Generation of iPSC-Derived Human Peripheral Sensory Neurons Releasing Substance P Elicited by TRPV1 Agonists

Abstract

Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro.

Neuromuscular actuation of biohybrid motile bots

“…Stem cell-derived neurons in culture have been shown to spontaneously develop networks with periodic bursting patterns (44). Advances in stem cell biology have enabled directed differentiation of pluripotent stem cells into neurons of spinal cord identity (25), cortical neurons (45), and, more recently, peripheral sensory neurons (46), thereby providing cell sources to possibly enhance functionalities of in vitro neural networks. These networks, in turn, may be leveraged to engineer autonomous biohybrid embodiments that exhibit adaptive motor patterns in response to environmental cues.…”

3D culture platform of human iPSCs-derived nociceptors for peripheral nerve modelling and tissue innervation

“…These differentiated nociceptors also released substance P in response to a noxious stimulus (RTX) (10 DIV cultures; Fig. 3B), denoting the presence of a functional TRPV-1 channel 26,35 . For these experiments, we used dissociated neuron cultures in order to improve the access of the electrical probe to single cells and ensure that substance P release was unhindered by the cell cluster.…”

“…1b and 2D). Contrarily, other nociceptor differentiation protocols culture cells on flat surfaces, producing disorganised and non-homogeneous cultures 19,35 . Before commencing differentiation, we adopted a strategy for cell synchronization via DMSO treatment, which has been reported as an effective method to arrest the cell cycle 36 .…”

Oligomeric α-Synuclein induces skin degeneration in a reconstructed human epidermis model

“…Accordingly, our hypothesis is that peripheral nerve terminals and melanocytes, which are the main cells expressing α-Syn in the skin, convey α-Syn to the epidermis. Interestingly, α-Syn is found in postmortem tissues of the peripheral nervous system from patients with α-synucleinopathy (Sumikura et al, 2015), in sensory nerve terminals in the skin (Akerman et al, 2019a), and in sensory neurons derived from human induced pluripotent stem cells (Guimarães et al, 2018). Therefore, simply by accumulation or via other factors, such as UV radiation (Carmo-Gonçalves et al, 2014), α-Syn aggregates into Oα-Syn, causing skin inflammation and degeneration (Figure 5).…”