A rapidly maturing, consistent and scalable isogenic system to study Huntington’s disease.
ioGlutamatergic Neurons HTT50CAG/WT are opti-ox™ precision reprogrammed glutamatergic neurons containing a genetically engineered heterozygous 50 CAG trinucleotide repeat expansion in exon 1 of the HTT gene. Human stem cells, within days, convert consistently into mature, functional glutamatergic neurons that express pan-neuronal and glutamatergic markers TUBB3, MAP2 and VGLUT2 by day 11, as well as the disease-relevant Huntingtin protein.
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 fast and easy-to-use next-generation model to investigate into the impact of gene function on disease progression.
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ioGlutamatergic Neurons HTT50CAG/WT generated by transcription factor-driven reprogramming 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 HTT50CAG/WT express neuron-specific markers with protein expression highly reminiscent to the isogenic control
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 HTT50CAG/WT compared to the isogenic control. 100X magnification.
ioGlutamatergic Neurons HTT50CAG/WT form structural networks by day 11
ioGlutamatergic Neurons HTT50CAG/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 HTT50CAG/WTdemonstrate gene expression of neuronal-specific and glutamatergic-specific markers following reprogramming
Gene expression analysis demonstrates that ioGlutamatergic Neurons HTT50CAG/WT (50CAG/WT) and the isogenic control (WT) at day 11 lack the expression of pluripotency makers (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 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/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 of heterozygous 50 CAG repeat expansion
(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 HTT50CAG/WT). PCR fragments at 395 bps 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 confirms the number of CAG repeats in wild type ioGlutamatergic Neurons (yellow) and ioGlutamatergic Neurons HTT50CAG/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 HTT50CAG/WT.
Industry leading seeding density
Do more with every vial
The recommended seeding density of our human iPSC-derived Huntington’s disease model and isogenic control has been optimised and validated making it possible to have a cost point of under £0.67 per well (~$0.79) (96 well plate, seeding density 30,000 cells/cm2, Large vial size).
This means scientists are able to do more with every vial and expand experimental design within budget without losing out on quality. Resulting in more experimental conditions, more repeats, and more confidence in the data.
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 plate.
One Large vial can plate a minimum of 3.6 x 24-well plate, 5.4 x 96-well plate, or 7.75 x 384-well plates.
Cells arrive ready to plate
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.