Research ArticleNEUROTOXICITY

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

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Science Translational Medicine  06 Apr 2016:
Vol. 8, Issue 333, pp. 333ra48
DOI: 10.1126/scitranslmed.aad0623
  • Fig. 1. Generation of forebrain progenitor cells with regional identity from human iPSCs.

    (A) Scheme of the RONA differentiation protocol showing generation of forebrain progenitors from human ESCs and iPSCs. Nog, noggin; SB, SB431542. (B) Rosettes were immunopositive for FOXG1 and NES at day 9 after initiation of neural differentiation. DAPI, 4′,6-diamidino-2-phenylindole. (C and D) With the prolongation of neural differentiation, highly proliferative rosettes started to pile up, resulting in three-dimensional (3D) columnar cellular aggregates, which were positive for FOXG1 and NES. (D) Phase-contrast image of RONA. Scale bar, 50 μm (B to D). (E to G) FOXG1 immunoreactive RONAs were located in the center (E) and demarcated clearly with surrounding CK8-positive cells. (F) Cells underneath the RONAs were FOXG1/CK8. (G) The white inset box in (F) is magnified (×2.5). (H to L) Expression and quantification of the early forebrain regionalization markers FOXG1 (H), PAX6 (I), LHX2 (J), NKX2.1 (K), and GSX2 (L) after 9, 16, and 18 days of differentiation. Data are presented as means ± SEM (n = 3). Scale bar, 20 μm. (M and N) RNA sequencing gene expression profiling of RONAs derived from the human ESC H1 line showing the enrichment of forebrain progenitors with regional identity. Data show the average RPKM (reads per kilobase of transcript per million mapped reads) value of two biological replicates of human ESC H1 cell line RNA sequencing experiments. ESRRB, estrogen-related receptor β; SMA, smooth muscle actin; HNF1B, hepatocyte nuclear factor 1 homeobox B; MGE, medial ganglionic eminence; CGE, caudal ganglionic eminence. (O) Spontaneous neuronal differentiation after isolation of RONA neural progenitors without the application of exogenous mitogens. Nuclei were counterstained with DAPI (blue). Colors are indicated in the images. Scale bar, 20 μm. Data are from the human ESC H1 cell line in (B) to (O). DCX, doublecortin.

  • Fig. 2. Timing of retinoic acid/SHH exposure determines differentiation of excitatory and inhibitory neurons.

    (A) Schematic summary of conditions for differentiation of appropriate balanced excitatory and inhibitory neurons. KoSR, knockout serum replacement; E/I, excitatory/inhibitory. (B) Immunocytochemical analysis of neuronal expression of the excitatory marker VGLUT and the inhibitory marker VGAT. Quantification of the percentages of VGLUT- and VGAT-positive cells with or without 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 neural differentiation. Quantification of the proportion of PSD95 puncta that were found associated with synapsin puncta. Data are presented as means ± SEM (n = 3). (D) Differentiation of inhibitory neurons expressing subtype markers. Colors are indicated in the images. Scale bar, 20 μm. (E) Quantification of inhibitory neurons with immunostaining analyses over 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 retinoic acid. Data from cortical neuronal cultures derived from the human ESC H1 cell line are presented as means ± SEM. (G) Developmental expression pattern of PV, SST, CR, nNOS, and CB in human cortical interneurons from mid-fetal stage, late fetal stage, to infant (60). Data from human cortical cultures are from the human ESC H1 cell line in (B) to (F).

  • Fig. 3. Generation of layer-specific human cortical neurons from FOXG1+ forebrain progenitor cells.

    (A) Immunocytochemical analysis of the cortical layer–specific markers TBR1, CTIP2, BRN2, and SATB2 in human neuronal culture differentiated from FOXG1+ neural progenitors derived from the human 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 human iPSC line SC1014. (C) Diagram of the 3D human cortical assembly assay. (D and E) Cross sections from 3D human cortical assemblies were immunostained 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. Scale bar, 50 μm. Data in (C) to (E) are from the human ESC H1 cell line.

  • Fig. 4. Development of a functional human cortical excitatory and inhibitory neuronal network.

    (A and B) Voltage-gated sodium (A) and potassium channels (B) present in human ESC H1 line–derived cortical neurons (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 neurons after 8 weeks of differentiation (n = 9). (D) Measurement of mIPSCs indicated the formation of a functional inhibitory network in human cortical neuronal culture. With 40 mM chloride ion in the pipette solution, the mIPSC current is inward (n = 11 for each group). (E) Homeostatic scaling of human ESC H1 cell line–derived cortical neurons. Measurement of mEPSCs from cultured human cortical neurons under control conditions (control), conditions of activity blockade [tetrodotoxin (TTX)], or conditions of activity enhancement with bicuculline (Bic). Forty-eight hours of activity blockade increased the amplitude of mEPSCs, whereas 48 hours of enhanced activity decreased the mEPSC amplitude. *P < 0.05, Mann-Whitney test, Control, n = 17; TTX, n = 21; Bic, n = 18. All data shown are from the human ESC H1 cell line.

  • Fig. 5. Characterizing human cortical neurons as a model for excitotoxicity.

    (A) Percentages of neuron death were assessed by propidium iodide (PI)/Hoechst staining 24 hours after different exposure times to 500 μM glutamate (Glut) and were quantified and expressed as means ± SEM (n = 3). (B) Percentages of neuron death were assessed by PI/Hoechst staining 24 hours after 30 min of glutamate exposure at different doses and were quantified and expressed as means ± SEM (n = 3). (C) MK801 (NMDA receptor antagonist) but not NBQX (antagonist of AMPA receptors and kainate receptors) abolished 500 μM glutamate–induced human cortical neuron death. Percentages of neuron death were quantified and expressed as means ± SEM (n = 3). Glutamate significantly killed neurons (***P < 0.001), and MK801 significantly protected the neurons against glutamate excitotoxicity (***P < 0.001), whereas NBQX had no effect on glutamate excitotoxicity. (D) Thirty minutes of treatment with NMDA at different concentrations. Percentages of neuron death were assessed by PI/Hoechst staining, quantified, and expressed as means ± SEM (n = 9). (E) Calcium imaging with Fura-2 of human cortical neuron cultures stimulated with 100 or 500 μM NMDA and 10 μM glycine, followed by MK801. Plots show the means ± SEM of 30 to 31 neurons. (F) Human ESC H1 cell line–derived cortical neurons were subjected to different lengths of oxygen-glucose deprivation. Percentages of neuron death were assessed by PI/Hoechst staining, quantified, and expressed as means ± SEM (n = 9). **P < 0.01 and ***P < 0.001, ANOVA with Bonferroni’s posttest. NT, not treated.

  • Fig. 6. Pathways involved in human cortical neuronal death induced by NMDA or oxygen-glucose deprivation.

    (A and B) NPLA (selective nNOS inhibitor) and DPQ (PARP inhibitor), but not Z-VAD (caspase inhibitor), NEC-1 (necroptosis inhibitor), or 3-MA (autophagy inhibitor), protected human ESC H1 cell line–derived cortical neuronal cultures from cell death due to (A) 30 min of exposure to 500 μM NMDA or (B) 2 hours of oxygen-glucose deprivation. Quantitative data from the PI/Hoechst-positive cell number ratio are shown as means ± SEM. For NMDA-treated neurons (A), n = 8 for the dimethyl sulfoxide (DMSO) control group and the NMDA plus DMSO group; for oxygen-glucose deprivation–treated neurons (B), n = 3 for the 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 and D) An nNOS inhibitor (NPLA), but not a PARP-1 inhibitor (DPQ), inhibited NO production in human ESC H1 cell line–derived cortical neuronal cultures after 30 min of treatment with 500 μM NMDA or 2 hours of oxygen-glucose deprivation. Data are shown as means ± SEM of NO units (fluorescence measured at excitation/emission wavelengths of 365 nm/450 nm) normalized to medium-only wells (n = 4). (E and F) Representative blots showing that treatment with a NOS inhibitor (NPLA or l-NAME) or a PARP inhibitor (DPQ) blocked PAR polymer formation (the product generated by PARP) after 30 min of treatment with 500 μM NMDA or 2 hours of oxygen-glucose deprivation in human ESC H1 cell line–derived cortical neuronal cultures; means ± SEM of PAR polymer/actin band density ratio (n = 3). (G and H) Lentiviral vector–based PARP-1 CRISPR/Cas9 sgRNAs protect human ESC H1 line–derived cortical cultures from NMDA- or oxygen-glucose deprivation–induced cell death. Data are shown as means ± SEM (n = 3). (I and J) PARP-1 inhibitors protect human ESC H1 line–derived cortical neuronal cultures from NMDA- or oxygen-glucose deprivation–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).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/333/333ra48/DC1

    Fig. S1. Comparison of human forebrain neuron differentiation method.

    Fig. S2. Neural induction of human iPSCs by dual inhibition of SMAD signaling enhances the efficiency of neural conversion via the RONA method.

    Fig. S3. Isolated FOXG1+ forebrain progenitors preserve excitatory and inhibitory neurogenic potential.

    Fig. S4. Development regulates the excitatory/inhibitory network formation and synaptic scaling.

    Fig. S5. NMDA induces human cortical neuron cell death.

    Fig. S6. PARP activation in male versus female human ESCs.

    Data file S1 (Excel file)

  • Supplementary Material for:

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

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

    *Corresponding author. E-mail: vdawson{at}jhmi.edu (V.L.W.); tdawson{at}jhmi.edu (T.M.W.)

    Published 6 April 2016, Sci. Transl. Med. 8, 333ra48 (2016)
    DOI: 10.1126/scitranslmed.aad0623

    This PDF file includes:

    • Fig. S1. Comparison of human forebrain neuron differentiation method.
    • Fig. S2. Neural induction of human iPSCs by dual inhibition of SMAD signaling enhances the efficiency of neural conversion via the RONA method.
    • Fig. S3. Isolated FOXG1+ forebrain progenitors preserve excitatory and inhibitory neurogenic potential.
    • Fig. S4. Development regulates the excitatory/inhibitory network formation and synaptic scaling.
    • Fig. S5. NMDA induces human cortical neuron cell death.
    • Fig. S6. PARP activation in male versus female human ESCs.

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    Other Supplementary Material for this manuscript includes the following:

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