Human Induced Pluripotent Stem Cell-Derived Nociceptors suitable for AutomatedPatch Clamp High Throughput Pain Drug Discovery
Patrick Walsh1, Aaron Randolph2 , Irene Lu2, Alison Obergrussberger3 , Rodolfo Haedo2, Vincent Truong1, Tim Strassmaier2, and Marc Rogers*
1Anatomic Inc., Minneapolis, MN, USA, 2Nanion Technologies Inc., Livingston, USA, 3Nanion Technologies GmbH, Munich, Germany

Introduction•Records both time course and voltage
dependence of compound application•

Calculation of crude Vhalf is possible for eachsweep

There is an unmet need for novel non-addictive pain analgesics as the•Tau of activation is also available for different
voltagesthroughout
the recording
opioid epidemic continues. Theabilityto screen
compounds on humansensory neuron nociceptors with high throughput would increase theefficiency and pace of preclinical pain drug discovery and improvetranslational success of new pain drug candidates. We have previously
demonstrated that human nociceptors can be generated in anaccelerated, scalable method from human induced pluripotent stem cells(hiPSCs), and that the hiPSC-derived nociceptors share similarities to human
dorsal root ganglia based on whole-transcriptome profiling and expressionof functional voltage- and ligand-gated channels important for nociception.
In this study, we utilise a novel dissociation method to enable automated
HCN2
Pharmacology
in HighThroughput
patch
clamp
electrophysiologicalrecordingsof multiple ion channels in
RealDRG™ cultures on the Patchliner and SyncroPatch 384 systems. The
functional expression and biophysical and pharmacological properties ofvoltage-gated sodium (Nav) and potassium ion channels (Kv), and ligandgated ionotropic GABA and P2X receptors were studied over 14, 21 and 28
days in culture, along with excitability properties and action potentials incurrent clamp.
The percentage of cells with at least one evoked action potential increased
from 42% to 77% over the course of maturation, with success rates
decreasing from 56 to 35% as the cells matured. There was also an increasein voltage-gated sodium (Nav) and potassium (Kv) currents over time inculture, with success rates ranging from 94-98% and 93-100%, respectively.
Most neurons had tetrodotoxin-resistant (TTXr) sodium currents, with a trendof increasing number of cells with Nav current and fraction of TTXr current
per cell. A ligand puff protocol was developed to reproducibly evoke
ligand-gated GABA-A and P2X receptor ion channels without
desensitisation. These findings demonstrate the ability to functionally screen
multiple analgesia drug targets in human iPSC nociceptors using highthroughput automated patch clamp systems

Cell size reveals neuron maturation and diversity

BB

Cell capacitance measurements of cell size reveals maturation anddiversityofRealDRG™
sensory
neuronphenotypes.
A: Single neuron capacitance measurements are indicative of cell size,
which increases as RealDRG™ sensory neurons mature during indicateddays in vitro (DIV). B: Plot of cell capacitance from 3 NPC-384 chiprecordings shows bimodal distribution typical of small and medium diameter
sensory neurons, with a small tail of larger diameter cells.

Voltage-gated currents during maturation

RealDRG™ sensory neurons:
Human stem cell-derived sensory neurons were rapidly differentiated andmatured from human induced pluripotent stem cells (hiPSCs) using avalidated protocol1 that works across multiple hiPS donor cell lines2. Neuralcrest is induced from primary ectoderm and then differentiated using small
molecules and growth factors, before sensory neurons are frozen andshipped to users for thawing and further maturation as desired. Of the key
173 evolutionarily-conserved human DRG-specific gene markers, over 90%
are expressed in RealDRG sensory neurons; the remaining 10% of genes
relate to myelination and immune cells and their absence reflects the factthat these iPSC-derived sensory neuron cultures are > 95% pure.
Automated patch clamp (APC) electrophysiology:
A novel dissociation method was used to prepare single cell suspensions
from RealDRG™ cell cultures at 14, 21 and 28 days in culture suitable for APC
electrophysiology on Nanion Patchliner and SyncroPatch384 platforms. Sealquality is excellent (>300 MW), enabling standard solutions and patch clamp
protocols to be used to record voltage- and ligand-gated ion channel
currents under voltage clamp, and record spontaneous and evoked action
potentials under current clamp.

High throughput success rate APC recordings
AAMethods

High quality, high throughput APC recordings from RealDRG sensory neurons.
A: NPC-384 plate view of a voltage clamp Nav-Kv experiment, with greenwells (cells) exceeding QC filters of >300 MW Rm and 2 pF Cm. Example
current traces on right from cells highlighted by black boxes. B: Summary ofexperimental success rates for cell capture, cells with Kv and Nav currents
and P2X and GABA-A ligand-gated ionotropic receptor responses, andaction potentials (AP), some of which increase with DIV maturation.

Ligand-gated ionotropic receptor currents
A: SyncroPatch 384

A20 mM ATPBC
B: Patchliner

DPatchliner
4 or 8 recording channels● www.nanion.de ● info@nanion.de ●● www.anatomic.tech●C: P2X current desensitisation

EP2X receptor currents activated using 20 µM ATP on the SyncroPatch 384(A) and Patchliner (B). A: Rapid activation and desensitisation of ATPevoked currents on SP384; inset at expanded time scale. Current density ofATP-activated currents decreased during sensory neuron maturation. B:P2X receptors rapidly activate and desensitise after application of 20 µM
ATP on Patchliner. C: Desensitisation of P2X currents evoked every 4 min.
This can be reduced to give consistent repeated responses in the same
cell if ATP applications are made every 10 minutes, or more rapidly in thepresence of hexokinase to prevent ATP degradation (data not shown).

SyncroPatch 384
384 recording channels

Differentiation of RealDRG™ sensory neurons
F

A: SyncroPatch 384
100 mM GABAG

B: Patchliner
C: GABA-A pharmacology

Differentiation of RealDRG™ from PSC progenitor through neuro-ectoderm
and neural crest to sensory neuron revealed by key gene markers.
Upper panels: Expression of classical markers for each cell type, culminatingwith sensory neuron markers TUJ1, ISLET1, Peripherin and BRN3A by day 7.
Lower: RealDRG™ neurons express 90% of key sensory neuron genes thatdefine the human DRG transcriptome3,5,6, only lacking the 10% of genes
related to myelination (Schwann cells) and immune cells (e.g. microglia).

A: Macroscopic single neuron recording showing ensemble of inward andoutward currents elicited by a series of increasing depolarized voltage
steps. B: KV current-voltage IV plot at 21 DIV and 28 DIV on the Patchliner.
C: Current density of KV current does not change during maturation (datafrom SyncroPatch 384). D: A family of inward TTX-resistant Nav current traces
evoked by increasing amplitude of depolarizing voltage steps. E: NaV
current density decreases during maturation (total NaV current). F: Navcurrent IV plot at 28 DIV in the absence (light blue) and presence of TTX(red). TTXr currents activate at more positive potentials (typical of Nav1.8channels4), and Vhalf of activation becomes more negative at later DIV.
G: The % cells with TTXr current increases with DIV (SP384 data), and 10-20%of total current is carried by TTXr channels (Patchliner data).

Ion channel expression in RealDRG™

Action potential recordings on APC platforms

A

A: Patchliner

B
Key Ion Channel ImmunocytochemistryTime-course RNAseq Expression

TUJ1
NaV1.7

PRPH

TUJ1
TRPV1

NaV1.8Relative Fold Expression to Human DermalFibroblasts

C

B: SyncroPatch384

1,000,000
100,000
10,0001,000100C:

101Chrono™ Sensory Neurons in Senso-MM D7

Human DRG (Normal)

Keynociceptorionchannels
areexpressedinRealDRG™.
A: Phase contrast morphology of RealDRG™ neurons, and IHC labelling withsensory neuron markers TUJ1, Peripherin, Nav1.7 and Nav1.8.
B: RNA sequencing data reveals rapid and sustained expression of genes
SCN9A, SCN10A and SCN11A (encoding NaV1.7, NaV1.8 and NaV1.9,respectively), and TRPV1 during 4 weeks of in vitro maturation. C:
Comparison of expression of ion channels in RealDRG™ sensory neurons withnative adult human DRG3,5,6.

Action potentials recorded on Patchliner (A) and SyncroPatch 384 (B).
A: Single and repetitive action potentials evoked by a 500 msec current stepon Patchliner; AP amplitude increases with DIV maturation. B: Spontaneous(left) and evoked APs (right) on SyncroPatch 384. C: Membrane excitability
assessed with a series of increasing current steps on SP384.

GABAA receptors activated by fast application of GABA on SyncroPatch 384(A) and Patchliner (B). There was a variable degree of desensitisation
between sensory neurons and platforms. Inset shows time course ofactivation on an expanded time scale. GABA current density increased at
28 DIV compared with 21 or 14 DIV. C: GABA currents were inhibited by thecompetitive GABAA antagonist Bicuculline (10 mM, blue trace).

Summary• RealDRG™ sensory neurons recapitulate the differentiation andmaturation of human DRG and express most of the characteristic
markers of A and C-fibre nociceptors and mechanoreceptors.
• RealDRG™ sensory neurons are well suited to automated patch clamp
as they produce large, smooth soma membranes with simple dendritic
processes for high quality sealing and good voltage control.
• RealDRG™ allow high success rate recordings on SyncroPatch 384 andPatchliner automated patch clamp devices.
• A TTX-sensitive NaV current was reliably recorded in almost all cells at14, 21 and 28 DIV.
• A TTX-resistant current was present in many cells, around 10-20% of total
inward current amplitude. Pharmacological dissection is ongoing.
• RealDRG sensory neurons exhibit low rates of spontaneous action
potential activity, typical of native rodent and human DRG neurons.
• Action potentials can be evoked with single or incrementing current
injections, for assessment of sensory neuron membrane excitability.
• The occurrence of action potentials increases with RealDRG maturation.
• Flexible liquid application capabilities allow study of fast ligand-gatedionotropic receptor responses typically found in sensory neurons.
• P2X-mediated currents were reliably recorded on SP384 after
desensitisation and ATP degradation were reduced. Responses
increased during maturation, but current density decreased.
• A GABA-activated current was recorded on SyncroPatch 384 andPatchliner, which was blocked by the GABA-A antagonist Bicuculline.
The % of cells with responses was maintained over 14, 21 and 28 DIV,and GABA-A current density increased as sensory neurons matured.
• RealDRG™ sensory neurons represent an affordable and scalable
solution for more predictive human translational analgesia studies.

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. Bennett et al. (2019) Physiol Rev 99: 1079–1151
4. Zhang et al. (2017) e-Life 6: e23235
5. Ray et al. (2018) Pain 159: 1325–1345
6. Tavares-Ferrara et al. (2022) Sci. Transl. Med. 14: eabj8186