Journal: Stem Cell Research & Therapy

Article Title: Activation of the G-protein coupled estrogen receptor 1 (GPER1) reduces transient receptor potential vanilloid 1 (TRPV1) activity and human iPSC-derived nociceptive neuron firing

doi: 10.1186/s13287-026-05174-3

Figure Lengend Snippet: Effect of G-protein coupled estrogen receptor (GPER1) activation by specific agonist G-1 on transient receptor potential vanilloid 1 (TRPV1) - mediated ion currents. Co-overexpression of TRPV1 and GPER1 (TRPV1/GPER1 OE) in human embryonic kidney (HEK293) cells compared to empty vector (EV) control was quantified by ( A ) Realtime-PCR and ( B , C ) western-blot analysis ( n = 3). Western-blot images have been cropped from the original blot as shown in additional Fig. 1. D Fluo-4-AM-based calcium assays revealed reduced relative fluorescence unit (RFU) values indicating reduced intracellular calcium levels after the application of 10 µM capsaicin (dotted line) in HEK293 cells overexpressing TRPV1 after the treatment with G-1, G-15 or the specific TRPV1 antagonist AMG517 compared to the respective vehicle control (VC). E Calculation of the area under curve (AUC) values of curves shown in ( D ) revealed significant reduction of intracellular calcium levels after the application of G-1, G-15 and AMG517 compared to VC (each n = 9). F For electrophysiological studies, exclusively green fluorescent protein (GFP) and mCherry double positive cells were chosen indicating TRPV1 (eGFP) and GPER1 (mCherry) co-overexpression (Scale bar: 200 μm). G Representative traces of TRPV1-mediated ion currents after the application of 10 µM capsaicin for 200 ms in the presence of G-1 or the corresponding amount of VC. Quantification of ( H ) current density, I up slope, J down slope and ( K ) time to repolarization of capsaicin-induced TRPV1 currents in TRPV1/GPER1 OE HEK293 cells in the presence of G-1 ( n = 33) or the respective amount of VC ( n = 32). Data were tested for normal distribution using Shapiro-Wilk test. Datapoint (n) represent independent measurements from three independent transfections. Means ± SEM (standard error of the mean) were statistically analyzed either by an unpaired ( A , C ) Student’s t-test, a non-parametric ( H–K ) Mann–Whitney test or ( E ) one-way ANOVA with Turkey’s multiple comparison test. (* p ≤ 0.05, ** p ≤ 0.01)

Article Snippet: TRPV1 inhibitor AMG517 (#HY-10634, MedChem Express, Sollentuna, Sweden), GPER1 agonist G-1 (#3577, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) and GPER1 antagonist G-15 (#3678, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) were dissolved in DMSO in order to get a 50 mM stock solution that were further diluted to the desired concentration.

Techniques: Activation Assay, Over Expression, Plasmid Preparation, Control, Western Blot, Fluorescence, Transfection, MANN-WHITNEY, Comparison

Journal: Stem Cell Research & Therapy

Article Title: Activation of the G-protein coupled estrogen receptor 1 (GPER1) reduces transient receptor potential vanilloid 1 (TRPV1) activity and human iPSC-derived nociceptive neuron firing

doi: 10.1186/s13287-026-05174-3

Figure Lengend Snippet: Generation of iPSC line BO-VC1 -derived nociceptive neurons. Differentiation of ( A ) human iPSC line BO-VC1 into nociceptive neurons was done by dual SMAD inhibition, suppressing TGF-ß and BMP4 signaling, in knockout serum replacement (KSR) medium (day 0–4). This was followed by an overlapping inhibition of glycogen synthase kinase-3ß (GSK-3ß), vascular endothelial growth factor (VEGF), and Notch signaling (day 2–12), with KSR medium incremented with N2/B27 medium every second day by 25% from day 4 on. After reseeding of the ( B ) immature sensory neurons, final maturation was performed in N2/B27 medium infused with the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) (day 13 - approx. 60) according to the protocols of Chambers and Schoepf leading to the differentiation of ( C ) intermediate and finally ( D ) mature nociceptors (Scale bar: 500 μm). Mature nociceptive neurons express ( E ) the neuron specific microtubule element βIII-tubulin (βIIITUB) and vGLUT2, the sensory neuron markers ( F ) insulin gene enhancer protein 1 (ISL1) as well as the brain-specific homeobox/POU domain protein 3 A (BRN3A). Additionally, iPSC-derived differentiated cells express the nociceptive markers ( G ) transient receptor potential cation channel subfamily V member 1(TRPV1), H voltage-gated sodium ion channel 1.9 (Na V 1.9) and I TWIK-related spinal cord potassium channel (TRESK). Moreover, co-expression of ( J ) the calcitonin gene-related peptide (CGRP), the nociceptive marker high affinity nerve growth factor receptor (TRKA) and the estrogen receptor G-protein coupled estrogen receptor 1 (GPER1) was confirmed (Scale bars: 50 μm) ( K ) Quantification of the percentage of positive cells for the respective markers was 95.96 ± 2,495% for CGRP, 89.68 ± 6,137% for GPER1, 91.67 ± 5.270% for Na V 1.9, 95.83 ± 4.167% for TRKA, 80.38 ± 7.143% for TRPV1 and 98.55 ± 0.2899% for TRESK. L Realtime-PCR analysis in order to quantify nociceptive neuron marker and GPER1 gene expression of HDFa-derived iPSCs (line BO-VC1) and corresponding nociceptive neurons relative to HDFa cell samples mean value. Means ± SEM (standard error of the mean) from three independent passages were statistically analyzed by one-way ANOVA with Turkey’s multiple comparison test (L). n = 3 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001)

Article Snippet: TRPV1 inhibitor AMG517 (#HY-10634, MedChem Express, Sollentuna, Sweden), GPER1 agonist G-1 (#3577, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) and GPER1 antagonist G-15 (#3678, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) were dissolved in DMSO in order to get a 50 mM stock solution that were further diluted to the desired concentration.

Techniques: Derivative Assay, Inhibition, Knock-Out, Expressing, Marker, Gene Expression, Comparison

Journal: Stem Cell Research & Therapy

Article Title: Activation of the G-protein coupled estrogen receptor 1 (GPER1) reduces transient receptor potential vanilloid 1 (TRPV1) activity and human iPSC-derived nociceptive neuron firing

doi: 10.1186/s13287-026-05174-3

Figure Lengend Snippet: Electrophysiological properties of iPSC line BO-VC1 -derived nociceptive neurons. A Nociceptive neurons show characteristic TRPV1-mediated ion influx after activation with 500 µM capsaicin for 200 ms at + 60 mV holding potential. Representative sweeps of ( B ) voltage-gated sodium and potassium currents activated by voltage steps from − 120 to + 60 mV for 500 ms with an increment of 10 mV at a holding potential of − 60 mV and ( C ) IV-relation of sodium currents from nociceptive neurons ( n = 23). D Quantification of maximum sodium currents of nociceptive neurons ( n = 30) and the ( E ) resting membrane ( n = 30) potential. F Trace from whole-cell current-clamp recordings of action potentials (AP) triggered by current steps from − 120 to + 360 pA for 500 ms with an increment of 20 pA showing maximum AP firing. G Quantification of AP numbers of nociceptive neurons ( n = 20) for each applied current step. H Quantification of the rheobase current ( n = 20), I firing rate of APs derived from recordings at 140 pA input current ( n = 19), J AP amplitude at 140 pA input current ( n = 21). Data shown as means ± SEM (standard error of the mean) from individually measured cells from three differentiations

Article Snippet: TRPV1 inhibitor AMG517 (#HY-10634, MedChem Express, Sollentuna, Sweden), GPER1 agonist G-1 (#3577, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) and GPER1 antagonist G-15 (#3678, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) were dissolved in DMSO in order to get a 50 mM stock solution that were further diluted to the desired concentration.

Techniques: Derivative Assay, Activation Assay, Membrane

Journal: Stem Cell Research & Therapy

Article Title: Activation of the G-protein coupled estrogen receptor 1 (GPER1) reduces transient receptor potential vanilloid 1 (TRPV1) activity and human iPSC-derived nociceptive neuron firing

doi: 10.1186/s13287-026-05174-3

Figure Lengend Snippet: Effect of GPER1-activation by G-1 in iPSC line BO-VC1 -derived nociceptive neurons. A Representative current-clamp recordings of human iPSC line BO-VC1 -derived nociceptive neurons at -120 and 140 pA input current for 500 ms treated with 5 µM G-1, 1 µM TRPV1-antagonist AMG517 or the respective vehicle control (VC). B Quantification of action potential (AP) firing rate at indicated input currents (VC: n = 17; G-1: n = 21; AMG517: n = 12) per 500 ms. C Quantification of the area under curve (AUC) shown in ( B ) (VC: n = 16; G-1: n = 17; AMG517: n = 12). D Firing rate (VC: n = 17; G-1: n = 21; AMG517: n = 12) and E rheobase current (VC: n = 17; G-1: n = 21; AMG517: n = 12) of VC, G-1 or AMG517-treated nociceptive neurons. F Fluo-4-AM-based calcium assay time course showing relative fluorescence units (RFU). Cells were pre-treated with VC ( n = 6), G-1 ( n = 6) or AMG517 ( n = 5) for 5 min prior to TRPV1 activation by the addition of 10 µM capsaicin after 20 min of baseline detection. G RFU was normalized to the baseline mean and analyzed by calculation of the area under curve (AUC) for statistics. Means ± SEM (standard error of the mean) were statistically analyzed by ( C , D , E , G ) one-way ANOVA or ( F ) mixed effect analysis with Tukey’s multiple comparisons test from individually measured cells/wells. (**** p < 0.0001)

Article Snippet: TRPV1 inhibitor AMG517 (#HY-10634, MedChem Express, Sollentuna, Sweden), GPER1 agonist G-1 (#3577, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) and GPER1 antagonist G-15 (#3678, Tocris, Bio-Techne GmbH, Wiesbaden, Germany) were dissolved in DMSO in order to get a 50 mM stock solution that were further diluted to the desired concentration.

Techniques: Activation Assay, Derivative Assay, Control, Calcium Assay, Fluorescence

Journal: Biomaterials Research

Article Title: Injectable Poloxamer and Hyaluronic Acid Hydrogel for Sustained Co-Delivery of Dexamethasone and Lidocaine Ameliorates Neuropathic Pain

doi: 10.34133/bmr.0373

Figure Lengend Snippet: TRPV1-mediated nociceptive sensitization in the DRG and spinal cord dorsal horn (SC) following CCI and treatment. (A) Schematic illustration of the proposed mechanism: Injury-induced TRPV1 ion channel activation triggers calcium influx and downstream CGRP release, which activates adenylate cyclase/PKA signaling to amplify neuropathic pain sensitization. (B) Representative immunofluorescence images of the DRG stained for TRPV1 (red), NeuN (green), and DAPI (blue). The Injury group shows marked up-regulation of TRPV1 in sensory neurons. Dex/Lid@PLX/HA treatment substantially reduces TRPV1 expression, restoring it to near-Naive levels. Scale bars, 200 μm (overview) and 50 μm (inset). (C) Immunofluorescence staining for TRPV1 (red) and NeuN (green) in the spinal dorsal horn. The dashed line indicates the dorsal horn boundary. Dex/Lid@PLX/HA significantly suppresses injury-induced central TRPV1 up-regulation. Scale bar, 200 μm. (D to F) Quantitative analysis of the relative TRPV1 + area in the DRG (top) and spinal cord (middle), and the SGC/neuron ratio. Dex/Lid@PLX/HA shows marked suppression of TRPV1 overexpression relative to the injury group. Data are presented as mean ± SEM. **** P < 0.0001, ** P < 0.01, * P < 0.05, ns: not significant.

Article Snippet: The sections were then incubated overnight at 4 °C with the following primary antibodies: TRPV1 (Alomone Labs, catalog number ACC-030-GP), Iba-1 (Abcam, catalog number ab5076), NeuN (Abcam, catalog number ab104224), CD68 (Abcam, catalog number ab31630), CD163 (Abcam, catalog number ab182422), CGRP (Abcam, catalog number ab47027), GFAP (Millipore, catalog number MAB360), and NF200 (Abcam, catalog number ab8135).

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Over Expression

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Immunoblot analysis shows TRPV1, TRPA1, and actin levels in WT BMDMs 48 h following treatment with or without IL-4 plus GMCSF (25 ng/ml). (B-C) Densitometric quantification of immunoblot data from (A) (n = 3 biological replicates; one-way ANOVA, **p < 0.01, ***p < 0.001). (D) Representative immunofluorescence images of WT BMDMs stained for TRPV1 (red) using anti-TRPV1 IgG (original magnification, 60 x; scale bar, 2 μm; n = 10 cells per condition). Statistical analysis by Student’s t-test, ***p < 0.001.

Article Snippet: C57BL/6 and TRPV1 KO mice were purchased from The Jackson Laboratory (ME, USA).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Representative Giemsa-stained images of multinucleated FBGCs in WT BMDMs left untreated or stimulated with IL-4 plus GMCSF (25 ng/ml, 96 h), in the presence or absence of the TRPV1 antagonist AMG. (B-D) Quantitative analysis of FBGC formation from (A): (B) number of FBGCs per high-power field, (C) percentage of fused BMDMs, and (D) average FBGC size. Data represent n = 3 biological replicates with 5 images per group; scale bar, 100 μm; Student’s t-test, ***p < 0.001, ****p < 0.0001. (E) Representative immunofluorescence images of WT BMDMs transfected with scramble or TRPV1-targeting siRNA, stained for TRPV1 (red) using anti-TRPV1 IgG (original magnification, 60x; scale bar, 2 μm). (F) Quantification of TRPV1 fluorescence intensity (n = 10 cells per condition; Student’s t-test, ***p < 0.001). (G) Immunoblot showing TRPV1 expression in WT BMDMs 48 h after transfection with scramble or TRPV1 siRNA. (H-J) Quantification of FBGC formation following TRPV1 knockdown: (H) number of FBGCs per high-power field, (I) percentage of fused BMDMs, and (J) average FBGC size. Data represent n = 3 biological replicates with 5 images per group; Student’s t-test, ***p < 0.001.

Article Snippet: C57BL/6 and TRPV1 KO mice were purchased from The Jackson Laboratory (ME, USA).

Techniques: Staining, Immunofluorescence, Transfection, Fluorescence, Western Blot, Expressing, Knockdown

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Representative images of FBGCs formed by WT BMDMs cultured on collagen-coated (10 μg/ml) PA hydrogels of defined stiffness (1 kPa and 50 kPa), left untreated or stimulated with IL-4 plus GMCSF (25 ng/ml, 96 h), in the presence or absence of the TRPV1 antagonist AMG. (B-C) Quantification of FBGC formation from (A): (B) number of FBGCs per high-power field and (C) percentage of fused BMDMs. (D) Representative images of FBGCs in WT and TRPV1 KO BMDMs cultured on Permanox slides under untreated or IL-4 plus GMCSF-stimulated conditions (25 ng/ml, 96 h). (E-F) Quantification of FBGC formation from (D): (E) number of FBGCs per high-power field and (F) percentage of fused BMDMs. (G) Representative images of FBGCs in WT and TRPV1 KO BMDMs cultured on collagen-coated (10 μg/ml) 50 kPa PA hydrogels, with or without IL-4 plus GMCSF stimulation (25 ng/ml, 96 h). (H-I) Quantification of FBGC formation from (G): (H) number of FBGCs per high-power field and (I) percentage of fused BMDMs. Data represent n = 3 biological replicates with 5 images per group. Scale bar, 20 μm; statistical analysis by one-way ANOVA, ***p < 0.001, ****p < 0.0001.

Article Snippet: C57BL/6 and TRPV1 KO mice were purchased from The Jackson Laboratory (ME, USA).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Spinning-disk confocal images of WT and TRPV1 KO BMDMs showing Ca 2+ influx (red) under untreated (UT) conditions or following IL-4 plus GMCSF stimulation (25 ng/ml) in response to the TRPV1 agonist capsaicin; scale bar, 50 μm. (B) Quantification of fluorescence intensity from (A) (n = 4 fields per condition). (C) FlexStation 3 measurements of Ca 2+ influx in TRPV1 KO BMDMs stimulated with the TRPV4-specific agonist GSK1016790A (GSK101) under untreated (buffer control) or IL-4 plus GMCSF-treated conditions (25 ng/ml). (D) Quantification of Ca 2+ responses from (C). Experiments were performed three times with quadruplicate measurements. RFU, relative fluorescence units. Statistical significance was determined by Student’s t -test (**p < 0.01, ***p < 0.001).

Article Snippet: C57BL/6 and TRPV1 KO mice were purchased from The Jackson Laboratory (ME, USA).

Techniques: Fluorescence, Control

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Immunoblot analysis shows TRPV1, TRPA1, and actin levels in WT BMDMs 48 h following treatment with or without IL-4 plus GMCSF (25 ng/ml). (B-C) Densitometric quantification of immunoblot data from (A) (n = 3 biological replicates; one-way ANOVA, **p < 0.01, ***p < 0.001). (D) Representative immunofluorescence images of WT BMDMs stained for TRPV1 (red) using anti-TRPV1 IgG (original magnification, 60 x; scale bar, 2 μm; n = 10 cells per condition). Statistical analysis by Student’s t-test, ***p < 0.001.

Article Snippet: Primary antibodies comprised anti-actin (cat# 4970S; Cell Signaling Technology, Danvers, MA) and anti-TRPV1 (cat# ACC-030), anti-TRPV2 (cat# ACC-039), and anti-TRPA1 (cat# ACC-037) from Alomone Labs. Species-appropriate secondary antibodies (goat, rabbit, and mouse) were obtained from Jackson ImmunoResearch.

Techniques: Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Representative Giemsa-stained images of multinucleated FBGCs in WT BMDMs left untreated or stimulated with IL-4 plus GMCSF (25 ng/ml, 96 h), in the presence or absence of the TRPV1 antagonist AMG. (B-D) Quantitative analysis of FBGC formation from (A): (B) number of FBGCs per high-power field, (C) percentage of fused BMDMs, and (D) average FBGC size. Data represent n = 3 biological replicates with 5 images per group; scale bar, 100 μm; Student’s t-test, ***p < 0.001, ****p < 0.0001. (E) Representative immunofluorescence images of WT BMDMs transfected with scramble or TRPV1-targeting siRNA, stained for TRPV1 (red) using anti-TRPV1 IgG (original magnification, 60x; scale bar, 2 μm). (F) Quantification of TRPV1 fluorescence intensity (n = 10 cells per condition; Student’s t-test, ***p < 0.001). (G) Immunoblot showing TRPV1 expression in WT BMDMs 48 h after transfection with scramble or TRPV1 siRNA. (H-J) Quantification of FBGC formation following TRPV1 knockdown: (H) number of FBGCs per high-power field, (I) percentage of fused BMDMs, and (J) average FBGC size. Data represent n = 3 biological replicates with 5 images per group; Student’s t-test, ***p < 0.001.

Article Snippet: Primary antibodies comprised anti-actin (cat# 4970S; Cell Signaling Technology, Danvers, MA) and anti-TRPV1 (cat# ACC-030), anti-TRPV2 (cat# ACC-039), and anti-TRPA1 (cat# ACC-037) from Alomone Labs. Species-appropriate secondary antibodies (goat, rabbit, and mouse) were obtained from Jackson ImmunoResearch.

Techniques: Staining, Immunofluorescence, Transfection, Fluorescence, Western Blot, Expressing, Knockdown