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: Nevertheless, incubation with the first antibodies CGRP (1:500; # 414 004, Synaptic Systems GmbH, Göttingen, Germany), TRKA (1:500; # MA515509 , Thermo Fisher Scientific, Darmstadt, Germany), GPER1 (1:500; # ab39742, Abcam, ), BRN3A (1:100, # ab245230, Abcam, Cambridge, UK), ISL1 (1:100, #H00003670-M05, Abnova, Taipeh, Taiwan), TRPV1 (1:1000, # SAB5700857, Sigma-Aldrich, Merck, Darmstadt, Germany), vGlut2 (1:400, # MAB5504, Sigma-Aldrich, Merck, Darmstadt, Germany), ßIIITUB (1:300, # PA1-41331, Thermo Fisher Scientific, Darmstadt, Germany or 1:250, #302 304, Synaptic Systems GmbH, Göttingen, Germany) and Na V 1.9 (1:100, # ab65160, Abcam, Cambridge, UK) was done at 4 °C overnight.
Techniques: Derivative Assay, Inhibition, Knock-Out, Expressing, Marker, Gene Expression, Comparison
Tocris is a verified supplier 
Tocris manufactures this product 
