iPSC-derived sensory neuron

We’ve used our expertise in neural differentiation to bring you human dorsal root ganglion (DRG) neurons derived from induced pluripotent stem cells (iPSCs).

Axol Human iPSC-Sensory Neuron Progenitors are derived from integration-free iPSCs and have been differentiated to neurons using small molecules. We also offer a fully optimized cell culture system including tailored Sensory Neuron Maintenance Medium and coating reagents to promote the viability and maturation of sensory neurons for endpoint assays on glass or plastic.

Our iPSC-derived sensory neurons express several voltage-gated sodium ion channels and transient receptor potential (TRP) ion channels that play a key role in nociception. These include sodium ion channels Nav1.7 and the DRG-specific, TTX-resistant channels, Nav1.8 and Nav1.9 as well as the temperature-sensitive, TRPV1 and TRPM8, and TRPA1, a sensor of pungency, bitterness and cold.

Axol iPSC-Derived Sensory Neuron Progenitors are available in large batch sizes for reliable and consistent results in high-throughput screening assays. The cells are also suitable for investigating disorders of the peripheral nervous system and chronic pain as well as drug targets for pain relief.

Products

RNA Sequencing Data

Axol has performed in-depth RNA-seq analysis and characterization of the mature Sensory neurons of a Nociceptor phenotype which are generated using the Axol Sensory neuron kit.

Sensory neurons are highly identified as having key roles in both Pain and Itch sensation and as such have been well characterized for their expressed gene sets.

Our RNA-seq data clearly shows the nociceptor neurons achieved and express typical canonical markers of sensory neurons such as TAC1 (substance P/neurokinin A precursor), RUNX1, SCN9A (NaV1.7), SCN10A (NaV1.8) and P2X3. As well as others.

The accompanying data shown characterizes the transcriptome of IPSC-sensory neurons during their differentiation from IPSC-Sensory progenitor to IPSC-sensory neuron and shows grouping and prevalence of key critical genes.

We note that statistically significant levels of SCN10A (NaV1.8) were not detected by the microarray, but it has been shown that a low level of expression can be observed by a more sensitive qPCR-based expression assay and electrophysiology methods.

The full primary data sets generated for our transcriptome analysis are available from Gene Expression Omnibus.

Phase contrast

Phase contrast images show the differentiation and maturation of Axol Human iPSC-derived Sensory Neuron Progenitors over two weeks after thawing and treatment with mitomycin C.

Phase contrast images of Axol Human iPSC-derived Sensory Neuron Progenitors The cells were plated on SureBond-XF in Neural Plating-XF Medium. The cells were then treated with mitomycin C two days after thawing and cultured in the supplemented Sensory Neuron Maintenance Medium. (iPSC-sensory neurons should be culture for a minimum of 3 weeks prior to performing endpoint assays.)

ReadyFect leads to 30-40% transfection efficiency. The image demonstrates transfection results using 3 µL of ReadyFect / 1 µg of a GFP-encoding vector (pVectOZ-GFP). GFP expression was evaluated two days post-transfection.

Sodium ion channel expression

Sodium channel RNA expression analysis by cDNA PCR

Axol iPSC-derived Sensory Neuron Progenitors show RNA expression of all three voltage-gated sodium ion channels, Nav1.7, Nav1.8 and Nav1.9.

cDNA from iPSC-derived Sensory Neurons was compared to cDNA from human tissue from the dorsal root ganglion (DRG). PCR analysis (40 cycles; 55oC ) confirmed the mRNA expression of SCN9A (82 bp, hNav1.7), SCN10A (149 bp, hNav1.8) and SCN11A (464 bp, hNav1.9) in Axol iPSC-derived sensory neurons. SCN5a (237 bp, hNav1.5) was included as a negative control. Data provided by Dr Edward Emery (University College London).

TTX-resistant Nav1.8 and Nav1.9

Nociceptive sensory neurons are unique in that they contain voltage-gated inward current sodium channels (Nav 1.8 and Nav 1.9) that are resistant to tetrodotoxin (TTX). The presence of these TTX-resistant ion channels was confirmed in Axol iPSC-derived sensory neurons.

Electrophysiological characterization of Axol Human iPSC-derived Sensory Neurons using patch clamp. A) Phase contrast image of iPSC-derived sensory neurons; B) Example of a sodium-current elicited by a voltage step from -100 mV to -25 mV in the presence of tetrodotoxin (0.5 mM); C) Current-voltage plot of averaged Na-currents recorded from iPSC-derived sensory neurons in the presence of TTX (n=9). Data provided by Dr Edward Emery (University College London).

TRPA1–mediated response to allyl isiothiocyanate

Axol iPSC-derived sensory neurons show a short burst of firing after the application of allyl isiothiocyanate (AIT), which indicates the presence of TRPA1 channels.

Axol human iPSC-derived sensory neurons show a short burst of firing after the application of 100 μM allyl isothiocyanate. The cells were cultured at 5.0 × 105 cells/cm2 on 64-channel MED-P515A MEA chips (Alpha MED Scientific). This rastor plot shows the firing frequency of sensory neurons before and after the application of 100 μM allyl isothiocyanate (AIT). A short burst of firing after the application of AIT is observed, as expected. Data provided by Prof. Ikuro Suzuki (Tohoku Institute of Technology).

Axol iPSC-derived sensory neurons have been shown to increase neuronal firing after the application of capsaicin, which indicates the presence of TRPV1 channels.

Axol human iPSC-derived sensory neurons show sustained firing after the application of 100 nM capsaicin. The cells were cultured at 5.0 × 105 cells/cm2 on 64-channel MED-P515A MEA chips (Alpha MED Scientific). This rastor plot shows the firing frequency of sensory neurons before and after the application of 100 nM capsaicin. Sustained firing is observed after the application of capsaicin. Data provided by Prof. Ikuro Suzuki (Tohoku Institute of Technology).

TRPV1-mediated response to temperature

Axol iPSC-derived Sensory Neurons subjected to an increase in temperature at day 14 and 21 results in an increase in neuronal firing. This suggests the expression of TRPV1.

Axol human iPSC-derived sensory neurons show an increase in neuronal firing in response to temperature The cells were cultured at 5.0 × 105 cells/cm2 in a 37°C , 5% CO2 atmosphere on 64-channel MED-P515A MEA chips (Alpha MED Scientific). Spontaneous and evoked extracellular field potentials were acquired at a sampling rate of 20 kHz/channel and signals were high-pass filtered at 100 Hz. Firing analyses and spike sortings were performed using Mobius software (Alpha MED Scientific) on days 14 (n=218) and 21 (n=510) (Alpha MED Scientific). Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology).

TRPM8-mediated response to menthol

Axol iPSC-derived sensory neurons show an increase in firing frequency after the application of menthol, which indicates the presence of TRPM8 channels.

Axol human iPSC-derived sensory neurons show an increase in firing frequency after the application of 100 μM menthol. The cells were cultured at 5.0 × 105 cells/cm2 on 64-channel MED-P515A MEA chips (Alpha MED Scientific). This rastor plot shows the firing frequency of sensory neurons before and after the application of 100 μM menthol. Data provided by Prof. Ikuro Suzuki (Tohoku Institute of Technology).

Electrical activity

Axol Human iPSC-derived Sensory Neuron Progenitors display electrical activity.

Axol Human iPSC-derived Sensory Neuron Progenitors show spontaneous extracellular field potential on a 64-channel MEA (Alpha MED Scientific) after 33 days in culture. Cells were seeded at 5×105 cells/cm2 . Data provided by Prof. Ikuro Suzuki (Tohoku Institute of Technology).

Drug treatment

Chemotherapy-induced neuropathic pain in the peripheral nervous system

Functional Characterization of Neuropathic Pain: Chemotherapy drugs, vincristine and oxaliplatin, can result in the toxic side effect of peripheral neuropathy. The application of both of these chemotherapy drugs to hiPSC-sensory neurons resulted in an acute increase in firing rate, the time taken for the hiPSC-sensory neurons to respond was slow in comparison to the application of capsaicin, menthol and AITC. This image shows the rastor plot and array-wide spike detection rate (AWSDR) before and after administration of A. vincristine 10μM and B. oxaliplatin 10μM. Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology).

Oxaliplatin-induced cold hypersensitivity

Characterization of Hypersensitivity Induced by Chemotherapy Drug Oxaliplatin

Characterization of Hypersensitivity Induced by Chemotherapy Drug Oxaliplatin: Oxaliplatin has further been implicated in the exacerbation of cold sensation. Here Oxaliplatin was shown to increase the firing rate of hiPSC-sensory neurons in response to AITC in a dose-dependent manner. Oxaliplatin results in a hypersensitivity to cold stimuli in hiPSC-sensory neurons. A. The response of hiPSC-sensory neuron to the application of AITC 50μM (control) B. hiPSC-sensory neuron treated with varying concentrations of oxaliplatin (10, 30 and 100μM), after 2 hours AITC 50μM was administered and the response was measured. C. Percentage increase of the number of firing spikes compared to the vehicle control. Treatment of oxaliplatin resulted in a dose-dependent increase in firing rate. (n=6, *p < 0.05, **p < 0.0005). Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology).

Menu