bitbio-logos–ioGluts_50CAG_transparent_one_line-1

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.

¹ Pawlowski et al., Stem Cell Report 2017

Product specifications

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 email info@bit.bio

Consistent

Batch to batch reproducibility and homogeneity create a stable human model for excitatory neuronal activity and disease.

Quick-orange-text

Ready for experimentation within days post revival.

Scalable-orange-text

Industrial scale quantities at a price point that allows the cells to be used from research to screening scale.

Easy-to-use-orange-text

Cells arrive programmed to rapidly mature upon revival. One medium required in a two-step protocol.

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 HTT50CAG/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.

 

ioGlutamatergic Neurons HTT50CAG/WT express neuron-specific markers with protein expression
highly reminiscent to the isogenic control

ioGlutamatergic Neurons HTT50CAG/WT

a.HTT50CAGWT_DAPI(B)

DAPI

a.HTT50CAGWT__TUBB3(G)

TUBB3

a.HTT50CAGWT__MAP2(R)

MAP2

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

MERGE

ioGlutamatergic Neurons - isogenic control

a.WT_DAPI(B)-croped

DAPI

a.WT__TUBB3(G)

TUBB3

a.WT__MAP2(R)

MAP2

a.WT_Overlay__TUBB3(G)_MAP2(R)_DAPI(B)_

MERGE

Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of pan-neuronal proteins (MAP2 and TUBB3) in ioGlutamatergic Neurons HTT50CAG/WT compared to the isogenic control. 100X magnification.

ioGlutamatergic Neurons HTT50CAG/WT

b.HTT50CAGWT_DAPI(B)-croped

DAPI

b.HTT50CAGWT_VGLUT2(G)

VGLUT2

b.HTT50CAGWT_MAP2(R)

MAP2

b.HTT50CAGWT_Overlay__VGLUT2(G)_MAP2(R)_DAPI(B)_

MERGE

ioGlutamatergic Neurons - isogenic control

b.WT_DAPI(B)

DAPI

b.WT_VGLUT2(G)

VGLUT2

b.WT_MAP2(R)

MAP2

b.WT_Overlay__VGLUT2(G)_MAP2(R)_DAPI(B)_

MERGE

Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of glutamatergic neuron-specific transporter (VGLUT2) 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

a.HTT50CAGWT_DAY1-croped

Day 1

a.HTT50CAGWT_DAY4

Day 4

a.HTT50CAGWT_DAY7

Day 7

a.HTT50CAGWT_DAY11-croped

Day 11

ioGlutamatergic Neurons - isogenic control

a.WT_DAY1

Day 1

a.WT_DAY4

Day 4

a.WT_DAY7

Day 7

a.WT_DAY11

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/WT demonstrate gene expression of neuronal specific and glutamatergic-specific markers following reprogramming

Gene_expression_day_11_orange_black-1

 

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.

Genotype validation of heterozygous 50 CAG repeat expansion

bit.bio_ioGlutamatergicNeuronsHTT50CAG_Confirmation of mutation Gel electrophoresis

 

(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

bit.bio_ioGlutamatergicNeuronsHTT50CAG_CAGrepeatconfirmation_25.4.22

 

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 

bit.bio_ioGlutamatergic Neurons HTT50CAGWT gene expression of HTT

 

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.

Cells arrive ready to plate

bit.bio-ioglutamatergic-neurons-htt-50cag-wt_horizontal bit.bio-ioglutamatergic-neurons-htt-50cag-wt_vertical
bitbio-ioGlutamatergic_neurons-HTT50CAG-WT-Square

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.

Cost effective and flexible

bitbio-well_plate_diagram_ioGlutamatergic _Neurons_HTT50CAGWT
ioGlutamatergic Neurons HTT50CAG/WT are compatible with plates ranging from 6 to 384 wells and are available in two vial sizes, tailored to suit your experimental needs with minimal waste. Recommended seeding density for ioGlutamatergic Neurons HTT50CAG/WT is 30,000 cells/cm2, compared to up to 250,000 cells/cm2 for other available 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 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.

Product specifications


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


Supporting documentation


 Download resources