NOCICEPTRA: Gene and microRNA Signatures and Their Trajectories Characterizing Human iPSC‐Derived Nociceptor Maturation

Abstract

Nociceptors are primary afferent neurons serving the reception of acute pain but also the transit into maladaptive pain disorders. Since native human nociceptors are hardly available for mechanistic functional research, and rodent models do not necessarily mirror human pathologies in all aspects, human induced pluripotent stem cell‐derived nociceptors (iDN) offer superior advantages as a human model system. Unbiased mRNA::microRNA co‐sequencing, immunofluorescence staining, and qPCR validations, reveal expression trajectories as well as miRNA target spaces throughout the transition of pluripotent cells into iDNs. mRNA and miRNA candidates emerge as regulatory hubs for neurite outgrowth, synapse development, and ion channel expression. The exploratory data analysis tool NOCICEPTRA is provided as a containerized platform to retrieve experimentally determined expression trajectories, and to query custom gene sets for pathway and disease enrichments. Querying NOCICEPTRA for marker genes of cortical neurogenesis reveals distinct similarities and differences for cortical and peripheral neurons. The platform provides a public domain neuroresource to exploit the entire data sets and explore miRNA and mRNA as hubs regulating human nociceptor differentiation and function.

Towards bridging the translational gap by improved modeling of human nociception in health and disease

“…Maturation of neurons is induced by co-administration of the nerve growth factor (NGF), the glial cell–derived neurotrophic factor (GDNF), BDNF, and NT-3. These more mature iPSC-derived sensory neurons fire single as well as trains of action potentials and are immune-positive for the peptide hormone precursors TAC1 (substance P, neurokinin A, neuropeptide K, and neuropeptide gamma) and CALCA (CGRP, calcitonin, and katacalcin), indicating that they adopt a peptidergic sensory neuron phenotype [ 61 , 68 , 69 ].…”

“…Although this protocol is considered the “gold standard” for iPSC-derived sensory neuron and nociceptor differentiation, several studies introduced modifications by inhibiting 3i until day 12 instead of day 10 of differentiation, replating cells before passaging, neglecting NT-3 as growth factor, adding ascorbic acid, changing media gradients, or adding proliferation inhibitors (such as AraC or MitoC), which overall can lead to an increased efficiency of differentiation [ 69 – 71 ]. Different timings of 3i inhibition, changes in growth factor administration (NGF, BDNF, GDNF, and NT-3), and addition of BMP-4 or cAMP produce different subtypes of sensory neurons such as proprioceptors, mechanoreceptors, as well as pruriceptors [ 72 – 74 ] (for an overview of the available differentiation protocols, see Table 1 ).…”

“…Mapping signatures of marker genes such as neuropeptides (CGRP, TAC1, SST), voltage-gated sodium channels (Na v 1.7, Nav 1 .8), TRP channels (TRPV1, TRPM8, TRPA1), and transcription factors (DRGX, MAF) suggest that most iPSC-derived nociceptor-like neurons represent an immature state of peptidergic nociceptors that express substance P, Na v 1.8, TRPV1, NEFH, and peripherin but also the P2X-purinoreceptor 3, which is a common marker for non-peptidergic sensory neurons (Fig. 1 , [ 22 , 67 , 69 ]). In addition, a trajectory analysis of all relevant and enriched human transcription factors (DRGX, PIRT, BRN3A, and TLX3) reveals that most of these factors are also enriched in iPSC-derived sensory neurons and abundant in human peptidergic sensory neurons.…”

“…In addition, a trajectory analysis of all relevant and enriched human transcription factors (DRGX, PIRT, BRN3A, and TLX3) reveals that most of these factors are also enriched in iPSC-derived sensory neurons and abundant in human peptidergic sensory neurons. Furthermore, iPSC-derived nociceptor-like neurons transcriptionally approach to hDRG throughout differentiation [ 69 ]. Likewise, iPSC-derived low-threshold mechanoreceptors (LTMRs), proprioceptors, as well as pruriceptors can be mapped to their human DRG orthologs, due to the abundant expression of TrkC and TrkB.…”

Nociception, Transcriptomics ET CETERA: NOCICEPTRA

“…Sensory neurons and signaling mechanisms mediating the perception of pain share many similarities but also substantial differences between mice and humans, and thus, translating the findings from rodents to humans is not always straightforward [7]. Although human iPSC-derived nociceptors cannot fully recapitulate the development of the human nociceptive pathway, the study by Zeidler et al [10], recently published in Advanced Science, tries to fill this gap. In their comprehensive, datafilled paper, the group of Michaela Kress (Institute of Physiology, Medical University Innsbruck) investigated iPSCderived human nociceptor (iDN) development in the dish.…”