ioGlutamatergic Neurons TDP-43 M337V

Human iPSC-derived glutamatergic neurons with a M337V mutation in the TDP-43 encoding TARDBP gene for the study of ALS and FTD.

ioEA1005 | Homozygous mutation
ioEA1006 | Heterozygous mutation

ioGlutamatergic Neurons TDP-43 disease model cells are induced pluripotent stem cell (iPSC)-derived glutamatergic neurons carrying a M337V mutation in the TARDBP gene, encoding TAR DNA binding protein 43 (TDP-43), which is associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The disease model cells are available with either a homozygous (M337V/M337V) or heterozygous (M337V/WT) genotype.

The cells have been reprogrammed from human iPSCs using our precision reprogramming technology: opti-ox™. Within days, these iPSCs convert consistently into mature, functional glutamatergic neurons providing a high-quality human model for studying FTD and ALS.

The M337V mutation has been introduced into one, or both TARDBP alleles using CRISPR/Cas9 genome editing, validated with sanger sequencing. With the exception of the M337V mutation, the disease model cells are identical to wild type ioGlutamatergic Neurons. As a result, the wild type cells form a genetically matched control for the disease model. This physiologically-relevant isogenic pairing offers a fast and easy-to-use system for investigations into the impact of the mutant TDP-43 protein on the disease progression.

Both the heterozygous and homozygous variants of the disease model have been characterised by ICC and RT-qPCR. By day 11 post-revival, the cells have been shown to express pan-neuronal and glutamatergic markers TUBB3, MAP2 and VGLUT2, as well as disease-relevant TARDBP.

Product specifications

Advantages

Genetically matched isogenic control
Highly characterised and defined
Ready for experimentation within days
Cost effective and flexible

Applications

– FTD and ALS research
– Drug discovery and development
– Neurodegenerative disease modelling
– High-throughput screening
– 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

Cells arrive cryopreserved and are ready for experimentation in just 11 days post revival.

Scalable

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

Easy-to-use

Cells arrive programmed to mature rapidly with a simple two-step protocol that requires a single open-source medium.

 

ioGlutamatergic Neurons TDP-43 disease model cells express neuron-specific markers
comparably to the isogenic control

ioGlutamatergic Neurons TDP-43M337V/WT

ICC showing MAP2 TUBB3 DAPI on the heterozygous TDP-43 disease model

ioGlutamatergic Neurons TDP-43M337V/M337V

ICC MAP2 TUBB3 DAPI TDP-43 hom ver 3

ioGlutamatergic Neurons - isogenic control

ICC showing MAP2 TUBB3 DAPI on the TDP-43 isogenic control

Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of pan-neuronal proteins (TUBB3 and MAP2) in ioGlutamatergic Neurons TDP-43M337V/WT and ioGlutamatergic Neurons TDP-43M337V/M337V compared to the isogenic control. 100X magnification.

ioGlutamatergic Neurons TDP-43M337V/WT

ICC showing MAP2 VGLUT2 DAPI on the heterozygous TDP-43 disease model

ioGlutamatergic Neurons TDP-43M337V/M337V

ICC MAP2 VGLUT2 DAPI TDP-43 hom ver 3

ioGlutamatergic Neurons - isogenic control

ICC showing MAP2 VGLUT2 DAPI on the TDP-43 isogenic control

Immunofluorescent staining on post-revival day 11 demonstrates a similar homogenous expression of glutamatergic neuron-specific transporter VGLUT2, and pan-neuronal protein MAP2 in ioGlutamatergic Neurons TDP-43M337V/WT and ioGlutamatergic Neurons TDP-43M337V/M337V compared to the isogenic control. 100x magnification.

ioGlutamatergic Neurons TDP-43 disease model cells form structural neuronal networks by day 11

ioGlutamatergic Neurons TDP-43M337V/WT 

ioGlutamatergic Neurons TDP-43 M337V/WT brightfield day 1

Day 1

ioGlutamatergic Neurons TDP-43 M337V/WT brightfield day 4

Day 4

ioGlutamatergic Neurons TDP-43 M337V/WT brightfield day 7

Day 7

ioGlutamatergic Neurons TDP-43 M337V/WT brightfield day 11

Day 11

ioGlutamatergic Neurons TDP-43M337V/M337V

ioGlutamatergic Neurons TDP-43 M337V/M337V brightfield day 1

Day 1

ioGlutamatergic Neurons TDP-43 M337V/M337V brightfield day 4

Day 4

ioGlutamatergic Neurons TDP-43 M337V/M337V brightfield day 7

Day 7

ioGlutamatergic Neurons TDP-43 M337V/M337V brightfield day 11

Day 11

ioGlutamatergic Neurons - isogenic control

isogenic control brightfield day 1

Day 1

isogenic control brightfield day 4

Day 4

isogenic control brightfield day 7

Day 7

isogenic control brightfield day 11

Day 11

ioGlutamatergic Neurons TDP-43M337V/WT and ioGlutamatergic Neurons TDP-43M337V/M337V 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 TDP-43 disease model cells demonstrate gene expression of
neuronal-specific and glutamatergic-specific markers following reprogramming

ioGlutamatergic Neurons TDP-43M337V/WT

key markers are expressed

 

ioGlutamatergic Neurons TDP-43M337V/M337V

TDP-43 hom expression data

 

Gene expression analysis demonstrates that ioGlutamatergic Neurons TDP-43 disease model cells and the isogenic control (WT) lack the expression of pluripotency makers (NANOG and OCT4) at day 11, 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 were assessed by RT-qPCR (data expressed relative to the parental hiPSC control (iPSC Control), normalised to HMBS). Data represents day 11 post-revival samples.

Disease-related TARDBP is expressed in ioGlutamatergic Neurons TDP-43 disease model cells

 

TARDBP expression het and hom

 

Gene expression analysis demonstrates that ioGlutamatergic Neurons TDP-43 disease model cells  and the isogenic control (WT) express the TARDBP gene encoding the TDP-43 protein at day 11. Gene expression levels were assessed by RT-qPCR. Data is expressed relative to the parental hiPSC control (iPSC Control), normalised to HMBS, and represents day 11 post-revival samples.

Cells arrive ready to plate

ioGluts TDP-43 M337V are ready in just 11 days ioGluts TDP-43 M337V are ready in just 11 days
A vial of ioGlutamatergic Neurons TDP-43 M337V/WT

The cells are delivered in a cryopreserved format and 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) where ioGlutamatergic Neuron TDP-43 disease model cells mature. Phases 1 and 2 are carried out at the customer site after revival of the cells.

Cost effective and flexible

How many wells you can seed with just one vial
ioGlutamatergic Neurons TDP-43 disease model cells 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 TDP-43M337V/WT and ioGlutamatergic Neurons TDP-43M337V/M337V is 30,000 cells/cm2, compared to up to 500,000 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-ox cellular reprogramming (Pawlowski, 2017)

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 (ICC), genotype (Sanger seq) 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
Homozygous (ioEA1005) or heterozygous (ioEA1006) M337V mutation in the TARDBP gene


Supporting documentation


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