Immunofluorescence staining of ioMotor Neurons; human induced pluripotent stem cell-derived motor neurons.

cat no | ioEA1027 Early Access

ioMotor Neurons

Human iPSC-derived motor neurons

ioMotor Neurons have been precision reprogrammed from human induced pluripotent stem cells (iPSC) using opti-ox™ technology. Within days, cells convert consistently to defined, functional motor neurons, showing the expression of key lower motor neuron marker genes MNX1(HB9), FOXP1, ISL2 and cholinergic markers CHAT & SLC18A3 (VAChT) by day 4.

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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.

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Benchtop benefits

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Functional

Functional neuronal networks are detected in co-culture with astrocytes from day 14.

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Quick and easy

Within 4 days post revival cells are ready for experimentation, displaying motor neuronal morphology without clumping.

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Defined

>80% cells express key lower motor neuron markers indicating a spinal motor neuron identity (cervical region). >99.9% neuronal population.

Technical data

Ready within days

ioMotor Neurons acquire a rapid motor neuronal phenotype, without clumping

ioMotor Neurons morphology 11 day V2

Time-lapse video capturing the rapid and homogeneous motor neuronal phenotype acquisition upon thawing of cryopreserved ioMotor Neurons, showing no signs of clumping. 11 day time course.

ioMotor Neurons form a homogenous neuronal network by day 4

iomotor_neurons_morphology_images_d1_d4_d11_d18
ioMotor Neurons mature rapidly and form homogenous populations over 18 days. Day 1 to 18 post thawing; 100X magnification.

Rapid gain of functional activity

Functional neuronal networks are detected in astrocyte co-culture from day 14
iomotor_neurons_MEA_mean_firing_rates

ioMotor Neurons are functional – showing activity in astrocyte co-culture that increases over time as networks mature. Mean firing rates (electrode spike count divided by the total time of the recording period) is shown to increase substantially throughout the course of the experiment, as demonstrated by multielectrode array activity (MEA).

image (4)
Spontaneous neuronal activity is exhibited from as early as day 14 and continues to increase up to the final measured timepoint, day 42.

Highly characterised and defined

Immunocytochemistry shows protein expression of key motor neuron markers

iomotor_neurons_ICC_panel2

Immunofluorescent staining on post-revival day 4 and day 11 demonstrates homogenous expression of the pan-neuronal protein TUBB3, motor neuron specific marker ISL2, the cholinergic marker ChAT and nuclear staining (DAPI).

iomotor_neurons_ICC_panel1

Immunofluorescent staining on post-revival day 4 and day 11 demonstrates homogenous expression of the pan-neuronal protein MAP2, motor neuron specific marker HB9, the cholinergic marker VAChT and nuclear staining (DAPI).

RT-qPCR shows gene expression of key motor neuron markers
iomotor_neuron_gene_expression_RTqPCR_data

RT-qPCR gene expression on post-revival days 1, 4, 11 & 18 demonstrates rapid acquisition of motor neuron genotype, shown by the expression of pan-neuronal, cholinergic & key lower motor neuron markers from as early as day 1. Pluripotency markers NANOG and OCT4 are swiftly downregulated.

Bulk RNA-sequencing exhibits a HOX gene signature indicative of a spinal motor neuron (cervical region) identity

Heatmap 22
Expression of HOX genes was evaluated using bulk RNA sequencing data. This heatmap shows expression of genes from the B cluster and expression of HOXC4 and HOXC5, although at lower levels. This data, together with the marker expression from single cell RNA sequencing, suggests that ioMotor Neurons have a spinal cord (cervical region) identity. Note, this data is from cells in continuous culture and not cryopreserved cells.
Single cell RNA-sequencing shows ioMotor Neurons form a pure population (>99.9%) of neurons
scRNA pan-neuronal markers NEW
Single cell RNA-sequencing analysis was performed with ioMotor Neurons at four timepoints: day 0 (iPSCs), 4, 7, and 14. Gene expression was assessed by 10x Genomics single cell RNA-sequencing. Note, this data is from cells in continuous culture and not cryopreserved cells. By day 14, the population has a distinct expression profile indicating a pure population (>99.9%) of post-mitotic neurons, demonstrated through the expression pan-neuronal markers MAP2 and TUBB3.

Single cell RNA-sequencing shows ioMotor Neurons express key spinal motor neuron markers, >80% of cells express MNX1 on day 14

 

scRNA motor markers NEW
Starting from day 4, the expression of the key spinal motor neuron marker genes MNX1 (HB9), FOXP1, and ISL2 is detected in the culture, with >80% of cells expressing MNX1 on day 14. These percentages are likely to be an underestimation due to limitation of single cell RNA sequencing, as ICC for HB9 & ISL2 shows homogeneous expression of these markers in our cultures
Single cell RNA-sequencing shows a high proportion of ioMotor Neurons express cholinergic markers by day 7 
scRNA cholinergic markers NEW
Within 7 days, the expression of the key cholinergic marker genes CHAT & SLC18A3 (VAChT) are detected in a high proportion of ioMotor Neurons.

Batch-to-batch consistency

Bulk RNA-sequencing demonstrates high batch-to-batch consistency of ioMotor Neurons

iomotor_neurons_bulk_rna_seq_PCA
Bulk RNA sequencing analysis was performed on three independent batches of ioMotor Neurons at three different time points throughout the reprogramming protocol. Principal component analysis represents the variance in gene expression between the batches of ioMotor Neurons. This analysis shows high consistency between each batch of ioMotor Neurons at each given timepoint. Populations of ioMotor Neurons with equivalent expression profiles can be generated consistently from every vial, allowing confidence in experimental reproducibility. Note, this data is from cells in continuous culture and not cryopreserved cells.

Industry leading seeding density

Do more with every vial

UPDATED ioMotor seeding graphic
The seeding density of our human iPSC-derived motor neurons has been optimised and validated to a recommended seeding density of 30,000 cells/cm2. This means scientists can 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.

Easy culturing

Cell arrive ready to plate

bit.bio_ioMotor Neurons_timeline_horizontal_withdoxycycline

ioMotor Neurons 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: 1. Stabilisation for 2 days. 2. Pre-maintenance for an additional 2 days. 3. Maintenance of cells according to the protocol and recommended media for the duration of assay requirements. 

Product information

Starting material

Human iPSC line

Seeding compatibility

6, 12, 24, 96 and 384 well plates

Shipping info

Dry ice

Donor

Caucasian adult male (skin fibroblast)

Vial size

Small: >1 x 10⁶ viable cells

Quality control

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

Differentiation method

opti-ox cellular reprogramming

Recommended seeding density

30,000 cells/cm²

User storage

LN2 or -150°C

Format

Cryopreserved cells

Product use

ioCells are for research use only

Applications

Neurodegeneration research
ALS disease modelling
Electrophysiological analysis
Drug development & discovery
Neuromuscular research
Neurotoxicology

Product resources

Empowering motor neuron disease research and drug discovery with a new class of functional, reproducible hiPSC-derived motor neurons Webinar
Empowering motor neuron disease research and drug discovery with a new class of functional, reproducible hiPSC-derived motor neurons
Tom Brown | Senior Product Manager | bit.bio
Marcos Herrera Vaquero, PhD | Senior Scientist | bit.bio
2024
Watch now
Rapid and consistent generation of functional motor neurons from reprogrammed human iPSCs using opti-ox™ technology Poster
Rapid and consistent generation of functional motor neurons from reprogrammed human iPSCs using opti-ox™ technology

Vaquero, et al

bit.bio

2023

Download
ioMotor Neurons™ – User Manual User manual
ioMotor Neurons™ – User Manual
V2
2023
bit.bio
Download
Mastering Cell Identity In A Dish: The Power Of Cellular Reprogramming Webinar
Mastering Cell Identity In A Dish: The Power Of Cellular Reprogramming

Prof Roger Pedersen | Adjunct Professor and Senior Research Scientist at Stanford University 

Dr Thomas Moreau | Director of Cell Biology Research | bit.bio

Watch now

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