Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) The mRNAs levels of endogenous SOX2, OCT3/4, LIN28 and NANOG in the iPSC lines were quantified and normalized relative to hESC line H9. ( B ) The amount of transcripts of the episomal reprograming vectors were quantified for the iPSC lines between passage number 12 and number 15, using human skin fibroblasts nucleofected with the same vectors as a positive control. DOI: http://dx.doi.org/10.7554/eLife.13073.005

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Positive Control

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) NKX2.1 protein expression was examined by immunocytochemistry in ventral telencephalic neural rosettes derived from a number of control and Dravet iPSC lines. Scale bars, 200 microns. ( B ) OLIG2 protein expression overlaps with NKX2.1 in ventral telencephalic progenitors derived from the hESC line H9. Scale bar, 100 microns. ( C ) Comparing NKX2.1, COUP-TFII and SP8 mRNA expression between dorsal and ventral telencephalic neural rosettes derived from control hESC line H9, Dravet iPSC line 6358–2, and Control iPSC line 8402–2 WT9. Data were quantified by the delta-delta Ct method, using GAPDH as the reference gene and undifferentiated H9 ESCs as the baseline control. It is known that SP8 is weakly expressed in progenitors of excitatory neurons in the developing dorsal telencephalon , which likely corresponds to the detection of SP8 in the dorsal rosette samples in this experiment. The samples represent two neural differentiation experiments (one for H9 and 6358–2 and one for 8402–2) that were independent of those described in . DOI: http://dx.doi.org/10.7554/eLife.13073.014

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Expressing, Immunocytochemistry, Derivative Assay, Control

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) An example for the immunostaining of the inhibitory neurotransmitter GABA (in green) and general neuronal marker MAP2 (in red) in differentiated inhibitory neurons (top) and excitatory neurons (bottom). The cell line in the images is hESC-H9. Scale bars, 50 microns. ( B ) Representative images showing a Calretinin + GAD67 + inhibitory neuron and a Somatostatin + GABA + inhibitory neuron. The cell line in the images is hESC-H9. ( C ) Percentage of Calretinin + or Somatostatin + inhibitory neurons from differentiation of hESC-H9 and control iPSC line 8858-C. ( D ) In inhibitory neuron cultures the expression of lentiviral reporter Dlxi1/2b-GFP overlapped with GABA immunostaining; on the contrary, in excitatory neuron cultures, CaMKII-GFP labeling and GABA immunostaining were mutually exclusive. Scale bars, 20 microns. ( E ) Around 65% of Dlxi1/2b-GFP + neurons were stained positive for GABA. Data represent two control iPSC lines 8858-C and 6593–7. ( F ) By single-cell RT-PCR paired with patch clamp recording, around 70% of Dlxi1/2b-GFP neurons express either GAD65 or GAD67, markers of inhibitory neurons. Data represent all Control (upper pie chart) and Dravet Syndrome (lower pie chart) pluripotent stem cell lines examined in this study. ( G ) Representative traces showing time-dependent maturation of the action potential firing pattern in inhibitory neurons. The cell line in this example is control iPSC 8858–3. ( H ) An example for the isolation of Gabazine-sensitive, spontaneous inhibitory synaptic currents (sIPSCs, insets in blue) and NBQX-sensitive, spontaneous excitatory synaptic currents (sEPSCs, insets in red) in cultures of differentiated inhibitory neurons. The detection of sEPSCs reflects the presence of a small fraction of excitatory neurons which likely arise from the low-percentage of PAX6 + progenitors in the ventral telencephalon patterning protocol . The cell line in this example is Dravet iPSC 6358–3. ( I ) Visualization of a hESC-H9-derived inhibitory neuron engrafted to the CA3 region of a rat hippocampal slice and co-cultured with the slice for 2 months. The human neuron was labeled with the lentiviral vector pLenti-Syn1-ChR2(H134R)-YFP. Rat neurons were visible by Ankyrin-G immunostaining (in red). Scale bar, 25 microns. ( J ) Under 470-nm light pulses, Gabazine-sensitive inhibitory postsynaptic currents (IPSCs, top trace) were detected in a rat neuron on a hippocampal slice engrafted with ChR2-YFP-expressing, hESC-H9-derived inhibitory neurons. Each trace represents the average of 20 consecutive trials (light pulses). DOI: http://dx.doi.org/10.7554/eLife.13073.015

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Immunostaining, Marker, Control, Expressing, Labeling, Staining, Reverse Transcription Polymerase Chain Reaction, Patch Clamp, Isolation, Derivative Assay, Cell Culture, Plasmid Preparation

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) Inhibitory neuron cultures derived from control iPSC line 8402–2 WT9 were fixed before plating onto rat astrocytes and stained for NKX2.1 (in green, using Novacastra antibody TTF‐1‐L‐CE), Calretinin (in red, using Swant antibody 7697) and nuclei (Hoechst, in blue). The arrows point to cells with neuronal morphology and co-expressing NKX2.1 and Calretinin. Scale bars, 50 microns. ( B ) The cells described in ( A ) were further differentiated in the presence of rat astrocytes and stained for GAD67 (in white, using EMD Millipore antibody AB5406) and SP8 (in red, using Sigma antibody HPA054006). In a total of 199 GAD67 + , neuronal-looking cells that we analyzed, 81% (161 out of 199) was negative for SP8 staining, and the remaining 19% (38 out of 199) showed a very weak signal (one example shown in ( B ), highlighted by open arrow head). Scale bar, 100 microns. ( C ) As a positive control for SP8 detection, we examined primary cultures of rat cortical neurons (three weeks in vitro) in the same experiment under identical staining and imaging conditions. Robust SP8 staining was seen in a subpopulation of GAD67 + inhibitory neurons (arrow head). Scale bar, 100 microns. Because the mutually exclusive expression of SP8 and NKX2.1 distinguishes between the CGE- and MGE-origin of inhibitory interneurons, this set of supplementary data further support that the human iPSC-derived inhibitory neurons in this study mostly resemble a MGE origin, and that Calretinin expression may arise in MGE-lineage inhibitory neurons within the time scale of the study. DOI: http://dx.doi.org/10.7554/eLife.13073.016

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Derivative Assay, Control, Staining, Expressing, Positive Control, In Vitro, Imaging

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) An example of I Na measured in an inhibitory neuron derived from a control iPSC line. The membrane depolarization steps were set between -92 mV and a range of values starting from -72 mV and ending at +38 mV, with 5 mV/step increments. ( B ) Complete blockade of I Na by 0.8 µM TTX. ( C and D ) I Na was indistinguishable between control and Dravet excitatory neurons. The example I Na traces in ( C ) were selected to represent the median values of all control excitatory neurons and all Dravet excitatory neurons, respectively. The current trace from the Dravet neuron was scaled relative to the control neuron according to whole-cell capacitance values. Panel ( D ) describes the current-voltage (I-V) relationships in control (n = 45 cells, 3 subjects) and Dravet (n = 45 cells, 2 subjects) excitatory neurons. p = 0.8225 by t-test of the maximal I Na amplitude. ( E and F ) I Na amplitude was reduced in Dravet inhibitory neurons relative to control inhibitory neurons. The example I Na traces in ( E ) were selected the same way as in ( C ). Panel ( F ) describes the I-V relationships of control (n = 28 cells, 4 subjects) and Dravet (n = 20 cells, 2 subjects) inhibitory neurons. p = 0.034 by t-test for the maximal I Na amplitude. All error bars are standard errors of the mean. See for detailed statistical analysis. DOI: http://dx.doi.org/10.7554/eLife.13073.020 10.7554/eLife.13073.021 Figure 4—source data 1. I Na values quantified in . DOI: http://dx.doi.org/10.7554/eLife.13073.021

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Derivative Assay, Control, Membrane

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: The pool of inhibitory neurons (n = 60) in this analysis were derived from hESC-H9 and control iPSC lines and co-cultured with rat astrocytes until around Day 90. Illustrated here are current clamp traces at the threshold current injection (i.e. the rheobase, in red) and at a supra-threshold current that is two or three times of the threshold current (in black). We also plotted the response to a−10 pA hyperpolarizing current (downward trace in black). All current injections are 1000-msec long. By their supra-threshold firing patterns, the inhibitory neurons are classified as accommodating, non-accomodating, stuttering, immature, and single-AP. Panel ( A ) is a pie chart summary of the percentage representation of the five patterns. Panels ( B ) through ( F ) are representative neurons for each pattern. DOI: http://dx.doi.org/10.7554/eLife.13073.018

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Derivative Assay, Control, Cell Culture, Injection

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) Differential expression of Na v 1.1 mRNA between cultures of telencephalic excitatory neurons and inhibitory neurons. Quantification of Na v 1.1 mRNA was based on real-time PCR and normalization to the auxiliary Na channel subunit NaVβ3. For control iPSC line 8858–3, n = 6 culture wells of excitatory neurons, n = 5 culture wells of inhibitory neurons. For the other cell lines, one culture well of excitatory neurons and one culture well of inhibitory neurons were assessed pairwise. ( B ) Visually identifying Dlxi1/2b-GFP neurons that co-express Na v 1.1-shRNAs with a tRFP tracer. Scale bar, 10 microns. Similar tracing was performed for CaMKII-GFP-labeled excitatory neurons co-expressing hNa v 1.1-shRNAs. ( C ) Visually identifying Dlxi1/2b-GFP neurons that co-express a doxycycline-inducible Na v 1.1 cDNA with a mCherry tracer. Scale bar, 25 microns. ( D ) Na v 1.1- shRNA1 and Na v 1.1-shRNA2 reduced Na + currents (I Na ) in hESC-H9 derived Dlxi1/2b-GFP + neurons, but not in CaMKII-GFP + neurons. Conversely, the Na v 1.1 cDNA restored the I Na in Dravet Dlxi1/2b-GFP + neurons (Cell line: 6358–2). F(7, 57) = 5.636, p<0.0001 by ANOVA for the entire dataset. Individual p values from post hoc Sidak’s multiple comparisons are indicated on the graph. ( E ) Na v 1.1-shRNAs suppressed action potential firing capacity in hESC-H9 derived Dlxi1/2b-GFP + neurons. F (1, 270) = 13.24, p = 0.0003 by two-way ANOVA for the effect of Na v 1.1-shRNAs on spike number over the range of current injections. ( F ) The Na v 1.1 cDNA restored action potential firing capacity in Dlxi1/2b-GFP + neurons derived from Dravet iPSC line 6358–2. F (1, 135) = 26.69, p<0.0001 by two-way ANOVA for the contribution of Na v 1.1 cDNA to spike number over the range of current injections. All error bars are standard errors of the mean. DOI: http://dx.doi.org/10.7554/eLife.13073.031 10.7554/eLife.13073.032 Figure 6—source data 1. I Na values quantified in , and action potential numbers quantified in . DOI: http://dx.doi.org/10.7554/eLife.13073.032

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Quantitative Proteomics, Real-time Polymerase Chain Reaction, Control, Labeling, Expressing, Derivative Assay

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) Immunocytochemistry for pluripotency markers in control iPSC lines 8858-C and 8858–3. OCT3/4 and NANOG are localized to the cell nucleus; TRA-1-60 and SSEA-3 are localized to the cell surface; TRA-2-49 is localized in the cytoplasm. Scale bars, 200 microns. ( B ) Immunocytochemistry for pluripotency markers in control iPSC line 8402–2 WT9. Upper row, co-expression of pluripotent stem cell markers OCT3/4 (in green) and TRA-1-60 (in red). Lower row, co-expression of pluripotent stem cell markers NANOG (in green) and SSEA-4 (in red, cell surface localization). Scale bars, 200 microns. ( C ) Summary of characterizations of all control iPSC lines with regard to the silencing of reprogramming vectors and genome integrity. DOI: http://dx.doi.org/10.7554/eLife.13073.007

Article Snippet: They also incorporated new data from a control iPSC line (8402-2 WT9) that was generated at Novartis from fibroblast cell line GW08402, in accordance with the conditions of the NIGMS Repository Samples, governed by the Coriell IRB in accordance with DHHS regulations, as outlined in the MTA between Coriell and Novartis.

Techniques: Immunocytochemistry, Control, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CACNA1A loss-of-function affects neurogenesis in human iPSC-derived neural models

doi: 10.1007/s00018-025-05740-7

Figure Lengend Snippet: Isogenic iPSC lines carrying CACNA1A loss-of-function mutations. ( A ) Cartoon showing Ca V 2.1 channel topology with the position of F1491S and Y1854X mutations. F1491S is located in a domain coded by a constitutive exon and therefore affects all CACNA1A isoforms. Y1854X is located in a C-terminal domain coded by the alternative exon 37a and selectively affects the Ca V 2.1[EFa] isoform. ( B ) Sanger sequencing results from iPSC lines in which the indicated mutations were introduced by CRISPR/Cas9 genome editing. Silent mutations in the gRNA binding site were also introduced to prevent re-cutting. ( C ) Representative confocal images (top) and summary graphs (bottom) showing the analysis of undifferentiated state markers in isogenic control and mutated iPSC lines. Scale bar: 10 μm. The typical stem cell markers OCT4, SSEA4, SOX2, and TRA-1–60 were quantified by immunofluorescence. At least 2000 cells were analyzed for each marker and sample at the cell passages preceding the neural induction. The bar graphs show data as mean ± SEM, while single biological replicates are superimposed as dots ( n = 3)

Article Snippet: For the generation of iPSC lines carrying CACNA1A variants we used a control iPSC line purchased from Applied StemCell, catalog number: ASE-9211.

Techniques: Sequencing, CRISPR, Binding Assay, Control, Immunofluorescence, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CACNA1A loss-of-function affects neurogenesis in human iPSC-derived neural models

doi: 10.1007/s00018-025-05740-7

Figure Lengend Snippet: CACNA1A mutations affect CACNA1A transcripts expression in iPSC-derived NPCs and neurons. ( A ) Representative brightfield images (top) and enlarged regions (bottom) showing typical morphology of control iPSC colonies, neural progenitors (NPCs), and neurons. Scale bar: 100 μm. ( B ) Quantification of CACNA1A mRNA by real time qPCR in control and mutated iPSCs, NPCs, and neurons (42 DIV). Data were normalized by the expression of PPIA and RPL13 A as reference genes, and with isogenic control iPSCs as reference sample. Data are presented as mean ± SEM (bars) and single replicates (dots). **, p < 0.01 vs control (Student’s t test, n = 4); #, p < 0.05; ###, p < 0.001 neurons vs NPCs (Student’s t test, n = 4). ( C ) Quantification of Ca V 2.1[EFa] (left) and Ca V 2.1[EFb] (right) alternative isoforms by real time qPCR using isoform-specific primers in control and mutated NPCs and neurons (42 DIV). Data were normalized by the expression of PPIA and RPL13 A as reference genes, and with control NPCs as reference sample. Data are presented as mean ± SEM (bars) and single replicates (dots). *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs control (Student’s t test, n = 4). #, p < 0.05; ##, p < 0.01; ###, p < 0.001 vs NPCs (Student’s t test, n = 4)

Article Snippet: For the generation of iPSC lines carrying CACNA1A variants we used a control iPSC line purchased from Applied StemCell, catalog number: ASE-9211.

Techniques: Expressing, Derivative Assay, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CACNA1A loss-of-function affects neurogenesis in human iPSC-derived neural models

doi: 10.1007/s00018-025-05740-7

Figure Lengend Snippet: CACNA1A mutations alter the migratory capacity of NPCs. ( A ) Representative confocal images of control and mutated iPSC-derived NPCs showing the expression of typical neural progenitors’ markers: SOX1, PAX6, Nestin, and SOX2. Cells were also counterstained with DAPI to label cell nuclei. Scale bar: 20 μm. (B, top) Dot plot graphs showing the relative expression of the indicated nuclear markers quantified from immunofluorescence images at single-cell level (at least 2000 cells were analyzed for each marker and sample during 3 cell passages, from P2 to P4). ( B , bottom) Bar graphs showing the percentage of positive NPCs for each marker and sample. Superimposed dots indicate biological replicates ( n = 3). ( C - D ) Analysis of cell migration by wound healing assay. Representative images ( C ) and analysis ( D ) of wounded areas of confluent neural progenitors at the indicated post-wounding time points. Wound edges, detected by image segmentation analysis, are outlined in green. Scale bar: 500 μm. *, p < 0.05, F1491S vs control; #, p < 0.05, Y1854X vs control (Student’s t test, n = 3). (E) Graph showing the analysis of cell proliferation by the BrdU assay. Data are presented as mean ± SEM (bars) and single replicates (dots). No statistical significance was found with the Student’s t test by comparing each mutation with respect to control ( n = 3)

Article Snippet: For the generation of iPSC lines carrying CACNA1A variants we used a control iPSC line purchased from Applied StemCell, catalog number: ASE-9211.

Techniques: Control, Derivative Assay, Expressing, Immunofluorescence, Marker, Migration, Wound Healing Assay, BrdU Staining, Mutagenesis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CACNA1A loss-of-function affects neurogenesis in human iPSC-derived neural models

doi: 10.1007/s00018-025-05740-7

Figure Lengend Snippet: CACNA1A loss-of-function caused by F1491S, but not by Y1854X, impairs neuronal polarization and maturation. ( A ) Representative confocal images of control and mutated iPSC-derived neurons at 10 DIV. Cells were labeled with antibodies directed against TUBB3 and NeuN (top images) or MAP2 and SMI-31 (bottom images) as markers of neuronal maturation. Cells were also counterstained with DAPI to label cell nuclei. Scale bar: 20 μm. ( B ) Zoomed detail from images in panel A (dotted rectangles) showing MAP2 and SMI-31 staining in control and Y1854X neurons. White arrows indicate examples of MAP2-negative and SMI-31-positive axons. ( C ) Graphs showing the quantification of NeuN-positive cells (%), neurites outgrowth (neurites area), and axon specification (SMI-31 area) at 10 DIV. Data are shown as mean ± SEM (bars) and single replicates (dots). **, p < 0.01; ***, p < 0.001 vs control with the Student’s t test ( n = 3). ( D ) Representative confocal images of control and mutated iPSC-derived neurons at 42 DIV. Cells were labeled with antibodies directed against MAP2, GFAP, and Ki67, as markers of neurons, glia, and proliferating cells, respectively. Cells were also counterstained with DAPI to label cell nuclei. Scale bar: 50 μm

Article Snippet: For the generation of iPSC lines carrying CACNA1A variants we used a control iPSC line purchased from Applied StemCell, catalog number: ASE-9211.

Techniques: Control, Derivative Assay, Labeling, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CACNA1A loss-of-function affects neurogenesis in human iPSC-derived neural models

doi: 10.1007/s00018-025-05740-7

Figure Lengend Snippet: CACNA1A loss-of-function caused by Y1854X mutation impairs neuronal network synchronization. ( A , B ) Raster plots (top) and array wide firing rate graph (bottom) showing 60 s of spontaneous activity recording at 28, 42 and 60 DIV in control ( A ) and Y1854X ( B ) neuronal cultures. The raster plot y-axis represents electrodes (from 0 to 4096), and each dot indicates a detected spike. The array wide firing rate quantifies the level of activity shown in the above raster plots and highlights the appearance of synchronous network events only in the control (green peaks at 42 and 60 DIV). ( C ) Graphs comparing the activity of control and mutated iPSC-derived neurons in terms of number of active electrodes, mean firing rate, mean bursting rate, mean burst duration, percentage of random spikes, percentage of bursting channels, network burst rate, and network burst duration. Data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01 vs control (Student’s t test, n = 3–6 replicates for each time points and sample)

Article Snippet: For the generation of iPSC lines carrying CACNA1A variants we used a control iPSC line purchased from Applied StemCell, catalog number: ASE-9211.

Techniques: Mutagenesis, Activity Assay, Control, Derivative Assay