Data from: Human dorsal root ganglia neuronal cell line to study nociceptive signaling: A new pipeline for pain therapy
Data files
Jun 17, 2026 version files 11.04 MB
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Figure_2C.csv
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Figure_2D.csv
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Figure_2E.csv
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Figure_2F.csv
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Figure_3B.csv
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Figure_3C.csv
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Figure_3D.csv
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Figure_3E.csv
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Figure_3G_Bottom.csv
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Figure_3G_Top.csv
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Figure_3H.csv
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Figure_3I.csv
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Figure_5B.csv
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Figure_5C.csv
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Figure_6A.csv
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Figure_6B.csv
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Figure_6C.csv
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Figure_6D.csv
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Figure_6E.csv
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Figure_6F.csv
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Figure_6G.csv
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Figure_6I.csv
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Figure_6J.csv
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Figure_6K.csv
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Figure_7C.csv
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README.md
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Abstract
Nociceptive afferent neurons within the dorsal root ganglion (DRG) detect and relay painful peripheral stimuli, and the malfunctioning of this process leads to sustained pain states. Animal model studies have been invaluable for demonstrating the importance of the DRG nociceptor in pain sensation and the development of related analgesic targets. However, a human in vitro model of nociception is essential to confirming the relevance of preclinical findings for therapeutic drug development. We characterized the nociceptive properties of differentiated cells from the human DRG-derived immortalized cell line HD10.6 and developed their use into an in vitro model of human nociceptive signaling and therapy. Within differentiated HD10.6 cells, we confirmed the abundance and function of machinery linked with pain sensation, including key ion channels (TRPV1, NaV1.7) and afferent peptides (CGRP, Substance P), by immunofluorescence and calcium influx assays. Through whole-cell patch clamp, we revealed baseline electrophysiological parameters of differentiated HD10.6 cells to be like those of human patient DRGs previously published. We found that differentiated HD10.6 cells express the mu opioid receptor 1 protein, and DAMGO, a mu agonist, blocks depolarization-evoked calcium influx in a naloxone-reversible fashion. Importantly, excitation and peripheral sensitization were induced within HD10.6 cells in response to an inflammatory cocktail, mirroring nociceptors in a pain state during and after tissue damage or inflammation. HD10.6 cells were also cultured into dual-chambered microfluidic devices to mirror the physiological anatomy of the nociceptor. Within this system, genetic therapy using adeno-associated virus was successfully taken up by the peripheral terminals and transported to the soma.
Dataset DOI: 10.5061/dryad.x95x69pz9
Description of the data and file structure
Figure 2C-F data come from calcium imaging performed on differentiated HD10.6 cells. Figure 2C demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). Capsaicin was added to differentiated HD10.6 cells and calcium influx was recorded over time. Figure 2D is derived from Figure 2C. Figure 2D demonstrates the maximum change in calcium influx value during treatment of cells with buffer only (“BUF.”), during or following capsaicin (“CAP.”), and after the addition of 50 mM KCl (“KCl”). Individual neuronal replicates fill in each row. Figure 2E demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). OD1 was added to differentiated HD10.6 cells and calcium influx was recorded over time. Figure 2F is derived from Figure 2E. Figure 2F demonstrates the maximum change in calcium influx value during treatment of cells with buffer only (“BUF.”), during or following OD1 (“OD1”), and after the addition of 50 mM KCl (KCl). Individual neuronal replicates fill in each row.
Figure 3B-E,G-I data come from whole-cell patch clamp/voltage clamp experimentation. Figure 3B demonstrates cell diameter (in micrometers) for individual differentiated HD10.6 cells. Figure 3C demonstrates cell capacitance (in pF) for individual differentiated HD10.6 cells. Figure 3D demonstrates resting membrane voltage (in mV) for individual differentiated HD10.6 cells. Figure 3E demonstrates resting membrane voltage (in mV) for an individual differentiated HD10.6 cell over time (rows). Figure 3D demonstrates resting membrane voltage (in mV) for individual differentiated HD10.6 cells. Figure 3G Bottom demonstrates resting membrane voltage (in mV) over time (rows) for an individual differentiated HD10.6 cell. Each trace of resting membrane voltage correlates with a respective current injection (Vm1 and I-com1, for example). Figure 3G Top demonstrates resting membrane voltage (in mV) over time (rows) for an individual differentiated HD10.6 cell. Each trace of resting membrane voltage correlates with a respective current injection (Vm1 and I-com1, for example). Figure 3H demonstrates sodium channel currents at over time at different represented resting membrane voltages for an individual differentiated HD10.6 cell. Figure 3I demonstrates sodium channel current density (pA/pF) for individual differentiated HD10.6 cells.
Figure 5B-C data come from calcium imaging performed on differentiated HD10.6 cells. Figure 5B demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). “DAMGO” replicates represent those treated with DAMGO only, “NALOX.” refers to those treated with DAMGO and Naloxone, and “VEHICLE” for those treated with vehicle only. Figure 5C is derived from Figure 5B. Figure 5C demonstrates the maximum change in calcium influx value during treatment of cells with 10 mM KCl for the three experimental conditions (DAMGO, NALOX., and VEHICLE, as described above). Individual neuronal replicates fill in each row.
Figure 6A-E data come from calcium imaging performed on differentiated HD10.6 cells. Figure 6F-G, I-K data come from electrophysiology (whole cell patch clamp, voltage clamp) performed on differentiated HD10.6 cells. Figure 6A demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). Inflammatory Soup was added to differentiated HD10.6 cells and calcium influx was recorded over time. Figure 6B is derived from Figure 6A. Figure 6B demonstrates the maximum change in calcium influx value during treatment of cells with buffer only (“BUF.”), during or following inflammatory soup (“IS”), and after the addition of 50 mM KCl (“KCl”). Individual neuronal replicates fill in each row. Figure 6C demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). “No Soup” replicates represent those pre-treated with vehicle only and “Soup” refers to those pre-treated with inflammatory soup for 4 hours. Figure 6D is derived from Figure 6C. Figure 6D demonstrates the maximum change in calcium influx value during treatment of cells with 10 mM KCl for the two experimental conditions (“No Soup” or “Soup”, as described above). Individual neuronal replicates fill in each row. Figure 6E demonstrates the percentage of differentiated HD10.6 cells per field of view that respond to 10 mM KCl treatment for the two experimental conditions (“No Soup” or “Soup”, as described above). Figure 6F demonstrates the resting membrane (mV) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above). Figure 6G demonstrates the rheobase (pA) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above). Figure 6I demonstrates the soma diameter (micrometers) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above). Figure 6J demonstrates the cell capacitance (pF) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above). Figure 6K demonstrates the sodium current density (pA/pF) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above).
Figure 7C comes from blinded counts taken following the performance of immunofluorescence on AAV-treated differentiated HD10.6 cells. E8 refers to cells treated with E8 genome copies of mCherry, and so on for E9 and E10. The mCherry-positive HD10.6 cells were quantified and are represented as a percentage.
Files and variables
File: Figure_2D.csv
Description: Figure 2D demonstrates the maximum change in calcium influx value during treatment of cells with...
Variables
- BUF.: buffer only
- CAP.: during or following capsaicin exposure
- KCl: during or following 50 mM KCl exposure
File: Figure_2C.csv
Description: Figure 2C demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). Capsaicin was added to differentiated HD10.6 cells and calcium influx was recorded over time.
Variables
- Times (seconds)
- Replicates: Individual neuronal replicates
File: Figure_2E.csv
Description: Figure 2E demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). OD1 was added to differentiated HD10.6 cells and calcium influx was recorded over time.
Variables
- Time (seconds)
- Replicates: Individual neuronal replicates
File: Figure_2F.csv
Description: Figure 2F demonstrates the maximum change in calcium influx value during treatment of cells...
Variables
- BUF.: with buffer only
- OD1: during or following OD1 exposure
- KCl: during or following 50 mM KCl exposure.
File: Figure_3B.csv
**Description: **Figure 3B demonstrates cell diameter (in micrometers) for individual differentiated HD10.6 cells.
Variables
- N/A
File: Figure_3C.csv
Description: Figure 3C demonstrates cell capacitance (in pF) for individual differentiated HD10.6 cells.
Variables
- N/A
File: Figure_3D.csv
Description: Figure 3D demonstrates resting membrane voltage (in mV) for individual differentiated HD10.6 cells.
Variables
- N/A
File: Figure_3F.csv
Description: Figure 3F demonstrates rheobase (in pA) for individual differentiated HD10.6 cells.
Variables
- N/A
File: Figure_3H.csv
Description: Figure 3H demonstrates sodium channel currents over time at different represented resting membrane voltages (mV) for an individual differentiated HD10.6 cell.
Variables
- Time (ms)
- 40 mV
- 30 mV
- 20 mV
- 10 mV
- 0 mV
- -10 mV
- -20 mV
- -30 mV
- -40 mV
- -50 mV
- -60 mV
- -70 mV
File: Figure_3G_Top.csv
Description: Figure 3G Top demonstrates resting membrane voltage (in mV) over time (rows) for an individual differentiated HD10.6 cell. Each trace of resting membrane voltage correlates with a respective current injection (Vm1 and I-com1, for example).
Variables
- Time (ms)
- Vm1
- Vm2
- Vm3
- Vm4
- Vm5
- Vm6
- Vm7
- Vm8
- Vm9
- Vm10
- Vm11
- I-com1
- I-com2
- I-com3
- I-com4
- I-com5
- I-com6
- I-com7
- I-com8
- I-com9
- I-com10
- I-com11
File: Figure_6B.csv
Description: Figure 6B is derived from Figure 6A. Figure 6B demonstrates the maximum change in calcium influx value during treatment of cells with buffer only (“BUF.”), during or following inflammatory soup (“IS”), and after the addition of 50 mM KCl (“KCl”). Individual neuronal replicates fill in each row.
Variables
- BUF.: buffer only
- IS: during or following inflammatory soup
- KCl: during or following 50 mM KCl
File: Figure_3E.csv
Description: Figure 3E demonstrates resting membrane voltage (in mV) for an individual differentiated HD10.6 cell over time (rows).
Variables
- Time (s)
- Vm (mV)
File: Figure_3I.csv
Description: Figure 3I demonstrates sodium channel current density (pA/pF) for individual differentiated HD10.6 cells.
Variables
- N/A
File: Figure_5C.csv
Description: Figure 5C is derived from Figure 5B. Figure 5C demonstrates the maximum change in calcium influx value during treatment of cells with 10 mM KCl for the three experimental conditions (DAMGO, NALOX., and VEHICLE, as described above). Individual neuronal replicates fill in each row.
Variables
- VEHICLE: Vehicle only
- DAMGO: Treatment with DAMGO
- NALOX.: Treatment with DAMGO and Naloxone
File: Figure_6D.csv
Description: Figure 6D is derived from Figure 6C. Figure 6D demonstrates the maximum change in calcium influx value during treatment of cells with 10 mM KCl for the two experimental conditions (“No Soup” or “Soup”, as described above). Individual neuronal replicates fill in each row.
Variables
- No Soup
- Soup
File: Figure_7C.csv
Description: Figure 7C comes from blinded counts taken following the performance of immunofluorescence on AAV-treated differentiated HD10.6 cells. E8 refers to cells treated with E8 genome copies of mCherry, and so on for E9 and E10. The mCherry-positive HD10.6 cells were quantified and are represented as a percentage.
Variables
- E8
- E9
- E10
- Mock
File: Figure_6F.csv
Description: Figure 6F demonstrates the resting membrane (mV) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above).
Variables
- No Soup
- Soup
File: Figure_6G.csv
Description: Figure 6G demonstrates the rheobase (pA) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above).
Variables
- No Soup
- Soup
File: Figure_5B.csv
Description: Figure 5B demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). “DAMGO” replicates represent those treated with DAMGO only, “NALOX.” refers to those treated with DAMGO and Naloxone, and “VEHICLE” for those treated with vehicle only.
Variables
- Time (seconds):
- DAMGO: Treatment with DAMGO
- NALOX.: Treatment with DAMGO and naloxone
- VEHICLE: Treatment with vehicle only
File: Figure_6E.csv
Description: Figure 6E demonstrates the percentage of differentiated HD10.6 cells per field of view that respond to 10 mM KCl treatment for the two experimental conditions (“No Soup” or “Soup”, as described above).
Variables
- No Soup
- Soup
File: Figure_6C.csv
Description: Figure 6C demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). “No Soup” replicates represent those pre-treated with vehicle only and “Soup” refers to those pre-treated with inflammatory soup for 4 hours
Variables
- Times (seconds): N/A
- No Soup: pre-treated with vehicle only 4 hours
- Soup: pre-treated with inflammatory soup for 4 hours
File: Figure_6A.csv
Description: Figure 6A demonstrates the change in calcium influx for individual neuronal replicate side-by-side (columns) over time (rows). Inflammatory Soup was added to differentiated HD10.6 cells and calcium influx was recorded over time.
Variables
- Time (seconds)
- Replicates
File: Figure_6I.csv
Description: Figure 6I demonstrates the soma diameter (micrometers) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above).
Variables
- No Soup
- Soup
File: Figure_6J.csv
Description: Figure 6J demonstrates the cell capacitance (pF) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above).
Variables
- No Soup
- Soup
File: Figure_6K.csv
Description: Figure 6K demonstrates the sodium current density (pA/pF) of differentiated HD10.6 cells pre-treated with the two experimental conditions (“No Soup” or “Soup”, as described above).
Variables
- No Soup
- Soup
File: Figure_3G_Bottom.csv
Description: Figure 3G Bottom demonstrates resting membrane voltage (in mV) over time (rows) for an individual differentiated HD10.6 cell. Each trace of resting membrane voltage correlates with a respective current injection (Vm1 and I-com1, for example).
Variables
- Time (ms)
- Vm1
- Vm2
- Vm3
- Vm4
- Vm5
- Vm6
- Vm7
- I-com1
- I-com2
- I-com3
- I-com4
- I-com5
- I-com6
- I-com7
Code/software
Data are stored in .csv files. Please use spreadsheet programs like Microsoft Excel, Google Sheets, or Apple Numbers.
Access information
Other publicly accessible locations of the data:
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Data was derived from the following sources:
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Immunofluorescence
HD10.6 cells on coated 12 mm circle glass coverslips were washed twice with 1X PBS and fixed for 10 minutes on ice with 4% paraformaldehyde (PFA). Cells were blocked using 2% fetal bovine serum (FBS), 0.2% Triton-X, and 2% serum from the appropriate host species (donkey or goat) of secondaries for 1 hour at room temperature. Cells were incubated with the primary antibody diluted in blocking buffer for 2 hours at room temperature. Following three 1X PBS washes, coverslips were incubated with secondary antibody diluted in blocking buffer for 1 hour at room temperature, covered from light. Following three 1X PBS washes, coverslips with cells were mounted on slides with ProlongGold containing DAPI (Thermo Fisher) and imaged using a Leica SP8 microscope. Images were analyzed using ImageJ, RRID: SCR_003070. Image acquisition and analysis were blinded.
Calcium Imaging
All calcium imaging experiments were performed on differentiated HD10.6 cells plated on coated 15 mm glass coverslips. HD10.6 cell cultures were loaded with the Ca2+ indicator Fluo-4 AM (5 μM; Invitrogen F23917) with PowerLoad Concentrate (Thermo Fisher P10020) in differentiation media without doxycycline for 1 hour at room temperature. Coverslips were placed in a laminar flow perfusion chamber (Warner Instrument Corp., UK) and continuously perfused with extracellular solution (in mM: NaCl 160; KCl 2.5; CaCl2 1; MgCl2 2; HEPES 10; glucose 10; pH 7.4). The perfusion system consisted of up to four individual compartments filled with treatment solutions, each one connected to a separate cannula that merged to one output cannula. The latter was inserted in the laminar flow perfusion chamber containing a glass coverslip with the HD10.6 cells. The flow was constant, and the HD10.6 cells were always covered in solution. Each treatment valve was opened manually by the experimenter. A suction pump connected to the outflow side of the chamber maintained a continuous perfusion. HD10.6 cells were recorded in an inverted Leica TCS SP5 confocal microscope. One field of view per coverslip was assessed. Cells were exposed to buffer, then stimulated with the drug diluted in buffer for the times of exposure as specified within the figure legends. All cultures were stimulated with 50 mM KCl for 5 seconds at the end of the experiments to confirm cellular viability. Neuronal Ca2+ responses were analyzed in 3–5 dishes per biological replicate by selecting individual cells as ROIs (Region of Interest) and calculating mean gray value variations on each individual cell using LAS AF version 2.7.3.9723 software. Data are presented as ΔF/F0, where F0 is the baseline, and the effect is quantified as maximal ΔF/F0. Randomization was applied during treatment. The experimenter was blinded to conditions during analysis.
Electrophysiological recordings
Whole-cell patch clamp was performed on intact, differentiated HD10.6 cells plated on coated 15 mm circle glass coverslips. Coverslips were transferred into the recording chamber with a continuous flow of extracellular solution containing 150 mM NaCl, 4.0 mM KCl, 2.0 mM CaCl2, 2.0 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). The extracellular solution was perfused at a rate of 1.5-2.0 mL/minute and heated to 30-32 °C with a temperature controller (TC-324B, Warner Instrument). The cells were allowed to rest in the chamber for 5-10 minutes before all recordings. HD10.6 cells were identified under the microscope of the recording rig by their small, oval-shaped cell body. Borosilicate glass pipettes (outer diameter: 1.5 mm, Warner, Hamden, CT) were pulled from a micropipette puller (Model P-97, Sutter). The recording electrodes had a resistance of 2-5 MΩ when filled with an internal solution containing 130 mM potassium gluconate, 1.0 mM MgCl2, 1.0 mM CaCl2, 10 mM HEPES, 4 mM MgATP, 0.3 mM Na2GTP, and 5.0 mM EGTA at pH 7.2. A MultiClamp 700B amplifier, an Axon Digidata 1550B, and Clampex 11 software (Molecular Devices, San Jose, CA, USA) were used for data acquisition, digitized at 2-10 kHz, and filtered at 2 kHz. The liquid junction potential was compensated for prior to forming the cell-attached mode for all recordings. A minimum of 2 GΩ seal resistance was required before rupturing the membrane for the whole-cell configuration. Membrane test in Clampex software was used to read the membrane capacitance (Cm), membrane resistance (Rm), and access resistance (Ra) immediately after whole-cell configuration. The cells were recorded in current clamp mode first to acquire resting membrane potential (resting Vm) without holding current, rheobase (10 ms duration of minimal current injection from 0 pA with 10 pA increment until the first action potential appears), and excitability (500 ms duration of -50 to +500 pA current injection steps with 50 pA increment). Then the cells were held at -70 mV in a voltage clamp. When the holding current stabilized, a voltage step protocol was applied to record the Na+ currents and K+ currents. Voltage steps were from -200 to +40 mV with 10 mV increments and 25 ms duration, and with the baseline holding at -80 mV. Only the cells with Ra <= Rm x 10% and less than 20% fluctuation of Ra were included for voltage clamp recordings. Multiple cells were recorded from each coverslip, and each coverslip was kept in the recording chamber for less than 2 hours to ensure that the cells were recorded in a healthy state. Randomization was applied during treatment.
All the data were analyzed in software ClampFit (Molecular Devices). For excitability analysis, the “Threshold search” function ClampFit was used; the depolarization peaks with overshoot above 0 mV were identified as action potentials, and only action potentials occurring during the current injection period were included for quantification of induced action potentials. For the cells showing spontaneous firing at rest, action potentials occurring outside the range of the current injection period were not included for induced action potential analysis. For Na+ currents analysis, leak current subtraction was performed in ClampFit to isolate depolarization steps induced by inward Na+ currents. The peak amplitude of each step was used to plot the I-V curve, and the maximum of the peak amplitude was used to calculate the current density. The outward currents induced by depolarizing steps near the end of voltage steps were used for the K+ current I-V plot, and the currents at the +40 mV step were used to calculate the K+ current density. The experimenter was blinded to conditions during analysis.
AAV9-mCherry Transduction
Within individual wells of a 24-well plate, 1E8, 1E9, or 1E10 vg (viral genomes) of AAV9-mCherry were added in 200 μL of differentiation media without doxycycline and incubated for 2 hours at 37°C and 5% CO2, following which 400 μL of media with doxycycline was added to reservoirs. Within microfluidic chambers, 1E9 vg were added in 5 μL of differentiation media without doxycycline into the axon-only compartment and incubated for 2 hours at 37°C and 5% CO2, followed by the addition of media with doxycycline to reservoirs. A 100 μL volume differential was always maintained between axon and soma compartments.
Statistical Analyses
Results were analyzed using GraphPad Prism v.10 software (RRID: SCR_002798, GraphPad®, San Diego, USA). Power analysis was used to determine the appropriate sample sizes for statistical analysis. The normal distribution of values in each analysis was performed with the Kolmogorov-Smirnov test according to the sample size. When a non-parametric statistical test was performed, the Mann-Whitney test was used to compare two means. When the comparison involved more than two means, the Kruskal-Wallis test was performed to determine if there were differences between the groups analyzed. If the values followed a normal distribution, parametric statistical tests were performed. The T-student test was selected when two means were compared. When the comparison involved more than two means, a one-way or two-way analysis of variance (ANOVA) was performed according to the experimental design followed. The level of significance adopted in each analysis of the results was p < 0.05. Specific analyses are included in the figure legends.