GABAergic Neurons

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opti-ox precision deterministically programmed ioGABAergic Neurons rapidly form a homogenous neuronal population

Time-lapse video capturing the rapid and homogeneous neuronal phenotype acquisition upon thawing of cryopreserved ioGABAergic Neurons (12 day time course).

Highly characterised and defined

ioGABAergic Neurons express key GABAergic neuron-specific markers

Immunocytochemistry showing ioGABAergic Neurons express key markers GABA and MAP2

Immunofluorescent staining of ioGABAergic Neurons at day 12 post-revival. The upper panel shows that ioGABAergic Neurons are positive for the pan-neuronal marker MAP2 (red), GABA (green), and the DAPI counterstain (blue). The lower panel shows that all MAP2 positive neurons have a GABAergic neuronal identity.

ioGABAergic Neurons show visible neuronal networks by day 10

ioGABAergic neurons show neuronal networks by day 10, shown in a 12 day time course

Upon reprogramming, rapid morphological changes are observed in the cells, with neurons identified by day 3 post-revival. Visible neuronal networks are observed by day 10 post-thaw. Images show day 1 to 12 post-thawing; 100X magnification.

Single cell RNA-sequencing shows ioGABAergic Neurons form a pure population (>99%) of GABAergic neurons

ioGABA UMAP showing expression profiles in a 14 day time course

Single cell RNA-sequencing analysis was performed with ioGABAergic Neurons at three specific timepoints (day 0, 7 and 14). By day 7, the population has a distinct expression profile indicating a pure population (>99%) of post-mitotic GABAergic neurons. Gene expression was assessed by 10x Genomics scRNA-sequencing. Note, this data is from cells in continuous culture, so minor variations may exist between this data and data from cryopreserved cells.

Single cell RNA-sequencing shows ioGABAergic Neurons express key GABAergic markers

Single cell RNA-sequencing analysis was performed with ioGABAergic Neurons at three specific timepoints (day 0, 7 and 14). By day 7, the expression of key GABAergic marker genes [GAD1, GAD2, SLC32A1 (VGAT), DLX2, DLX5], together with the pan-neuronal marker MAP2, could be detected in post-mitotic GABAergic neurons. Gene expression was assessed by 10x Genomics scRNA-sequencing. Note, this data is from cells in continuous culture, so minor variations may exist between this data and data from cryopreserved cells.

Single cell RNA-sequencing shows ioGABAergic Neurons display minimal expression of markers indicative of other neuronal lineages

Single cell RNA-sequencing analysis was performed with ioGABAergic Neurons at three specific timepoints (day 0, 7 and 14). By day 14, the expression of markers of non-GABAergic neuronal lineages [TH - dopaminergic, TPH1 - serotonergic, CHAT - cholinergic, SLC17A6 (VGLUT2) - glutamatergic, SLC17A7 (VGLUT1) - glutamatergic, SLC18A3 (VACHT) - cholinergic] are largely absent. Gene expression was assessed by 10x Genomics scRNA-sequencing. Note, this data is from cells in continuous culture, so minor variations may exist between this data and data from cryopreserved cells.

Single cell RNA-sequencing indicates that ioGABAergic Neurons are of the SST sub-type

Single cell RNA-sequencing analysis was performed with ioGABAergic Neurons at three specific timepoints (day 0, 7 and 14). By day 7, cells appear to largely express the somatostatin (SST) marker indicating that this population is of the SST sub-type. Gene expression was assessed by 10x Genomics scRNA-sequencing. Note, this data is from cells in continuous culture, so minor variations may exist between this data and data from cryopreserved cells.

Whole transcriptome analysis demonstrates high lot-to-lot consistency of ioGABAergic Neurons

io1003 ioGABAergic neurons bulk RNAseq lot consistency figure

Bulk RNA sequencing analysis was performed on three technical replicates of three independent lots of ioGABAergic Neurons at different time points throughout the reprogramming protocol. Principal component analysis represents the variance in gene expression between the lots of ioGABAergic Neurons. This analysis shows high consistency between each lot of ioGABAergic Neurons at each given timepoint. Differential gene expression analysis shows only 9 or less differentially expressed genes between lots, less than <1% of the total 25,000 genes within a human cell, at day 12 post-thaw. Pure populations of ioGABAergic Neurons with equivalent expression profiles can be generated consistently from every vial, allowing confidence in experimental reproducibility.

Rapid gain of functional activity

Calcium imaging of ioGABAergic Neurons demonstrates spontaneous activity

Video showing spontaneous activity of ioGABAergic Neurons, subjected to calcium imaging at day 16 post-revival.

Functional co-cultures

ioGABAergic Neurons form functional neuronal networks and modulate network activity in tri-cultures with ioGlutamatergic Neurons and astrocytes

MEA data showing ioGABAergic Neurons form stable functional neuronal networks and display network modulation in tri-cultures with ioGlutamatergic Neurons and astrocytes_FINAL

Tri-cultures containing increasing numbers of ioGABAergic Neurons, and a fixed number of ioGlutamatergic Neurons and hiPSC-derived astrocytes were thawed and seeded together into a 48-well CytoView MEA plate (Axion Biosystems). Control co-cultures consisted of either ioGABAergic Neurons and hiPSC-derived astrocytes or ioGlutamatergic Neurons and hiPSC-derived astrocytes. Analysis was performed on an Axion Maestro Pro MEA platform over a time period of 64 days in vitro (DIV). A. Co-cultures containing excitatory ioGlutamatergic Neurons and astrocytes show the strongest synchronised network activity, indicated by the highest number of spikes per network burst. The addition of increasing numbers of inhibitory ioGABAergic Neurons to the tri-cultures reduces this synchronised network activity of the excitatory ioGlutamatergic Neurons, as expected. The co-culture of ioGABAergic Neurons and astrocytes shows no network bursts, indicating the absence of excitatory neurons and that the population of ioGABAergic Neurons is highly pure. B. Both co-culture and tri-culture conditions show increasing spontaneous activity, as measured by an increase in the number of active electrodes up to 25 DIV, followed by a plateau, indicating sustained activity over the time period of 64 DIV. This data was generated in partnership with Charles River.

ioGABAergic Neurons exert an inhibitory effect on the excitatory ioGlutamatergic Neurons within the tri-cultures leading to a higher network burst rate

ioGABA MEA co-culture raster plots_Fig 2-1

The effect of adding increasing numbers of inhibitory ioGABAergic Neurons to the tri-cultures was investigated by MEA analysis at 53 DIV, alongside the control co-cultures. Representative raster plots displaying the activity of 16 electrodes over a time period of 300 seconds are shown. Each horizontal row of the raster plot represents the activity of an electrode, within which each vertical black dash indicates a firing event, a blue dash indicates a single electrode burst, and a pink box indicates a network burst event. The histogram trace on top of the raster plot is a measure of the number of spikes per network burst. The co-culture with ioGlutamatergic Neurons and astrocytes shows the strongest network bursts as indicated by the increased number of spikes per network burst and shows a lower network burst rate (NBR) compared to the tri-cultures. The addition of increasing numbers of inhibitory ioGABAergic Neurons to the tri-cultures reduces the number of spikes per network burst and leads to an increased NBR. This indicates that ioGABAergic Neurons are having an inhibitory effect on the excitatory ioGlutamatergic Neurons. The co-culture of ioGABAergic Neurons and astrocytes shows no network bursts, indicating the absence of excitatory neurons and that the population of ioGABAergic Neurons is highly pure. Analysis was performed on an Axion Maestro Pro MEA platform. This data was generated in partnership with Charles River Laboratories.

Bicuculline compound response

Addition of bicuculline, a competitive antagonist of GABA_A receptors, to the tri-cultures releases the inhibitory effect of the ioGABAergic Neurons

Addition of bicuculline to tri-cultures releases the inhibitory effect of the ioGABAergic Neuron_FINAL2

The effect of adding bicuculline, a competitive antagonist of GABA_A receptors, to the tri-cultures and control co-cultures was investigated by MEA analysis at 65 DIV. Representative raster plots displaying the activity of 16 electrodes over a time period of 300 seconds are shown before and 20 minutes after the addition of bicuculline. Each horizontal row of the raster plot represents the activity of an electrode, within which each vertical black dash indicates a firing event, a blue dash indicates a single electrode burst, and a pink box indicates a network burst event. The histogram trace on top of the raster plot is a measure of the number of spikes per network burst. A and B. The addition of bicuculline does not affect either co-culture condition, as expected. C. The antagonistic effect of bicuculline on GABA_Areceptors releases the inhibitory effect of the ioGABAergic Neurons within the tri-culture, thereby reducing the NBR and leading to an increase in the number of spikes per network burst. D. Graphs displaying the number of spikes per network burst over time after the addition of bicuculline show no change in either control co-culture condition (left and centre graphs). A dose dependent increase in the number of spikes per network burst is seen in the tri-culture (right graph). This data was generated in partnership with Charles River Laboratories.

Diazepam compound response

Addition of diazepam, a positive allosteric modulator of GABA_A receptors, to the tri-cultures increases the inhibitory effect of the ioGABAergic Neurons

Addition of diazepam to tri-cultures increases the inhibitory effect of the ioGABAergic Neurons_FINAL2

The effect of adding diazepam, a positive allosteric modulator of GABA_A receptors, to the tri-cultures and control co-cultures was investigated by MEA analysis at 65 DIV. Representative raster plots displaying the activity of 16 electrodes over a time period of 300 seconds are shown before and 20 minutes after the addition of diazepam. Each horizontal row of the raster plot represents the activity of an electrode, within which each vertical black dash indicates a firing event, a blue dash indicates a single electrode burst, and a pink box indicates a network burst event. The histogram trace on top of the raster plot is a measure of the number of spikes per network burst. A and B. The addition of diazepam does not affect either co-culture condition, as expected. C. The inhibitory effect of ioGABAergic Neurons on the tri-cultures is increased by the positive modulatory effect of diazepam, shown by the sharp decrease in the number of spikes per network burst and a decrease in the NBR. D. Graphs displaying the number of spikes per network burst over time after the addition of diazepam shows no change in either control co-culture condition (left and centre graphs). The number of spikes per network burst decreases to zero and makes all bursting disordered in the tri-culture (right graph). Analysis was performed on an Axion Maestro Pro MEA platform. This data was generated in partnership with Charles River Laboratories.

Cells arrive ready to plate

bit.bio_ioGABAergic Neurons_timeline_horizontal_withoutdox

ioGABAergic Neurons are delivered in a cryopreserved format and are programmed to rapidly mature upon revival in the recommended media. The protocol for the generation of these cells is a two-phase process: Induction, which is carried out at bit.bio, Stabilisation for 3 days (Phase 1), and Maintenance (Phase 2) during which the ioGABAergic Neurons mature. Phases 1 and 2 after revival of cells are carried out at the customer site.