after transgene induction was sufficient to improve the complexity of the neuron morphology in terms of the max process length and the total neurite outgrowth (e, g), continuous DAPT treatment from day 2 onwards was necessary to further increase the number of branches (d).

Supplementary Figure 6 Ascl1 and Dlx2 induction generates mature neurons that form functional inhibitory synapses.

(a) Spontaneous action potentials (APs) recorded from AD-iN cells, as replated on mouse glia at one week post-induction (WPI) and co-cultured for additional 4 (red, left) to 6 (blue, right) weeks (i); Representative traces (left) and average values (means ± SEM, n = 10 cells, right) of voltage-gated Na+ and K+ currents recorded from AD-iN cells at 6 weeks after glia co-culture (ii). (b) Immunoblot analyses of proteins extracted from AD-iN cells co-cultured with mouse glia cells or mouse glia only. Proteins are identified on the left (b-actin, GFAP, MAP2, Gephyrin, VELI, Munc18, Cask and SYT1 (Synaptotagmin-1). (c) Representative image showing vGAT and Synapsin 1 (SYN1) expression in AD-iN cells co-cultured with mouse glia. (d) Representative traces of sIPSCs (blue trace, i) and extracellular stimulation-induced evoked IPSCs (blue trace, ii), as recorded from AD-iN cells 7 WPI, and were subsequently blocked by 50 μM picrotoxin (red traces, i and ii), a pharmacological blocker of GABAARs.

Supplementary Figure 7 Continuous expression of AMD for 14 days was sufficient to induce neuronal cells with complex morphology and expressing synaptic marker SYN1.

AMD-treated human ES cells where co-cultured with mouse glia from 7 days after AMD-induction. Doxycycline was withdrawn at different time (days after AMD-induction as indicated in the upper-left corner of each image). Immunofluorescence analysis was performed on day 28.

Supplementary Figure 8 Long term stability of the GABAergic fate and continuous maturation of AD-iN cells in vivo.

(a,b) Representative image of an AD-iN cell graft in the mouse cortex 2 weeks post transplantation (w.p.t.). Transplanted cells were detected with EGFP (green) and human nuclei antibodies (blue) and expressed high levels of DCX (magenta). (b) Individual human DCX-positive cells show migrating morphologies. (c-e) After 3 months post transplantation (m.p.t.) we observed robust GABA and NeuN staining in the grafted cells. We also detected an increased number of NeuN+ cells over time.

Supplementary Figure 9 AMD-induced inhibitory neurons for human neurological disease modeling.

(a) Sequence of shRNAs targeting human Collybistin mRNA. (b) Patch-clamp configuration for postsynaptic recordings performed on iN cells expressing Collybistin shRNAs (EGFP-positive). Collybistin KD iN cells were co-cultured with Ngn2-induced human neurons (EGFP-negative, black arrowheads). Left, bright-field; Middle, EGFP-fluorescence view; and Right, as both views merged. Rec: recording electrode; Scale bars: 15 μm. The lower panel shows representative traces of AMPAR-mediated sEPSCs recorded from control (Ctrl, black) vs. Collybistin shRNA #4 –expressing (Sh#4, brown) neurons. (c) Cumulative plots (left) and average graphs (right) representing mean ± SE values of sEPSC amplitude (i, top) and event frequency (ii, bottom), for control vs. Collybistin shRNA #4 –infected neurons. (d) Cumulative plots (left) and average values (mean ± SE, right) of intrinsic membrane properties: membrane capacitance (Cm, i), input resistance (Rm, ii), and resting membrane potential (Vm, iii), as recorded from control (Ctrl, black) vs. Collybistin shRNA #4 expressing (Sh# 4, brown) human neurons. For cumulative plots, color-matched connected circles represent average values of corresponding parameters recorded from individual cells. For all experimental approaches, the numbers inside average bar-graphs indicate total number of cells recorded / number of independent batches. Statistical significance was tested by two-tailed, unpaired, Student’s t-test (ns = not significant, P > 0.05).

Supplementary Figure 10 Analysis of different transcription factor combinations confirmed that Ascl1 and Dlx2 are sufficient to generate SST-, CALB1(CB)-, CALB2(CR)-, VIP- and RELN-expressing cells.

qRT-PCR analysis was performed for indicated genes using AD-iN cells or iN cells generated by AD plus indicated factors. Expression levels (expressed as Ct values) are color coded as shown. In general, all the “AD plus 1” conditions generated GABAergic neuron populations similar to those induced by AD or AD with Myt1l. All conditions generated iN cells that robustly expressed SST, CALB1, SYN1 and vGAT. Whereas the expression levels of PVAL and NPY are less prominent and more variable.

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Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–2. (PDF 2486 kb)

Supplementary Protocol

A step-by-step protocol for AD-iN differentiation. (PDF 250 kb)

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Yang, N., Chanda, S., Marro, S. et al. Generation of pure GABAergic neurons by transcription factor programming. Nat Methods 14, 621–628 (2017). https://doi.org/10.1038/nmeth.4291

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