a.HTT50CAGWT_Overlay__TUBB3(G)_MAP2(R)_DAPI(B)_ (1)

cat no | ioEA1004

ioGlutamatergic Neurons
HTT 50CAG/WT

Human iPSCderived 

Huntington’s disease model

A rapidly maturing, consistent and scalable isogenic system to study Huntington’s disease.

ioGlutamatergic Neurons HTT 50CAG/WT are opti‑ox deterministically programmed glutamatergic neurons containing a genetically engineered heterozygous 50 CAG trinucleotide repeat expansion in exon 1 of the huntingtin (HTT) gene. 

Place your order

Confidently investigate your phenotype of interest across multiple clones with our disease model clone panel. Detailed characterisation data (below) and bulk RNA sequencing data (upon request) help you select specific clones if required.

per vial

Benchtop benefits

phenotype_0

Disease-related phenotype

Microelectrode array analysis reveals delayed neuronal network formation and decreased spontaneous activity compared to the wild‑type control.

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Scalable

Industrial scale quantities are available with industry-leading seeding densities, and at a price point that allows the cells to be used from research to high throughput screening.

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Make True Comparisons

Be confident in your data. ioDisease Model Cells can be paired with ioWild Type Cells to provide a genetically matched, highly characterised background for the precise analysis of gene function.

Technical data

Ready within days

ioGlutamatergic Neurons HTT 50CAG/WT generated by transcription factor-driven deterministic programming of iPSCs using opti-ox technology

Video capturing the rapid morphological changes of the ioGlutamatergic Neurons HTT 50CAG/WT upon revival of the cryopreserved product over an 11-day culturing period. The observed rapid morphological changes are enabled by opti-ox precision reprogramming.

Highly characterised and defined

ioGlutamatergic Neurons HTT 50CAG/WT express neuron-specific markers with protein expression highly reminiscent to the isogenic control

ioGlutamatergic_neurons-HTT-50CAG-WT-ICC-VGLUT2

Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of pan-neuronal proteins MAP2 and TUBB3 (upper panel) and glutamatergic neuron-specific transporter VGLUT2 (lower panel) in ioGlutamatergic Neurons HTT 50CAG/WT compared to the isogenic control. 100X magnification.

ioGlutamatergic Neurons HTT 50CAG/WT form structural networks by day 11

ioGlutamatergic_neurons-HTT-50CAG-WT-morphology-min

ioGlutamatergic Neurons HTT 50CAG/WT mature rapidly and form structural neuronal networks over 11 days, when compared to the isogenic control. Day 1 to 11 post thawing; 100X magnification.

ioGlutamatergic Neurons HTT 50CAG/WT demonstrate gene expression of neuronal-specific and glutamatergic-specific markers following deterministic  programming

ioGlutamatergic_neurons-HTT-50CAG-WT-rt-qPCR

Gene expression analysis demonstrates that ioGlutamatergic Neurons HTT 50CAG/WT (50CAG/WT) and the isogenic control (WT) at day 11 lack the expression of pluripotency markers (NANOG and OCT4) whilst robustly expressing pan-neuronal (TUBB3 and SYP) and glutamatergic specific (VGLUT1 and VGLUT2) markers, as well as the glutamate receptor GRIA4. Gene expression levels assessed by RT-qPCR (data expressed relative to the parental hiPSC control (iPSC Control), normalised to HMBS). Data represents day 11 post-revival samples; n=2 biological replicates.

Disease-related Huntingtin (HTT) is expressed in ioGlutamatergic Neurons HTT 50CAG/WT

Disease-related Huntingtin (HTT) is expressed in ioGlutamatergic Neurons HTT50CAG_WT -min

RT-qPCR analysis demonstrates similar expression level of the Huntingtin gene in both wild type ioGlutamatergic Neurons (WT) and ioGlutamatergic Neurons HTT 50CAG/WT (50CAG/WT) at day 11 post-revival (n=2 replicates). cDNA samples of the parental iPSC line
(iPSC control) were included as a reference.

Genotype validation

Genotype validation of heterozygous 50 CAG repeat expansion

ioGlutamatergic_neurons-HTT-50CAG-WT-genotype_validation-A

(A) Successful on-target integration into one HTT allele confirmed by gel electrophoresis. Genotyping primers flanking the endogenous HTT CAG repeat expansion region produce a band at approximately 320 bps, by PCR, in both isogenic control (ioGlutamatergic Neurons) and disease model (ioGlutamatergic Neurons HTT 50CAG/WT). PCR fragments at 395 bps detect on-target gene editing and introduction of a 50 CAG repeat expansion in ioGlutamatergic Neurons HTT 50CAG/WT only. (B) Amplicon PCR of the plasmid donor reveals no random integration in genomic DNA from targeted colonies via gel electrophoresis. Off-target random insertion of the donor template (used to introduce the 50 CAG repeat expansion at the WT HTT locus) is detected by PCR amplification of the donor vector backbone. This is not detected in the samples from ioGlutamatergic Neurons HTT 50CAG/WT.

Genotype validation of the number of CAG repeats

ioGlutamatergic_neurons-HTT-50CAG-WT-genotype_validation-B

NGS-amplicon sequencing confirms the number of CAG repeats in wild type ioGlutamatergic Neurons (yellow) and ioGlutamatergic Neurons HTT 50CAG/WT (orange). The number of CAG repeats shows a peak at the normal physiological range of 24 for both the wild type and mutant cells. The 50 CAG repeat was detected only in the mutant cells (orange) confirming the successful introduction of a heterozygous 50 CAG repeat expansion in ioGlutamatergic Neurons HTT 50CAG/WT.

Disease-related phenotype

ioGlutamatergic Neurons HTT 50CAG/WT demonstrate a significant decrease in network activity compared to wild-type control by MEA analysis

bitbio-single-cell and network_development_HTT50CAG_WT

Functional characterisation of ioGlutamatergic Neurons HTT 50CAG/WT by Charles River Laboratories using MaxWell’s MaxTwo high-density microelectrode array (MEA) platform. Comparison of wild-type ioGlutamatergic Neurons (WT) and Huntington’s disease model (HD) in single-cell and network development. The Activity Scan captures spontaneous action potentials of cells and reveals the spatial distribution of the electrical activity from the cell cultures over the electrode array. Map of the Firing Rate distribution over 26,400 electrodes at DIV 38 for (A) WT and (B) HD. Network Firing Rate at DIV 38, recorded for 300 sec. for (C) WT, and (D) HD. (E) Mean Firing Rate recorded from 26,400 electrodes on each well. Data shows results from WT and HD at DIV 14, 21, 38. (F) Mean Burst Frequency recorded from 26,400 electrodes on each well. Data shows results from WT and HD at DIV 35 and 38. Scale bar: 1mm, *p<0.05 (Mann Whitney U Test). The wild-type cells show a higher spontaneous activity than the disease model cells (A, B, E); both cultures show synchronous and spontaneous network activity (C, D, F). The data demonstrate significant HD relevant differences at the network levels between wild-type and disease-model cells.

Single-cell analysis showing significant Huntington’s disease related differences between ioGlutamatergic Neurons HTT 50CAG/WT and wild-type control

bitbio-Single-cell analysis-HTT50CAG_WT

Functional characterisation of ioGlutamatergic Neurons HTT 50CAG/WT by Charles River using MaxWell’s MaxTwo high-density MEA platform. Single-cell analysis showing differences between ioGlutamatergic Neurons (WT) and ioGlutamatergic Neurons HTT 50CAG/WT (HD). The Axon Tracking assay reveals the spatial propagation of the neuronal action potential from the soma to distant axonal branches. Map showing spatial distribution of the action potential amplitude for selected tracked neurons at DIV 32 for (A) WT, and (B) HD. (C-F) Mean Neuron Conduction Velocity, Total Axon Length, Firing Rate, and Amplitude recorded from 26,400 electrodes on each well. Data shows results from WT and HD at DIV 32. Scale bar: 1mm, *p=0.05, ****p<0.0001 (Mann Whitney U Test). The data demonstrate significant HD relevant differences at single-cell level between wild-type and disease-model cells. 

Cells arrive ready to plate

ioGlutamatergic_Neurons_and_disease_models_timeline

ioGlutamatergic Neurons HTT 50CAG/WT are delivered in a cryopreserved format and are programmed to mature rapidly upon revival in the recommended media. The protocol for the generation of these cells is a two-phase process: Phase 1, Stabilisation for 4 days; Phase 2, Maintenance, during which the neurons mature. Phases 1 and 2 after revival of cells are carried out by the customer.

Industry leading seeding density

Do more with every vial
ioGlut-HTT50CAG_WT-well_plate-2

The recommended minimum seeding density is 30,000 cells/cm2, compared to up to 250,000 cells/cm2 for other similar products on the market. One small vial can plate a minimum of 0.7 x 24-well plate, 1 x 96-well plate, or 1.5 x 384-well plates. One large vial can plate a minimum of 3.6 x 24-well plates, 5.4 x 96-well plates, or 7.75 x 384-well plates. This means every vial goes further, enabling more experimental conditions and more repeats, resulting in more confidence in the data.

Product information

Starting material

Human iPSC line

Karyotype

Normal (46, XY)

Seeding compatibility

6, 12, 24, 48, 96 & 384 well plates

Shipping info

Dry ice

Donor

Caucasian adult male (skin fibroblast)

Vial size

Small: >1 x 10 viable cells
Large: >5 x 10 viable cells

Quality control

Sterility, protein expression (ICC), gene expression (RT-qPCR) and genotype validation

Differentiation method

opti-ox deterministic cell programming

Recommended seeding density

30,000 cells/cm2

User storage

LN2 or -150°C

Format

Cryopreserved cells

Genetic modification

Heterozygous - HTT 50 CAG repeat expansion

Applications

Huntington’s disease research
Drug discovery
Disease modelling
Electrophysiological assays (MEA)
Co-culture studies

Product use

ioCells are for research use only

Product resources

ioGlutamatergic Neurons Wild Type and related disease models | User Manual User manual
ioGlutamatergic Neurons Wild Type and related disease models | User Manual

V11

bit.bio

2024

Download
Modelling neurodegeneration: Human isogenic system to study FTD & ALS Poster
Modelling neurodegeneration: Human isogenic system to study FTD & ALS

Oosterveen, et al

bit.bio & Charles River Laboratories

2023

View
Precision Cellular Reprogramming for Scalable and Consistent Human Neurodegenerative Disease Models Talk
Precision Cellular Reprogramming for Scalable and Consistent Human Neurodegenerative Disease Models

Madeleine Garrett | Field Application Specialist | bit.bio

Watch now
How to culture ioGlutamatergic Neurons HTT 50CAG/WT Video tutorial
How to culture ioGlutamatergic Neurons HTT 50CAG/WT

Madeleine Garrett | Field Application Scientist | bit.bio

Watch now
Introducing ioGlutamatergic Neurons HTT 50CAG/WT | A next-generation approach to study Huntington's disease Video
Introducing ioGlutamatergic Neurons HTT 50CAG/WT | A next-generation approach to study Huntington's disease

bit.bio

Watch
Improving Huntington’s disease drug discovery with new reproducible disease models Webinar
Improving Huntington’s disease drug discovery with new reproducible disease models

Dr Emma V Jones | Senior Scientist | Medicines Discovery Catapult

Dr Tony Oosterveen | Senior Scientist | bit.bio

Watch now
Modelling human neurodegenerative diseases in research & drug discovery Webinar
Modelling human neurodegenerative diseases in research & drug discovery

Dr Mariangela Iovino | Group Leader | Charles River

Dr Tony Oosterveen | Senior Scientist | bit.bio

Watch now

Developing next-generation in vitro phenotypic assays for Huntington’s disease by combining a precision reprogrammed hiPSC-derived disease model with high-density microelectrode arrays

Read the Application Note to discover how Charles River Laboratories functionally characterised ioGlutamatergic Neurons HTT 50CAG/WT and ioGlutamatergic Neurons developed by bit.bio using the MaxTwo high-density microelectrode array from MaxWell Biosystems.

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Wild Type and Isogenic Disease Model cells: A true comparison.

Further your disease research by pairing our wild type cells with isogenic disease models.

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