Batch to batch reproducibility and homogeneity create a stable human model for excitatory neuronal activity and disease.
Human iPSC-derived glutamatergic neurons HTT50CAG/WT
ioEA1004 | Early Access
ioGlutamatergic Neurons HTT50CAG/WT are ioGlutamatergic Neurons carrying the disease-relevant 50 CAG trinucleotide repeat expansion, associated with Huntington’s disease. ioGlutamatergic Neuron HTT50CAG/WT have been reprogrammed from human iPSCs using our precise reprogramming technology: opti-ox™1 (optimised inducible overexpression). Using CRISPR/Cas9 genome editing we have introduced an abnormal expansion of 50 CAG repeats in the first exon of the Huntingtin gene. Human stem cells, within days, convert consistently into mature, functional glutamatergic neurons providing a high quality human model for the study of Huntington’s disease.
bit.bio’s wild type ioGlutamatergic Neurons form the genetically matched control for the ioGlutamatergic Neurons HTT50CAG/WT disease model. This physiologically-relevant isogenic pairing offers a powerful next generation model to study Huntington’s disease in research and drug discovery.
ioGlutamatergic Neurons HTT50CAG/WT express pan-neuronal and glutamatergic markers TUBB3, MAP2 and VGLUT2 by day 11, as well as the disease-relevant Huntingtin protein.
This disease model offers a fast and easy-to-use system for investigations into the impact of gene function on disease progression against an isogenic control.
Advantages
– Highly characterised isogenic control
– Ready for experimentation within days
– Highly characterised and defined
– Easy culturing
– Genotype validation
Applications
– Academic research
– Drug development
– Drug discovery
– High-throughput screening
– Disease modelling
– Electrophysiological assays (MEA)
– Co-culture studies with astrocytes
For more information download our brochure
For more information email info@bit.bio
Batch to batch reproducibility and homogeneity create a stable human model for excitatory neuronal activity and disease.
Ready for experimentation within days post revival.
Industrial scale quantities at a price point that allows the cells to be used from research to screening scale.
Cells arrive programmed to rapidly mature upon revival. One medium required in a two-step protocol.
ioGlutamatergic Neurons HTT50CAG/WT
DAPI
TUBB3
MAP2
MERGE
ioGlutamatergic Neurons - isogenic control
DAPI
TUBB3
MAP2
MERGE
ioGlutamatergic Neurons HTT50CAG/WT
DAPI
VGLUT2
MAP2
MERGE
ioGlutamatergic Neurons - isogenic control
DAPI
VGLUT2
MAP2
MERGE
ioGlutamatergic Neurons HTT50CAG/WT form structural networks by Day 11
ioGlutamatergic Neurons HTT50CAG/WT
Day 1
Day 4
Day 7
Day 11
ioGlutamatergic Neurons - isogenic control
Day 1
Day 4
Day 7
Day 11
ioGlutamatergic Neurons HTT50CAG/WT demonstrate gene expression of neuronal specific and glutamatergic-specific markers following reprogramming
(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 320bp, by PCR, in both isogenic control (ioGlutamatergic Neurons) and disease model (ioGlutamatergic Neurons HTT50CAG/WT). PCR fragments at 395bp detect on-target gene editing and introduction of a 50 CAG repeat expansion in ioGlutamatergic Neurons HTT50CAG/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 HTT50CAG/WT.
Genotype validation of the number of CAG repeats
NGS-Amplicon Sequencing was employed to confirm the number of CAG repeats in both the ioGlutamatergic Neurons HTT50CAG/WT (orange) and isogenic control (black). The number of CAG repeat reads peak at 24 for both the isogenic control and ioGlutamatergic Neurons HTT50CAG/WT. The 50 CAG repeat expansion was detected only in the ioGlutamatergic Neurons HTT50CAG/WT (orange) confirming a successful introduction of a heterozygous
50 CAG repeat expansion.
Disease-related Huntingtin (HTT) is expressed in ioGlutamatergic Neurons HTT50CAG/WT
RT-qPCR analysis demonstrates similar expression level of the Huntingtin gene in both wild type ioGlutamatergic Neurons (WT) and ioGlutamatergic Neurons HTT50CAG/WT (50CAG) at day 11 post-revival (n=2 replicates). cDNA samples of the parental iPSC line
(iPSC control) were included as a reference.
ioGlutamatergic Neurons HTT50CAG/WT 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 three-phase process: Induction, which is carried out at bit.bio (Phase 0), Stabilisation for 4 days (Phase 1), and Maintenance (Phase 2) during which the ioGlutamatergic Neurons HTT50CAG/WT mature. Phases 1 and 2 after revival of cells are carried out at the customer site.
Starting material
human iPSC line
Donor
caucasian adult male
(skin fibroblast)
Differentiation method
opti-oxTM cellular reprogramming
Karyotype
Normal (46, XY)
Vial size
Small: >1 x 106 viable cells
Large: >5 x 106 viable cells
Recommended seeding density
30,000 cells/cm2
Seeding compatibility
6 to 384 well plates
Quality control
Sterility, protein expression (IF) and gene expression (RT-qPCR)
User storage
LN2 or -150°C
Shipping info
Dry ice
Product use
These cells are for research use only
Genetic modification
Heterozygous - HTT 50 CAG repeat expansion