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Sensory Neuron Progenitors Brochure Human iPSC-derived Sensory Neuron Progenitors Phase Contrast Sodium Ion Channel Expression Transient Receptor Potential Ion Channel Expression Drug Treatment ax0055 hiPSC-derived Sensory Neuron Progenitors (Male) ax0057 hiPSC-derived Sensory Neuron Progenitor (Kit) Protocol Human iPSC-derived Sensory Neuron Progenitors We’ve used our expertise in neural differentiation to bring The cells were plated on SureBond-XF in Neural Plating-XF Medium. The cells were then treated with mitomycin C two days you dorsal root ganglion (DRG) neurons derived from induced after thawing and cultured in Sensory Neuron Maintenance Medium containing growth factors (GDNF, NGF, BDNF and NT-3) pluripotent stem cells (iPSCs). Axol Human iPSC-Sensory for two weeks. (iPSC-sensory neurons should be cultured for a minimum of 6 weeks prior to performing endpoint assays.) Neuron Progenitors are derived from integration-free iPSCs of a healthy male donor and have been differentiated to neurons using small molecule inhibitors. We also offer a fully Sodium Ion Channel Expression optimized cell culture system including tailored Sensory Neuron Maintenance Medium and coating reagents to Sodium channel RNA expression analysis by cDNA PCR promote the viability and maturation of sensory neurons for endpoint assays on glass or plastic. Axol’s iPSC-Derived Sensory Neuron Progenitors show RNA expression of all three voltage-gated sodium ion channels, Nav1.7, Nav1.8 and Nav1.9. 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’s 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. Phase Contrast Phase contrast images show the maturation of Axol Human iPSC-Derived Sensory Neuron Progenitors over two weeks after thawing and treatment with mitomycin C. cDNA from iPSC-Derived Sensory Neuron Progenitors cultured for 8 weeks 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). 01 02 Human iPSC-derived Sensory Neuron Progenitors Sodium channel-mediated response to potassium chloride Nav1.7 immunocytochemistry Axol iPSC-Derived Sensory Neuron Progenitors have been shown to elicit a calcium response to the treatment of KCl at the Axol Human iPSC-Derived Sensory Neuron Progenitors express the nociceptive voltage-gated sodium ion channel Nav1.7 soma signifying the presence of sodium channels. after 63 days culture in Sensory Neuron Maintenance Medium on a multi-electrode array (MEA) (Alpha MED Scientific). Nav1.7 (red), β-tubulin III (green), Hoechst (blue). Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology). Somal response to 15 mM potassium chloride in iPSC- derived sensory neurons Each trace represents the change in Fluo-4 fluorescence intensity from one iPSC- Average peak response to potassium chloride Each bar derived sensory neuron to the bath application of 15 mM illustrates peak fluorescence at baseline and during a 60 Nav1.7 and Nav1.8 immunocytochemistry KCl at the soma. Data collected from n=4 coverslips 4 second bath application of 15 mM KCl at the soma. Data Mature iPSC-derived sensory neurons express the sodium ion channels, Nav1.7 and Nav1.8, and the maturation marker, weeks post plating. collected from n=4 coverslips 4 weeks post plating. doublecortin (DCX) after 6 weeks in culture. Data provided by Dr Ramin Raouf and Natasha Rangwani (King’s 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. Axol Human iPSC-Derived Sensory Neuron Progenitors show positive staining for the nociceptive voltage-gated sodium ion channels A) Nav1.7 (green), Nav1.8 (red), DAPI (blue) after 38 days in culture and B) Nav1.8 (red) and doublecortin (green) Electrophysiological characterization of Axol Human iPSC-Derived Sensory Neuron Progenitors using patch clamp after after 42 days in culture. 56 days in culture A) Phase contrast image of iPSC-derived sensory neurons at 8 weeks; 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). 03 04 Human iPSC-derived Sensory Neuron Progenitors Transient receptor potential ion channel expression TRPA1 immunocytochemistry Axol Human iPSC-Derived Sensory Neuron Progenitors express TRPA1, the ion channel involved in sensing cold, pungency and bitterness. TRPV1 immunocytochemistry Axol Human iPSC-Derived Sensory Neuron Progenitors express TRPV1, the ion channel involved in sensing heat. Axol Human iPSC-Derived Sensory Neuron Progenitors express the TRPV1 ion channel after 63 days culture in Sensory Neuron Maintenance Medium on a multi-electrode array (MEA) (Alpha MED Scientific). TRPV1 (yellow), β-tubulin III (green), Hoechst Axol Human iPSC-Derived Sensory Neuron Progenitors express the TRPA1 ion channel after 63 days culture in Sensory (blue). Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology). Neuron Maintenance Medium on a multi-electrode array (MEA) (Alpha MED Scientific). TRPA1 (purple), β-tubulin (green), Hoechst (blue). Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology). TRPA1 immunocytochemistry 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) coated with SureBond+ReadySet for 7 weeks to obtain mature sensory neurons. 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). 05 06 Human iPSC-derived Sensory Neuron Progenitors TRPV1-mediated response to capsaicin 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 iPSC-Derived Sensory Neuron Progenitors have been shown to elicit a calcium response to the treatment of capsaicin at both the soma and the axon. This indicates that the iPSC-Derived Sensory Neurons express TRPV1 and can sense heat. Somal response to 500 nM capsaicin in iPSC-derived sensory neurons Each trace represents the response from one iPSC- derived sensory neuron to 500 nM capsaicin (CAP) using calcium dye Fluo-4 at the soma. Data collected from n=3 coverslips 4 weeks post plating. Axonal response to 500 nM capsaicin Axol human iPSC-derived sensory neurons show sustained firing after the application of 100 nM capsaicin. The in iPSC-derived sensory neurons. Each cells were cultured at 5.0 × 105 cells/cm² on 64-channel MED-P515A MEA chips (Alpha MED Scientific) coated with trace represents the change in Fluo-4 SureBond+ReadySet for 6 weeks to obtain mature sensory neurons. This raster plot shows the firing frequency of sensory fluorescence intensity in the axons from neurons before and after the application of 100 nM capsaicin. Sustained firing is observed after the application of capsaicin. iPSC-derived sensory neuron in response to Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology). bath application of 500 nM capsaicin (CAP). Data collected from n=2 coverslips 4 weeks post plating. Average peak response to capsaicin. Each bar illustrates peak fluorescence at baseline and during a 90 second bath application of capsaicin (500nM) at the soma. Data collected from n=3 coverslips 4 weeks post plating. Data provided by Dr Ramin Raouf and Natasha Rangwani (King’s College London). 07 08 Human iPSC-derived Sensory Neuron Progenitors 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) coated with SureBond+ReadySet. 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. 37°C 42.6°C 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 5x105 cells/cm² and treated with 2.5 µg/ml mitomycin C. Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology).
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