Induction of Human Pruriceptors from Pluripotent Stem Cells via Transcription Factors
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Abstract
Pruriception is crucial for defense against external stimuli but can lead to chronic pruritus, a debilitating condition affecting millions worldwide. Our understanding of the cellular and molecular mechanisms behind the sensation of itch has been hindered by the lack of functional human models. Here, we address this limitation by developing a protocol to generate induced pruriceptors (iPruriceptors) from human pluripotent stem cells (hPSCs). We compared two differentiation approaches: a direct method via forced expression of transcription factors (TFs) in hPSCs, and a 2-step process through expression of TFs in hPSC-derived neural crest cells (NCCs). The 2-step protocol proved superior in inducing a transcriptional program that closely resembles that of human pruriceptors. Our optimized protocol employs forced expression of NGN1 and ISL1 to drive differentiation from NCCs into pruriceptors, enhancing the expression of known pruritogen receptors such as IL31RA, which pairs with OSMR, and HRH1. The induction of this transcriptional program leads to functional maturation of iPruriceptors. Accordingly, iPruriceptors exhibit robust responses to itch stimuli and in vivo -like itch pharmacology such as treatment with ABT-317, a JAK1 inhibitor tool compound, similar to those targeting intensive pruritus in atopic dermatitis (AD). Importantly, iPruriceptors can be generated without viral vectors or safe-harbor gene editing, using a PiggyBac-based transfection method that simplifies scalability. Our protocol offers a robust platform for investigating itch biology, modeling chronic pruritus, and enabling high-throughput screening for therapeutic target discovery.
Highlights
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NGN1 and ISL1 forced expression in NCCs induces rapid differentiation to iPruriceptors
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iPruriceptors share transcriptional profile of primary human pruriceptors
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iPruriceptors have electrophysiological responses to known pruritogens
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iPruriceptors have JAK1-dependent IL-31/-13 responses blocked by ABT-317
Article activity feed
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Finally, MEA recordings revealed functional responses to histamine and hIL-31
I'm not sure if these data are available, but is it possible to compare the increase in mean firing rate with HA or IL-31 treatment in iPruriceptors vs primary human pruriceptors?
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Notably, the choice of protocol (1-step vs. 2-step) had a greater impact on differentiation outcomes than the identity of the overexpressed TF as shown by the principal component analysis
Wow, very interesting. Why do you think this is true?
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HRH1 receptor
H4 too, right?
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Together, these results demonstrate that functional iPruriceptors can be efficiently generated from hPSCs
Have you tested the neuronal responses of iPruriceptors to other pruritogens, like serotonin or TSLP? If not, does the expression pattern of differentiated iPruriceptors indicate that the receptors for other known pruritogens are present on iPruriceptors?
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MEA recordings revealed functional responses to histamine and hIL-31
I'm curious if you have performed the multielectrode array assay on iPruriceptors in parallel with hDRGs for direct comparison?
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Representative images showing neuronal morphology in 1-step NGN1, 1-step NGN2, 2-step NGN1, 2-step NGN2 on Day 21.
Congratulations on developing this wonderful resource for studying itch! I have a few questions. Were the two groups of images being compared (2 left panels vs. 2 right panels) captured using similar microscopy techniques? At first glance, the left panels appear to have been captured using possibly DIC microscopy and the right panels captured using brightfield?
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