Journal: bioRxiv

Article Title: A highly efficient method to differentiate CGRP-expressing peptidergic nociceptors from human induced pluripotent stem cells

doi: 10.1101/2025.04.29.651263

Figure Lengend Snippet: a) (Left panel) RUNX1 expression increases while cMET expression decreases from post-mitotic placodal stage to day 40 of neuronal maturation, scale bar 10µm. Dashed box reveals small population that has RUNX1 - /cMET + peptidergic identity at day 40, scale bar 25µm. (Right panel) Gene expression profiling by qRT-PCR also reveals significant increase in non-peptidergic RUNX1 marker while peptidergic-specific cMET and CGRP marker expression is significantly reduced (p<0.05, n = 3 from three independent iPSC differentiation rounds). b) (Left panel) Immunocytochemistry reveals that day 60 mature TG nociceptors express TRPV1, TRKA and RET while CGRP continues to be lowly expressed, scale bar 50µm. (Right panel) Validation by immunocytochemistry for nociceptive markers PERIPHERIN, ISL1, BRN3A, neuronal marker TUJ1 and mature neuronal marker SYNAPTOPHYSIN which further confirms their mature sensory neuronal identity, scale bar 10µm. c) Calcium imaging demonstrates that TG-placodal isolated nociceptors respond robustly to TRPA1 agonist mustard oil followed by high dose of TRPV1-agonist capsaicin giving them a non-peptidergic identity (n = 67 cells, pooled from three independent rounds of iPSC differentiation).

Article Snippet: Primary antibodies used were rabbit anti-PAX6 (1:200, Biolegend), goat anti-SIX1 (1:100, SCBT), rabbit anti-CASPASE3 (1:200, ), goat anti-RUNX1 (1:200, SCBT), rabbit anti-cMET (1:200, Abcam), goat anti-PERIPHERIN (1:100, SCBT), rabbit anti-ISLET1 (1:200, Abcam), rabbit anti-BRN3A (1:100, Abcam), mouse anti-TUJ1 (1:1000, Biolegend), rabbit anti-SYNAPTOPHYSIN (1:100, Abcam), rabbit anti-TRPV1 (1:100, Alomone Labs), rabbit anti-5HT 1D (1:100, Alomone labs), mouse anti-CGRP (1:100, Abcam), rabbit anti-TRKA (1:100, Alomone Labs), rabbit-anti RET (1:200, Alomone Labs), rabbit anti-NEUN (1:500, Milipore), chicken anti-GFAP (Abcam) and mouse anti-TUJ1 (Abcam).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Marker, Immunocytochemistry, Biomarker Discovery, Imaging, Isolation

Journal: bioRxiv

Article Title: A highly efficient method to differentiate CGRP-expressing peptidergic nociceptors from human induced pluripotent stem cells

doi: 10.1101/2025.04.29.651263

Figure Lengend Snippet: a) Workflow for generation of CGRP-GFP reporter line. b) CGRP-GFP expressed in mature TG nociceptors in the absence of placodal isolation, with or without replating (bottom panel, scale bar 5µm). c) Immunocytochemistry reveals co-expression of CGRP and TRPV1 while RET expression is absent suggesting peptidergic subtypes, scale bars 5µm, 50µm. d) Functional analysis by calcium imaging and ELISA on non-GFP iPSC lines, for placodal non-isolated non-replated (left panel, n = 54 cells) placodal non-isolated replated (right panel, n = 35 cells). Calcium imaging shows that these nociceptors are TRPA1/TRPV1-responsive to low dose capsaicin, characteristic of peptidergic sensory neurons. Three independent rounds of differentiation were performed. They also undergo significant CGRP release upon stimulation by inflammatory soup (I.S.) (n = 3 dishes per stimulation per differentiation round, fold change quantified relative to basal release per stimulation, p<0.05). e) Immunocytochemistry and analysis by flow cytometry for placodal non-isolated replated cultures demonstrates co-localization of mature neuronal marker NEUN with peptidergic marker CGRP, further validating their nociceptive identity, scale bars 5µm, 50µm. f) (Top panel) CGRP release quantified by ELISA upon stimulation with 80mM KCl and 1µM PACAP-38. (Bottom panel) CGRP release in response to cAMP (FK, PACAP-38) and cGMP (SNAP, GTN, 8-bromo-cGMP) agonists (n = 3 dishes per stimulation per differentiation round, fold change quantified relative to basal release per stimulation, p<0.05)

Article Snippet: Primary antibodies used were rabbit anti-PAX6 (1:200, Biolegend), goat anti-SIX1 (1:100, SCBT), rabbit anti-CASPASE3 (1:200, ), goat anti-RUNX1 (1:200, SCBT), rabbit anti-cMET (1:200, Abcam), goat anti-PERIPHERIN (1:100, SCBT), rabbit anti-ISLET1 (1:200, Abcam), rabbit anti-BRN3A (1:100, Abcam), mouse anti-TUJ1 (1:1000, Biolegend), rabbit anti-SYNAPTOPHYSIN (1:100, Abcam), rabbit anti-TRPV1 (1:100, Alomone Labs), rabbit anti-5HT 1D (1:100, Alomone labs), mouse anti-CGRP (1:100, Abcam), rabbit anti-TRKA (1:100, Alomone Labs), rabbit-anti RET (1:200, Alomone Labs), rabbit anti-NEUN (1:500, Milipore), chicken anti-GFAP (Abcam) and mouse anti-TUJ1 (Abcam).

Techniques: Isolation, Immunocytochemistry, Expressing, Functional Assay, Imaging, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Marker