Human iPSC-derived Sensory Progenitors

Phase Contrast Sodium Ion Channel Expression Transient Potential Ion Channel Expression Drug Treatment ax0055 hiPSC-derived 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 (DRG) 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 (TRP) ion channels that play a key role in . These include sodium ion channels Nav1.7 and the DRG-specific, TTX-resistant channels, Nav1.8 and Nav1.9 as well as the -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 and chronic 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 (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 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).

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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 . This indicates that the iPSC-Derived Sensory Neurons express TRPV1 and can 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 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).

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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).

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) 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 menthol. Data provided by Prof Ikuro Suzuki (Tohoku Institute of Technology).

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Drug Treatment Oxaliplatin-induced cold hypersensitivity 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 Chemotherapy-induced in the peripheral nervous system response to AITC in a dose-dependent manner. Oxaliplatin results in a hypersensitivity to cold stimuli in hiPSC-sensory Functional Characterization of Neuropathic Pain: Chemotherapy drugs, vincristine and oxaliplatin, can result in the toxic side neurons. A. The response of hiPSC-sensory neuron to the application of AITC 50μM (control) B. hiPSC-sensory neuron effect of . The application of both of these chemotherapy drugs to hiPSC-sensory neurons resulted treated with varying concentrations of oxaliplatin (10, 30 and 100μM), after 2 hours AITC 50μM was administered and the in an acute increase in firing rate, the time taken for the hiPSC-sensory neurons to respond was slow in comparison to the response was measured. C. Percentage increase of the number of firing spikes compared to the vehicle control. Treatment application of capsaicin, menthol and AITC. This image shows the raster plot and array-wide spike detection rate (AWSDR) of oxaliplatin resulted in a dose-dependent increase in firing rate. (n=6, *p < 0.05, **p < 0.0005). Data provided by Prof Ikuro before and after administration of A. vincristine 10μM and B. oxaliplatin 10μM. Data provided by Prof Ikuro Suzuki (Tohoku Suzuki (Tohoku Institute of Technology). Institute of Technology). A. B.

C.

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Products Protocol

Human iPSC-derived sensory neuron progenitors (ax0055) Preparation of reagents

Axol Human iPSC-Derived Sensory Neuron Progenitors Neural Plating-XF Medium derived from a healthy male donor. Axol’s induced pluripotent stem cells (iPSCs) were differentiated to • Upon receipt, store Neural Plating-XF Medium at or below -80°C protected from . sensory neuron progenitors using small molecule • When ready to use, thaw Neural Plating-XF Medium overnight at 4°C in the dark. inhibitors. For endpoint assays on plastic, SureBond-XF • Once thawed, Neural Plating-XF Medium must be used and cannot be refrozen. should be used for coating cell culture surfaces. For endpoint assays on glass, SureBond+ReadySet coating reagent should be used. After thawing and plating, the Sensory Neuron Maintenance Medium progenitors need to be growth arrested and then should be matured for approximately 6 weeks prior to endpoint • Upon receipt, aliquot and store Sensory Neuron Maintenance Medium at or below -80°C protected from light. assays. The expression of Nav1.7 should occur at around • When ready to use, thaw an aliquot of Sensory Neuron Maintenance Medium overnight at 4°C in the dark. 4-5 weeks of maturation and Nav1.8 is expressed after • Prepare 10μg/mL stock solutions of each growth factor by resuspending the lyophilized powder in PBS (1x) supplemented approximately 6-8 weeks of maturation in Sensory with 0.05% human serum albumin (HSA). Neuron Maintenance Medium. • The growth factors can be aliquoted and stored at 4°C for up to 1 week or -20°C for longer storage/ • Prepare Sensory Neuron Maintenance Medium by adding the following growth factors:

Growth Factor Stock concentration Final concentration Volume to add in 20 mL Recombinant Human Glial-Derived Neurotrophic Factor (GDNF) (ax139855) 10 μg/mL 25 ng/mL 50 μL Recombinant Human -Derived Neurotrophic Factor (BDNF) (ax139800) 10 μg/mL 25 ng/mL 50 μL Recombinant Human Growth Factor 10 μg/mL 10 ng/mL 20 μL Human iPSC-derived sensory neuron progenitors kit (ax0057) (NGF) (ax139789) Recombinant Human -3 (NT-3) (ax139811) 10 μg/mL 10 ng/mL 20 μL

• Growth factors should be added fresh each time an aliquot of Sensory Neuron Maintenance Medium is thawed. • A thawed and supplemented aliquot of Sensory Neuron Maintenance Medium can be stored at 4°C for 1 week.

We have put together our hiPSC-Sensory Neuron Progenitors with our optimized media and growth factors in one kit with an easy-to-follow protocol to help you generate the best sensory neuron culture for your research.

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Mitomycin C SureBond • Dilute the SureBond stock solution (1 mg/ml) in D-PBS (1x) (without calcium or magnesium) to a final concentration of 20 • Prepare a 0.5 mg/mL stock concentration of mitomycin C by solubilizing 2 mg in 4 mL of ddH2O. Make 50-100 μL aliquots µg/mL e.g. 120 μL in 6 mL. of mitomycin C (0.5 mg/mL), protect from light and store in a dark box at 4°C. Stored at 4°C, mitomycin C is stable for up to • Coat the surface of your culture vessel with the SureBond 1x working solution. We recommend coating at a volume of 200 8 weeks. μL per cm2. • Incubate overnight at 37ºC. • Remove the SureBond from the culture dish prior to seeding. Do not wash the culture vessel after coating with SureBond. Sensory Neuron Maintenance Medium containing Mytomycin C • Do not let the SureBond coating dry out before seeding the cells.

• Prepare medium containing 2.5 μg/mL of mitomycin C by adding 100 μL of the 0.5 mg/mL stock of mitomycin C to 20 mL of Sensory Neuron Maintenance Medium. Culture of Human iPSC-Derived Sensory Neuron Progenitors • This medium should then be filter sterilized prior to use using a 0.22 μM filter. Thawing and Plating

SureBond-XF Coating Solution • Thaw Neural Plating-XF Medium overnight at 4°C. This coating solution is primarily used for culture of iPSC-derived sensory neuron progenitors on a plastic culture vessel. • Prepare culture vessels by coating with either SureBond-XF 4 hours prior to thawing cells or SureBond+ReadySet on the previous day. • Upon receipt, store SureBond-XF at 4°C. • Pre-warm all media and culture vessels to 37°C before use. • Calculate the total surface area that requires coating. • To thaw the cells – transfer the vial of cells from storage by transporting the vial buried in dry ice. Remove the vial from dry • Dilute the SureBond-XF stock solution (200x) in Dulbecco’s-PBS (1x) (D-PBS without calcium or magnesium) to make 1x ice and transfer it to a 37°C water bath. working solution e.g. 30 μL in 6 mL. • Quickly thaw the vial of cells in a 37°C water bath. Do not completely submerge the vial (only up to 2/3rd of the vial). • Coat the surface of your culture vessel with the SureBond-XF 1x working solution. We recommend coating at a volume of Remove the vial before the last bit of ice has melted, after 1-2 minutes. 200 μL per cm² however, please optimize for your experiments. • Do not shake the vial during thawing. • Incubate for 4 hours at 37°C. • Take the vial of cells to a biological safety cabinet, spraying the vial and hood thoroughly with 70% ethanol and wiping with • Remove the SureBond-XF from the culture dish prior to seeding. Do not wash the culture vessel after coating with an autoclaved paper towel before placing the vial in the hood. SureBond-XF.

SureBond+ReadySet Coating Solution This coating solution is primarily used for culture of iPSC-derived sensory neuron progenitors on a glass culture vessel or multi-electrode array plate.

• Upon receipt store SureBond at or below -80°C and store ReadySet at 4°C. • Thaw the SureBond coating solution overnight at 4°C. • Calculate the total surface area that requires coating. • If using glass coverslips, clean coverslips thoroughly before coating with SureBond+ReadySet.

ReadySet • Pre-coat your culture vessel with ReadySet at a volume of 250 μL per cm². • Incubate at 37ºC for 45 minutes. • Wash the plate thoroughly four times using an equal volume of sterile distilled H2O (e.g. if 250 µL of ReadySet, use 250 µL sterile distilled H2O). During each wash rock the dish to ensure thorough washing. • Do not let the ReadySet dry out following washing, proceed straight to coating with SureBond.

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• Using a P1000 pipette, transfer the cell suspension into a 15 mL sterile conical tube. Gently wash the cryogenic vial with 1 mL of warm Neural Plating-XF Medium and transfer this to the 15 mL sterile conical tube. • Centrifuge cells at 200 x g for 5 minutes at room temperature. • Aspirate and discard the supernatant carefully with a pipette. • Using a P1000 pipette, gently resuspend the cell pellet in 1 mL of Neural PlatingXF Medium until they are in a single cell suspension. • Perform a cell count to ensure optimal seeding density. • Plate the resuspended cells drop-wise and evenly at a density ranging from 100,000 - 150,000 cells/cm². • Gently rock the culture vessel back and forth to ensure an even seeding density. • Incubate the cells at 37°C, 5% CO2. • The day after plating, replace the medium with fresh pre-warmed, 37°C, Sensory Neuron Maintenance Medium supplemented with GDNF (25 ng/mL), NGF (25 ng/mL), BDNF (10 ng/mL), NT-3 (10 ng/mL).

Growth Arrest and Purification

• Two days after thawing the Human iPSC-Derived Sensory Neuron Progenitors remove all of the culture medium and replace with Sensory Neuron Maintenance Medium containing 2.5 μg/mL of mitomycin C. • Incubate the cells for 2 hours at 37ºC, 5% CO2. • After the incubation period, remove the Sensory Neuron Maintenance Medium containing 2.5 μg/mL of mitomycin C from the culture and gently wash the cells once with D-PBS (1x) (without calcium or magnesium). • After washing, add pre-warmed, 37ºC, Sensory Neuron Maintenance Medium supplemented with GDNF (25 ng/mL), NGF (25 ng/mL), BDNF (10 ng/mL), NT-3 (10 ng/mL). The effect of mitomycin C treatment is not immediate. Non-neuronal cell death will not occur until 4-5 days after treatment. Full effects will be apparent after 7 days.

Maintenance of Human iPSC-Derived Sensory Neuron Progenitors

• To maintain a healthy culture, replace half the volume of medium with fresh prewarmed, 37ºC, Sensory Neuron Maintenance Medium supplemented with GDNF (25 ng/mL), NGF (25 ng/mL), BDNF (10 ng/mL), NT-3 (10 ng/mL) every 3-4 days. • Maintain the neurons in Sensory Neuron Maintenance Medium supplemented with GDNF (25 ng/mL), NGF (25 ng/mL), BDNF (10 ng/mL), NT-3 (10 ng/mL) for a minimum of 6 weeks prior to performing endpoint assays. • After 4-6 weeks of maturation, the sodium channel Nav1.7 should be expressed and after 6-8 weeks of maturation, Nav1.8 should be expressed by the sensory neurons.

Important! Axol Neural Cell Culture Media

DOES NOT contain antibiotics or antifungal agents. Axol Bioscience does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin. Antimicrobial agents should not be necessary if proper aseptic technique is adopted.

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