Application Note

Ion Channels:

High throughput APC recordings fromRealDRGTM iPSC sensory neurons for
analgesia drug discovery

NaV, CaV, K VCells:

RealDRGTM hiPSCderived sensory neuronsTools:
SyncroPatch 384
Patchliner

Nanion Technologies GmbH, Munich.
Anatomic Incorporated, MN, USA.

Summary
There is great need for more effective, non-addictive• Ionic currents can be reliably studied using complex voltage

analgesics due to poor efficacy and narrow therapeutic

clamp protocols

index of current treatments, and the desire to replace opioids
due to the ongoing addiction and abuse crisis. Analgesia
drug discovery still relies on animal models that poorly predict• This technique allows current clamp recordings of single
action potentials and membrane excitability

efficacy in human patients, leading to costly clinical failures forvalidated pain targets. Access to human dorsal root ganglion(DRG) reagents promises superior translation, but donor human

RealDRGTM human iPSC sensory neurons rapidly mature totissue is limited and expensive. In contrast, human iPSC sensory

express nociceptor markers such as TRPV1, P2X3, NaV1.7,

neurons offer a scalable, flexible and cost-effective analgesia
drug discovery reagent amenable to many screening
platforms1,2. These human DRG reagents will also help meet
3Rs guidelines to reduce animal use and are compatible withNaV1.8 and NaV1.92. APC voltage clamp recordings on thePatchliner and SyncroPatch 384 revealed voltage-gated

KV and TTX-sensitive (TTXs) and TTX-resistant (TTXr) inward NaV
channels, and overshooting single and repetitive action

the FDA Modernization Act 2.0 that will allow drug approvalpotentials under current clamp. This validation data confirms

without animal testing.

that Nanion APC platforms and Anatomic’s hiPSC sensory
neurons can be combined for reliable and scalable highHere we demonstrate that hiPSC sensory neurons are ideallythroughput patch clamp studies of human pain signallingsuited for automated patch clamp (APC) recordings, as:targets and peripheral neurotoxicity screening.

• Their large soma and simple dendritic architecture areretained after dissociation and cell suspension• Capacitance measurements allow categorization ofdifferent sensory neuron phenotypes by cell size
• High throughput sampling from large numbers of cells
enables statistical analysis of sensory cell types• High fidelity recordings provide excellent voltage control insingle patch-clamped cells

Figure 1: Ion channel expression in RealDRGTM.
A Immunocytochemistry characterization ofRealDRG™. B Comparison of expression of ionchannels in RealDRG™ with human DRG. C RNA
sequencing data reveals expression of genes
SCN9A, SCN10A and SCN11A encoding NaV1.7,
NaV1.8 and NaV1.9, respectively, along with TRPV1.

Results
Human stem cell-derived sensory neurons offer manyAβ and silent nociceptors).

translational and ethical advantages over current preclinicalanimal tissue and in vivo models and human donor tissue,

RealDRGTM hiPSC sensory neurons can produce highas they offer easy and affordable access to highly pure,throughput, high quality recordings on APC platforms withconsistent, transferable and scalable cell-based assays withexcellent success rates, surpassing that seen with native

commercial freedom-to-operate. RealDRGTM hiPSC sensory

rodent DRG neurons. Seal quality is excellent (>300 MΩ), andneurons can be rapidly differentiated and matured using aoverall quality control pass rates range between 35-60% forvalidated protocol and manufacturing process that works

cells matured over 14-28 days in vitro (DIV), meaning that highacross multiple hiPS donor cell lines1. Neural crest is induced

quality data from over 100 sensory neurons can be obtained infrom primary ectoderm and then differentiated using small

less than an hour from each NPC-384 chip on the SyncroPatchmolecules and growth factors, before the sensory neurons are384. Voltage-gated inward NaV and outward KV currents andfrozen and shipped to users for thawing and further maturation
as desired. Extensive molecular validation shows that expected
sensory neuron markers such as ISLET1, TUJ1, BRN3A, PRPH andfunctional P2X and GABAA ligand-responses are seen in 80-

100% of cells, while action potential firing increases from 40 to80% of cells over 3 weeks (Table 1).

various TRK neurotrophin receptors are prominently expressedby Day 7, alongside key analgesia ligand- and voltagegated ion channels such as TRPV1, P2X3, and various NaV1.x

and CaV3.x ion channels whose expression is maintained for
4 weeks in vitro and which achieve expression levels similar
to that seen in adult human DRG (Fig. 1). Of the key 173

DIVQC

NaV

KV

TTXr

P2X

GABA

AP

14

56

94

93

80

91

88

42

21

43

96

98

91

79

94

63

28

35

98

100

96

92

92

77

evolutionarily-conserved human DRG-specific gene markers,
over 90% are expressed in RealDRGTM iPSC sensory neurons,

Table 1: Amount of successful wells (%) at different time points (14, 21

whereas only 63% are present in mouse DRG, highlighting theand 28 DIV).

species differences in this ganglia3. The remaining 10% of genes
relate to myelination and immune cells, and their absencereflects the fact that these sensory neuron cultures are > 95%

DRG sensory neurons express a wide range of voltage-gatedpure (allowing users to add native or iPSC astrocytes andinward NaV and CaV , and outward KV currents that control

microglia to their end-stage assays). RealDRGTM hiPSC neurons
recapitulate the developmental patterns and maturationof DRG into varied types of sensory neurons4, including
characteristic gene expression patterns typical of human Aand C-fibres (e.g. Aβ and Aδ mechanoreceptors, and PENK+,

pain transmission, which can be accurately measured undervoltage clamp using APC. TTX-sensitive NaV1.7 and TTX-resistant
NaV1.8 and NaV1.9 channels are of particular interest for

analgesia, as mutations in these targets are associated withvarious pain states in human patients5. RealDRGTM sensory
neurons express SCN9A and SCN10A RNA and protein,with lower levels of SCN11A, and IV curves applied on theSyncroPatch 384 and Patchliner show large TTX-sensitive
currents activating at negative potentials, and a significant(~10 - 20%) component of TTX-resistant inward current that
activates at more positive potentials, characteristic of NaV1.8

channels (Fig. 3). The kinetics of TTX-resistant currents in

RealDRGTM iPSC sensory neurons, and their relative contribution
to macroscopic INa, are similar to those recorded from adulthuman DRG6 but differ from that in rat DRG neurons, illustrating

Figure 2: Success rates on the SyncroPatch 384. A
Screenshot of an experiment showing >50% of wells
have a cell captured. B Cell capacitance increases
with time in culture (DIV). C Success rates for cell
capture, recordings of ion channel currents andeliciting action potentials (AP) at 14, 21 & 28 DIV.

Voltage (mV)
20 40 60-80 -60 -40 -20
Current Density(pA/pF)

SyncroPatch 384. B Raw current traces from an
example cell in the presence of TTX. C NaV current
density decreases during maturation (total NaVcurrent), and the Vhalf of activation of the TTXr
current becomes more negative (D).

-100-150

28 DIV Control,
Vhalf = -55.9 mV-200

28 DIV 500 nM TTX,
Vhalf = -37.6 mV

0DIVDIV
14

importance of using human cells for target validation andCurrent Density (pA/pF)

the species differences in many sensory neuron responses and-50-250CB

0.2 nA2 ms

D28

and presence of TTX (red) recorded on theA

DIVneurons. A IV plot at 28 DIV in the absence (blue)

21Figure 3: NaV currents in RealDRGTM sensory-50-100-150-200-250-300-350

translation.
RealDRGTM sensory neurons also express TTX-sensitive NaV1.2and NaV1.6 channel genes which are found in maturerodent and human DRG neurons, as well as low levels of
the embryonic NaV1.3 channel which is up-regulated afterDiscussion

nerve injury5. NaV1.5 is the third TTX-resistant channel normally

These data demonstrate the utility of RealDRGTM human iPSC

expressed in neonatal and adult DRG neurons (especially insensory neurons to bridge the translational gap in analgesia

human Aδ mechanoreceptors), and is downregulated during

R&D, and their compatibility with APC platforms. High quality

in vitro maturation of RealDRGTM iPSC sensory neurons as

recordings of NaV currents can be obtained with highsuccess, with NaV channel biophysics and pharmacology

occurs in vivo.
DIV

Spontaneous APs (% cells)

Elicited APs (% cells)

140.4

4221

3.3

6328

3.5

77

indicating the presence of large TTXs currents (likely NaV1.7),
alongside a significant component of TTXr current whichtranscriptome and pharmacology data suggest is carriedby NaV1.8 channels. NaV1.7 and NaV1.8 are importantanalgesia drug discovery targets based on human pain
channelopathies and remain the focus of industry efforts

Table 2: Spontaneous action potentials were observed in <5% of cells

to develop sub-type selective small molecules, mRNA andbut could be elicited in >40% of cells, increasing to 77% with culture

biologic ligands to treat pain. It is also possible to recordtime.

firing of single and repetitive action potentials on APC
platforms, and assess changes in sensory neuron membraneIt is also possible to use APC to make high throughput current

excitability with complex current clamp protocols whichclamp recordings from hiPSC sensory neurons, even in thecan be useful for phenotypic analgesia drug screening. The

same cells used for voltage clamp experiments to correlate

low rate of spontaneous AP firing, together with the lack ofionic current expression and pharmacology with neuronalan extensive dendritic network and synaptic connections,

excitability. Baseline membrane potential recordings show

makes it more difficult to achieve high throughputa moderate resting membrane potential and a low level ofrecordings from sensory neurons on multi-electrode arrays

spontaneous action potential (AP) firing, as expected for DRG

compared to CNS neurons, making APC a promisingneurons (Table 2). Spontaneous firing is very low after 14 DIV,

platform for analgesia drug testing and neurotoxicity

but increases to occur in 3% of sensory neurons after further

screening with RealDRGTM hiPSC sensory neurons. The

maturation. Single and repetitive APs can be elicited withability to achieve good voltage control and quickly

depolarizing current injections on the SyncroPatch 384 andobtain complex datasets from hundreds of single cells will

Patchliner (Fig. 4), enabling categorization of single sensory

facilitate mechanistic understanding of drug actions, andneurons by their AP shape, pharmacology and evoked AP

the plasticity of RealDRGTM hiPSC sensory neurons means

trains. Application of current steps or ramps can also be usedthat novel maturation protocols can be applied to direct

to assess changes in sensory neuron excitability, a key driver ofthe development of specific DRG sensory neuron subtypes

pain signalling.

relevant to analgesia research.

Figure 4: Action potentials in RealDRGTM sensory
neurons. A Current step protocol to elicit action
potentials. B Spontaneous APs recorded on theSyncroPatch 384. C Elicited APs using a current
step to 40 pA. D & E Elicited APs on the Patchliner.
In some cells a single AP was elicited (D) whilst inothers a train of APs was elicited during the 500 mscurrent pulse.

References
1. Walsh et al. (2020) Stem Cells 38 (11): 1400–1408
2. Li et al. (2021) Front Cell Dev Biol 14 (9): 796960
3. Ray et al. (2018) Pain 159: 1325–1345
4. Tavares-Ferrara et al. (2022) Sci. Transl. Med. 14: eabj8186
5. Bennett et al. (2019) Physiol Rev 99: 1079–1151
6. Zhang et al. (2017) e-Life 6: e23235

Methods
Cells
RealDRGTM iPSC sensory neurons were differentiated according
to manufacturers protocols and frozen after 7 days.
Electrophysiology
Cells were thawed and plated into T75 flasks coated withiMatrix 511-SILK at a seeding density of 40,000 cells per cm2and matured in Senso-MM before being gently dissociatedand resuspended in external recording solution and stored
in the CellHotel on the Patchliner or SyncroPatch 384 APC
platform, before being dispensed into each well of the NPC16 or NPC-384 chip, respectively. Standard patch clamp
electrophysiology internal (KF-based) and external solutions
were used to record inward NaV and outward KV currents andaction potentials, and NaV currents were isolated by using aCsF-based internal solution. Standard voltage and current
clamp protocols were used to set holding potential andmeasure resting membrane potential, and voltage-gatedcurrents and action potentials were evoked with step voltage
or current pulses.

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