Robust induction of neural crest cells to derive peripheral sensory neurons from human induced pluripotent stem cells

Abstract

Because intractable itch reduces quality of life, understanding the fundamental mechanisms of itch is required to develop antipruritic treatments. Itch is mediated by peripheral sensory neurons, which originate from the neural crest (nc) during development. itch-associated signaling molecules have been detected in genetically engineered animals and in cultures of peripheral neurons from dorsal root ganglia (DRG). Ethical difficulties collecting peripheral neurons from human DRG have limited analysis of itch in humans. This study describes a method of differentiating peripheral neurons from human induced pluripotent stem cells (hipScs) for physiological study of itch. this method resulted in the robust induction of p75 and HNK1 double-positive NC cells from hiPSCs. The expression of NC markers TFAP2A, SOX10 and SNAI1 increased during NC induction. The induction efficiency was nearly 90%, and human peripheral neurons expressing peripherin were efficiently differentiated from hiPSC-derived NC cells. Moreover, induced peripheral neurons expressed the sensory neuronal marker BRN3A and the itch-related receptors HRH1, MRGPRX1, IL31R and IL-4R. Calcium imaging analyses indicated that these peripheral neurons included sensory neurons responsive to itch-related stimuli such as histamine, BAM8-22, IL-31 and IL-4. These findings may enable detailed analyses of human DRG neurons and may result in new therapies for intractable itch. Somatic sensations, including itch and pain are mediated by primary sensory neurons, which have cell bodies in dorsal root ganglia (DRG) or trigeminal ganglia. These neurons differ in somal size, expression of ion channels and receptors, innervation territories, and electrophysiological properties 1. Small-diameter DRG neurons with unmyelinated axons (C fibers) are the major types that mediate itch and pain 1,2. Similar to pain, chronic and intractable itch reduces quality of life in patients with skin diseases such as atopic dermatitis (AD), which are frequently resistant to conventional treatments, such as histamine H 1 receptor antagonists (antihistamines). Itch in atopic skin is elicited by noxious stimuli, with barrier dysfunction caused by itch-induced scratching, which are associated with the itch-scratch cycle 3. Understanding the fundamental mechanisms of itch is required for the development of antipruritic treatments. Studies in genetically engineered animals and rodent DRG neuron cultures have revealed signaling molecules and receptors that contribute to the induction and transmission of itch. Mas-related G protein-coupled receptors (Mrgprs), especially MrgprAs, MrgprB4, MrgprB5, MrgprC11 and MrgprD, have been reported involved in histamine-independent itch pathways in mice, with the expression of these Mrgprs restricted to small-diameter DRG neurons 4. In humans, MRGPRXs are also selectively expressed in DRG neurons 5. The antimalarial drug chloroquine and bovine adrenal medulla peptide 8-22 (BAM8-22) activate MrgprA3 and MrgprC11, respectively, and elicit itch in mice through a distin...

Exosomes derived from differentiated human ADMSC with the Schwann cell phenotype modulate peripheral nerve-related cellular functions

“…These genes are related to sodium channels with the function of transmitting pain signals. The expressions of all these genes indicate that some of these cells were differentiated into nociceptive neurons [ 83 , 84 ]. IF staining further confirmed the positive expressions of β-3 tubulin and TRPV1, which exist in sensory neurons, in the hiPSC-SNs after induction ( Fig.…”

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

“…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 ). Neuronal induction media composed of DMEM, N2, NEAA, and heparin, followed by administration of retinoic acid and BMP4, successfully induce TrkA + nociceptors from human embryonic stem cells [ 75 ].…”

“…While proprioceptors show strong expression of TrkC, mechanoreceptors are either positive for TrkC, TrkB, parvalbumin, and the transcription factor short stature homeobox 2 (SHOX2) [ 72 ]. iPSC-derived pruriceptor expression of itch-related molecules such as IL31R, IL-4R, and the histamine receptor HRH1 is highly correlated with non-peptidergic itch-related human DRG neurons [ 74 ].…”

Cellular models of pain: New technologies and their potential to progress preclinical research

“…Despite evidence for a C-fibre identity, neurons are readily myelinated when co-cultured with Schwann cells ( Clark et al, 2017 ), making it difficult to rule out an Aδ-nociceptive identity. Several groups have adopted the Chambers protocol and report congruous findings ( Eberhardt et al, 2015 , McDermott et al, 2019 , Mis et al, 2019 , Pettingill et al, 2019 , Schwartzentruber et al, 2018 ), however, there are also reports of the same protocol generating neurons with gene expression profiles resembling other sensory neuron types, such as pruriceptors ( Umehara et al, 2020 ), C-low threshold mechanoreceptors ( Guimarães et al, 2018 ) and proprioceptors ( Dionisi et al, 2020 ). Such differences in resultant cell type likely result from minor protocol modifications and illustrate the differentiation sensitivity, especially when small molecules and defined media are employed.…”