The use of induced pluripotent stem cells (iPSCs) in disease modeling and drug screening has been widely spread in biomedicine (Omole & Fakoya, 2018) but mainly as a tool for the study of neurodegenerative and neurodevelopmental disorders probably because it somehow allows access to the central nervous system (CNS), permitting the exploration of molecular pathways of certain subtypes of active human neurons and other neural cells (Marchetto et al., 2011; Russo et al., 2018). Additionally, iPSCs keep the genetic background of the donor, which is especially important to study polygenic and multifactorial disorders like autism spectrum disorder (ASD) (Marchetto et al., 2009). Human iPSCs have been used to generate several neuronal and glial subtypes (Tcw et al., 2017; Bianchi et al., 2018; Jerber et al., 2021; Johns & Maragakis, 2022; Xu et al., 2022), as well as whole-brain organoids (Lancaster et al., 2013) or organoids from specific brain regions (Qian et al., 2018; Jacob et al., 2020), to develop a basis for understanding neurological diseases.

Despite the numerous advances in iPSC-derived nerve cells, there are few protocols to produce sensory neurons, which are the cells that capture environmental or visceral stimuli and transmit them to the CNS (Chambers et al., 2012; Eberhardt et al., 2015; Bear et al., 2016; Guimarães et al., 2018; Schwartzentruber et al., 2018; Mis et al., 2019; Umehara et al., 2020). In addition, sensory neurons have recently been described as very much affected during viral infections, like in the coronavirus disease of 2019 (COVID-19) pandemic, in which 80% of infected people manifested alterations in senses related to the peripheral nervous system (PNS), like ageusia (loss of sense of taste) and anosmia (loss of sense of smell) (Cooper et al., 2020; Patel et al., 2020; Cunhados et al., 2021). Once again, sensorial stimuli seem to be affected in neurodevelopmental disorders, such as ASD and attention-deficit hyperactivity disorder (ADHD), in neuropathic pain, and specific sensory disorders, like sensory processing disorder (SPD) (Koziol et al., 2011; Weiland et al., 2011; Hazen et al., 2014; Labau et al., 2022).

Neurons from the sensory system still face difficulties in differentiation protocols, which impact their use for in vitro modeling (Labau et al., 2022). Sensory neuron differentiation protocols (Chambers et al., 2012; Eberhardt et al., 2015; Guimarães et al., 2018; Schwartzentruber et al., 2018; Mis et al., 2019; Nickolls et al., 2020; Umehara et al., 2020) have been tested on more than 100 different human iPSC (hiPSC) strains, all derived from pulmonary, dermal fibroblasts, or blood, which are cells of mesenchymal origin (LeBleu & Neilson, 2020). It is important to consider that sensory neurons belonging to the PNS originate in the neural crest, derived from ectoderm differentiation during the neurulation process (Huang & Saint- Jeannet, 2004). The neural crest cells act in the formation of several parts of the vertebrate body: in the formation of pigmented cells, originating the ganglia of the peripheral nervous system and composing part of the cartilages and bones of the face; in the cardiac formation (cells that form septa and valves, and muscle cells); and in tooth structure, originating cells forming dentin and, in mammals, pulp, cementum, periodontal ligament, and alveolar bone (Hall, 2009; Szobó and Mayor, 2018). In the pulp of human exfoliated deciduous teeth, there is a population of postnatal multipotent stem cells called stem cells from human exfoliated deciduous teeth (SHED), which have a high proliferation rate and the ability to form clusters, and are osteoinductive in vivo (Miura et al., 2003). Because they share the same embryonic origin as some neural cells, SHED express neuronal and glial markers (Miura et al., 2003), and the dental pulp cells produce neurotrophic factors and interact with peripheral nervous system neurons in vitro (Nosrat et al., 2001). Considering that even after reprogramming hiPSCs maintain a particular transcriptional signature from the donor cell (Marchetto et al., 2009), it is essential to choose for reprograming the cell that is derived from the same germ layer as the final differentiated cell, which is the target of the study. Here, we have established a protocol to obtain functional peripheral sensory neurons (fPSNs) derived from SHED to use as a platform for studying sensory alterations or diseases that affect this system.

Our group was the first to establish the protocol to produce fPSNs derived from SHED-iPSC. fPSNs were produced based on a protocol previously established for iPSCs derived from fibroblasts (Guimarães et al., 2018). The protocol is represented in Figure 1.

SHED were obtained from neurotypical individuals. Cellular reprogramming was highly effective, with small iPSC colonies being observed 25 days after transduction (Figures 2A–G), which were collected and established in feeder-free culture conditions (without a supporting cell layer) (Figure 2H). The iPSC colonies showed a delimited border with a phase-bright center, and the cells had a prominent nucleus and less cytoplasm (Figure 2I). The expression of pluripotent cell markers (Sox2, NANOG, and Oct4) was analyzed by immunofluorescence assays, confirming the success of cell reprogramming (Figures 2J–L).

The iPSC colonies must be small (∼500µm) to start the NCPC differentiation (Figure 3A) since this makes the factors reach the colony’s interior and prevents them from growing uncontrollably until day 11. Around day 3, the colony’s border is no longer delimited, and it becomes possible to observe cytoplasmic expansions on it (Figure 3B). When the culture is only supplemented with CHIR-99021, on day 6, some cells begin to migrate out of the colonies (Figure 3C), and on day 8, colonies may show overgrowth and an increased risk of detachment (Figure 3D). After transferring the cells to PO-lam-treated plates, it may take a few days to become established, and cells can form cell aggregates at this stage (Figure 3E). As soon as the NCPC (Figure 3F) reached 70% confluence, expansion and storage of these cells were performed. Cellular characterization was performed after the fourth passage. The NCPC showed expression for nestin and peripherin, an intermediate filament protein from proliferation cells and peripheral nervous system neurons, respectively (Figure 3G).

From the NCPCs (Figure 4A), the PSN already assumed a neuronal morphology after 20 days of differentiation (Figure 4B), and, as expected, the neurite branches increased after the addition of HEK-conditioned medium during the last 10 days of the protocol (Figure 4C).

The protocol allowed the differentiation of fPSNs with long neurites of more than 300 μm. fPSN characterization was performed with MAP2, a neuron marker; peripherin (Figure 4D), a marker for peripheral nervous system neurons; and Islet 1 (Figure 4E), highly expressed in fPSNs but not in NCPCs (Guimarães et al., 2018), concluding that the cells obtained using this protocol are specific neurons from the peripheral system. In order to confirm the need to use the conditioned medium to allow the neurons to mature, three culture conditions were tested as follows: 1) using the HEK-conditioned medium during the last 5 days of the protocol; 2) using the HEK-conditioned medium during the last 10 days of the protocol, and 3) without the HEK-conditioned medium during the last 10 days of the protocol.

There was an increase in the number of pre-synaptic markers when 75% of the HEK-conditioned medium was used, in contrast to the protocol using only the 3 N medium (Figures 5B,C,G). Although a variation in the expression of peripherin appears, it is not significant when compared to the use of the HEK-conditioned medium for 5 or 10 days (Figures 5A,B,G). The expression of pre-synaptic markers does not vary significantly between the three culture conditions, although image analysis seems to show a subtle increase in the protocol’s original condition (using a conditioned medium) (Figures 5D–F,H). It lacks other evaluation methods to verify how sensitive this protocol is to changes.

In this study, we established a protocol of differentiation to obtain NCPCs and fPSNs from iPSCs derived from SHED, aiming at their use in studies related to sensory disorders.

SHED were isolated from the dental pulp after exfoliation. Therefore, it is non-invasive, easy to obtain and isolate, and has an optimal application for studies, in which a patient’s skin biopsy would be impracticable and potentially traumatic, for example, in cases of pediatric diseases or syndromes.

This protocol is similar to the dual-SMAD inhibition method described by Chambers et al. (2012), in which what triggers neuronal differentiation are small inhibitory molecules such as LDN-193189, SB431542, and CHIR-99021, but this method and those with little modifications (Eberhardt et al., 2015; Schwartzentruber et al., 2018; Mis et al., 2019; Umehara et al., 2020) use fibroblast- or blood-derived iPSC lines. The other differentiation protocols for human sensory neurons, those that do not use this dual-SMAD method, start from hiPSCs derived from fibroblasts (Nickolls et al., 2020), from human embryonic stem cells (hESCs) (Bajpai et al., 2010; Schrenk-Siemens et al., 2014; Alshawaf et al., 2018; Hulme et al., 2020) or from murine fibroblasts (Wainger et al., 2014; Blanchard et al., 2015). We were the first group to establish this protocol using human SHED-iPSCs, whose cell source comes from accessible tissues.

This whole protocol takes around 2 months without going through a suspension culture stage, which includes three principal phases: NCPC induction, NCPC maintenance and expansion, and PSN induction and maturation; the latter is divided into two stages, one with only 3 N medium and supplements for 20 days and the other with the addition of the HEK-conditioned medium for 10 days, which seems to be essential for neuronal maturation (Guimarães et al., 2018). To start the protocol, iPSCs should have approximately 60% confluence composed of small colonies as failure to comply with this determination increases the risk of plate detachment, inadequate NCPC differentiation, and low densities in the initial plating in the dual-SMAD inhibition method, and promotes differentiation to neural crest cells (Chambers et al., 2009). In the first passages, NCPC differentiation is not fully established and may show slight changes in morphology and characterization, probably depending on the genetic background of the cell lineage.

Keratinocytes stimulate axonal growth in sensory neurons via the release of NGF and BDNF (Ulmann et al., 2007). In our protocol, these two factors were added exogenously, independent of the presence of the conditioned medium produced from the keratinocytes, and probably were sufficient to promote axonal growth despite the time and the presence of the conditioned medium. It would be interesting to test a co-culture model with sensory neurons and keratinocytes to improve PSN differentiation because recent studies have shown synaptic communication between them (Talagas et al., 2020).

Sensory neurons capture and transmit environmental or visceral stimuli to the CNS (Bear et al., 2016). In addition to the entire PNS, sensory neurons differentiate from neural crest cells during embryogenesis (Hall, 2009; Szobó& Mayor, 2018) from a portion of cells at the edge of the neural plate that starts to separate and migrate during the formation of the neural tube, leading to the origin of CNS cells (de Lahunta et al., 2016). Our protocol differentiates sensory neurons from somatic cells that also originated from the neural crest (Labau et al., 2022), and, as cells after reprogramming still carry their transcriptional signature, it is important that when working with iPSC technology, whenever possible, using the same embryonic origin of the cell source and the cell target of the study after the final differentiation is considered. An advantage presented is that the PSNs generated by this method do not present significant changes in their functionality according to variations in the final phase of the protocol, indicating that they are not as sensitive as those originating from fibroblasts (Schwartzentruber et al., 2018; Umehara et al., 2020), although additional analyses are necessary to confirm this result. Moreover, another benefit of using cells from the dental pulp of the exfoliated deciduous tooth is the low age of the donor, probably with fewer mitochondrial DNA mutations, which can alter the metabolism of the iPSCs (Kang et al., 2016).

This work showed the production of NCPCs and PSNs from SHED, cells with the same ontogenetic origin. Obtaining sensory neurons in vitro can open a wide variety of applications consistently, such as studies of pain or pruritus, the effects of peripheral inflammation and infections, drug testing, and altered sensitivity alterations in diseases or syndromes. Last, starting from the NCPCs established here, it is possible to develop new differentiation protocols to achieve other of its various cellular destinations.