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Sensory Neurons That Behave Like Sensory Neurons

When you work with sensory neurons in vitro, you want – need, them to behave like sensory neurons do in vivo. Thankfully, when you use human iPSC-derived sensory neurons from us, that’s exactly what you get. They’re also mature and responsive in under four weeks when you culture them with Sensory Neuron Maturation Maximizer Supplement.

Sensory Neurons With Proper Levels of the Right Channels

Sensory neurons sense. To do that, they need to have transient receptor potential (TRP) and voltage-gated sodium (NaV) channels. Well, our sensory neurons do. And they express TRPV1, NaV 1.7 and NaV 1.8 after 24 days in vitro (DIV) or post-plating (Figure 1). To check levels, iPSC-derived sensory neurons cDNA was compared with cDNA from human dorsal root ganglion (DRG) tissue (Figure 2).

As you can see, there’s good expression of these essential channels.

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Figure 1. ICC from Axol human iPSC-derived sensory neurons cultured with Sensory Neuron Maturation Maximizer Supplement. 24 days in vitro (DIV)/post-plating ICC data showing NaV 1.7, NaV 1.8, TRPV1, and TUJ1 expression.

cDNA from iPSC-derived Sensory Neurons compared to cDNA 
Figure 2. cDNA from iPSC-derived Sensory Neurons 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).

Okay but what about the voltage-gated calcium (CaV) channels? And all the other NaV and TRP channels? We looked into that as well. In fact, we ran a load of RNAseq to check (Figure 3).

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Figure 3. RNAseq analysis of Axol human iPSC-derived sensory neurons for CaV (top), NaV (middle) and TRP (bottom) channels compared with human DRG tissue and other cell types.

Sensory Neurons Respond to Hot and Cold

So, the channels are there, but are they working? They sure are. We ran thermoception assays, where we exposed the sensory neurons to both cooling as heating. Looking at multi-electrode array (MEA) data, you can clearly see the firing responses to the changes in temperature (Figure 4). 

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Figure 4. DIV23 and 24 thermoception data show how human iPSC-derived sensory neurons respond to both cooling and heating.

Sensory Neurons Respond to Classic Compounds

The classic sensory challenges: capsaicin, menthol, and mustard oil. We subjected our sensory neurons, after fewer than four weeks with Sensory Neuron Maturation Maximizer Supplement, to all of these classic compounds and generated MEA data around burst firing and waveform analysis. Our sensory neurons demonstrated their relevant functionality by responding to these in a codependent manner (Figures 5–7). Waveform recordings also nicely show how the action potential waveform is unaffected by exposure to any of these compounds.

What you also see is desensitization to a compound following repeated exposure (tachyphylaxis) (Figure 8). This is how sensory neurons behave in vivo so it’s important to see that behaviour here in vitro.

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Figure 5. Capsaicin treatment and electrophysiological characterization of Axol human iPSC-derived sensory neurons cultured with Sensory Neuron Maturation Maximizer Supplement. A) DIV21 MEA data showing functional characterization of sensory neurons grown in maintenance media without (left) and with (right) Maximizer Supplement. B) DIV21 averaged waveform analysis, recorded over a 4 minutes showing that capsaicin has no effect on the action potential waveform (confirmed further in C).

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Figure 6. Menthol treatment and electrophysiological characterization of Axol human iPSC-derived sensory neurons cultured with Sensory Neuron Maturation Maximizer Supplement. A) DIV27 MEA data showing functional characterization of sensory neurons grown in maintenance media without (left) and with (right) Maximizer Supplement. B) DIV27 averaged waveform analysis, recorded over 4 minutes, showing that menthol does not affect the action potential waveform (confirmed further in C). 

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Figure 7. Mustard oil treatment and electrophysiological characterization of Axol human iPSC-derived sensory neurons cultured with Sensory Neuron Maturation Maximizer Supplement. A) DIV22 MEA data showing functional characterization of sensory neurons grown in maintenance media with Maximizer Supplement. B) DIV22 averaged waveform analysis, recorded over 4 minutes, showing that mustard oil does not affect the action potential waveform (confirmed further in C). 

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Figure 8. Menthol desensitization (tachyphylaxis) experiments an electrophysiological characterization of Axol human iPSC-derived sensory neurons cultured with Sensory Neuron Maturation Maximizer Supplement. A) DIV21 MEA data from exposure to a menthol analog. B) DIV21 data showing how repeated exposure to menthol caused a significant reduction in the total number of spikes. C) DIV21 time series data reinforcing the reduction in spike number with repeated application of menthol.

Sensory Neurons That Behave Like Sensory Neurons

So there you have it: human iPSC-derived sensory neurons that really do behave like sensory neurons. That they’re mature and behaving in the manner you’ve seen in these data in under four weeks when cultured with Sensory Neuron Maturation Maximizer Supplement is just an added bonus!

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