Rapid generation of human sensory neurons from iPSC for modeling of peripheral neuropathies

Abstract

Background

Genetic and acquired forms of small fiber neuropathies lead to debilitating health conditions, resulting in pain or loss of sensation. These neuropathies are caused by dysfunction or degeneration of unmyelinated peripheral sensory axons: c fibers. Rodent models have been used to investigate molecular mechanisms leading to neuropathies and search for potential therapeutic targets. Although these studies have advanced our understanding of small fiber neuropathies, rodent studies don’t fully recapitulate human diseases. Sensory neurons derived from human induced pluripotent stem cells (iPSC) hold promise towards advancing the field of small fiber neuropathies.

New method

Here, we report a quick and efficient protocol to generate human sensory neurons from iPSC within ten days.

Results

The generated neurons expressed generic sensory markers (Brn3A, Islet 1, and neurofilament M) as well as the sensory neuron-associated sodium channel subtypes NaV1.7 and NaV1.8. Additionally, ~80% of generated sensory neurons were positive for transient receptor potential vanilloid 1 (TRPV-1), a marker for unmyelinated sensory axons.

Comparison with other methods

Previous investigators have generated sensory neurons from iPSCs, with a protocol length range of up to 42 days. Most methodologies either employ a small molecule differentiation or an “accelerated” method, whereas our new model is generated by utilizing mRNA.

Conclusion

This novel protocol enables rapid generation of human sensory neurons from human iPSCs, achieving 60–70% efficiency. A shorter protocol can improve modeling of small-fiber neuropathies — for example, diabetic peripheral neuropathy and toxic conditions like chemotherapy-induced peripheral neuropathy.

1. Introduction

Advances in cellular reprogramming have made it possible to derive patient-specific neurons from iPSCs^1,2. Patient-derived iPSCs are powerful resources for both investigating the pathogenesis pathways of human diseases and testing potential therapeutic agents^3–9. Patient-derived iPSCs have become “game changers” for genetically linked forms of neurological disorders and have led to discoveries of potential therapies^10–13.

Recently, iPSC-derived neurons from sporadic cases of multiple neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis, have been generated and display pathological features of these diseases^13–15. Furthermore, for various neuropathies with no known genetic causes, such as diabetic peripheral neuropathy (DPN) and chemotherapy induced peripheral neuropathy (CIPN), efficient generation of human sensory neurons which can be subjected to toxic agents or metabolic stressors could serve as an effective platform to investigate these disorders.

We surveyed previous studies on iPSC-modeling of induced and inherited neuropathies and their respective cell line sources in Table 1. Specifically, studies investigating CIPN typically use iPSCs derived from healthy individuals to observe the neurotoxic impacts of chemotherapeutic agents and elucidate CIPN mechanisms. Multiple chemotherapeutic agents have been tested, including bortezomib, cisplatin, paclitaxel, and vincristine^16–23. In one study, iPSCs derived from peripheral blood monoculture cells (PBMC) of breast cancer patients were used to model paclitaxel impact. Furthermore, SARS-CoV-2 induced neuropathy was modeled by infecting a healthy iPSC line with multiple strains of the SARS-CoV-2 virus²⁴.

Table 1.

Modeling neuropathies by iPSC-derived sensory neurons

Study/disease	iPSC source & differentiation protocol information	Citation
Chemotherapy-induced peripheral neuropathy (CIPN)
Bortezomib	Three iPSC lines derived from dermal fibroblasts obtained from Mayo Clinic Biotrust.	Hrstka et al., 202116
Paclitaxel	WTC11 cell line. Differentiation protocol.	Xiong et al., 202117
Paclitaxel	Control cell line from dermal fibroblast: BIHi005-A. Breast cancer patient cell lines from PBMC: BIHi264-and BIHi263-A. Differentiation protocol65.	Huehnchen et al., 202218
Bortezomib	NIH stem cell line: LiPSC-GR1.1. Differentiation protocol41.	Snavely et al., 202219
Vincristine	PBMC-derived iPSC lines: NTUH-iPSC-02-02, IBMS-iPSC-02-07, TVGH-iPSC-02-07. Differentiation protocol53.	Tsai et al., 202220
Paclitaxel	ThermoFisher cell line: A18945. UCSF cell line: WTC-11. Differentiation protocol53.	Mortensen et al., 202366
Paclitaxel and vincristine	ThermoFisher cell line: A18945. Differentiation protocol66.	Mortensen et al., 202421
Paclitaxel, vincristine, bortezomib, cisplatin	Female donor cell lines: BIHi264-A and BIHi265-A. Differentiation protocol18.	Hew et al., 202522
Diabetic peripheral neuropathy
Pain	Human iPSC-derived sensory neurons: RealDRG (Anatomic Incorporated).	Singh et al., 202531
Itch
Atopic dermatitis; itch sensory neuron-like cells	Fibroblast-derived iPSCs. Modified differentiation protocol from67.	Guo et al., 202151
Histamine-dependent itch model	BJ fibroblast-derived cell line: BJ-iPS. Differentiation protocol68.	Tay et al., 202452
Inherited neuropathies & sensory axon injury
Hereditary sensory neuropathy type 1 (HSN1)	Control cell lines: AD2-01, AD3-01, NFDF-1, AH017-7. HSN1 cell lines: 997-01, 998-01, 999-01. Lines from StemBANCC Consortium.	Clark et al., 202111
Spinocerebellar/Friedreich's ataxia (FRDA)	Lines derived from dermal fibroblasts. Control lines: HEL46.11, HEL24.3. FRDA lines: HEL135.2, ULBi004FA4, ULBi005FA1. Differentiation protocol69	Dionisi et al., 202035
Friedrich’s ataxia	Fibroblast-derived iPSCs. Differentiated into dorsal root ganglion organoid.	Mazzara et al., 202012
Frederich ataxia	Fibroblast-derived FRDA cell line: FA10.	Viventi et al., 202132
Friedrich’s ataxia	FRDA patient and isogenic control line from male patient.	Yang et al., 202533
Axon regeneration; spinal cord injury	iPSC-derived sensory neurons41.	Cheah et al., 202334
Injury & regeneration	iPSC-derived sensory neurons41.	Xie et al., 202370
Pain & nociception
Pain	Fibroblast-derived iPSC from a healthy individual71. Modified differentiation protocol53.	Eberhardt et al., 201537
Inherited erythromelalgia (IEM)	PBMC-derived iPSC lines from five IEM subjects. Differentiation protocol53;72.	Cao et al., 201646
Inherited erythromelalgia (IEM)	IEM patient lines: UKAi0005-A and UKAi0006-A. Healthy non-IEM line: Ctrl 2. All iPSC lines are derived from fibroblasts. Differentiation protocol53;37.	Meents et al., 201947
Inherited erythromelalgia (IEM)	IEM patient lines: P301 and P300. Control line: P303. Lines derived from PBMC. Modified differentiation protocol from53	Mis et al., 201948
Inflammatory pain	iPSC sensory neuron progenitors: #ax0055 (AXOL Bioscience).	Chase et al., 202238
Pain	iPSC line: Sigma iPSC0028 (Si28).	Holzer et al., 202239
Pain	iPSC lines: LiPSC-GR1.1, NCRM5, CMT2A1.1, CMT2A2.1. CMT2A3.1.	Deng et al., 202341
Sickle cell disease (SCD); acute & chronic pain	3 SCD lines and 3 healthy control iPSC lines (WiCell).	Allison et al., 202410
Fabry disease	Dermal fibroblast-derived iPSCs. Differentiation protocol37.	Klein et al., 202449
Pain	Mesenchymal stromal cell-derived iPSC line (Uniklinik RWTH Aachen): B1 clone 102.2.	Liu et al., 202442
Peripheral analgesia	iPSC-derived sensory neurons from Anatomic Incorporated.	Shin et al., 202443
Pain	iPSC-derived sensory neurons from Anatomic Incorporated: female and male subjects.	Zurek et al., 202444
Chronic pain	iPSC-derived nociceptors: RealDRG (Anatomic Incorporated). Differentiation protocol73.	Fofie et al., 202545
Inherited erythromelalgia (IEM); small fiber neuropathy; parozysmal extreme pain disorder	iPSC lines derived from PBMC. Modified differentiation protocol from53.	Shim et al., 202550
Rare neuropathies
Rare sensory disorder of touch & proprioception	Dermal fibroblast-derived iPSC lines: C1, C2, P1, P2, P2C, KO. Differentiation protocol74.	Nickolls et al., 202036
SARS-CoV-2 neuropathy	Coriell Institute line: AG07095.	Flamier et al., 202324

hiPSC-derived sensory neurons have also been used to model DPN. DPN pathology is characterized by degenerative changes—nerve fiber swelling and tortuosity—and ultimately by loss of peripheral nerve terminals. These features are easily quantified via epidermal nerve fiber density of skin biopsies^25–30. The development of reproducible skin biopsy protocols has aided the accuracy of assessing diabetic neuropathy progression in human patients. iPSC-derived sensory neurons might result in axonal dysfunction or degeneration when challenged with glucose, supporting the notion that diabetic conditions induce degeneration. Additionally, faithful modeling of DPN, particularly the interaction of diabetic patient-derived sensory neurons with skin, has not been previously explored. By utilizing a co-culture of diabetic skin and axons, it could asses axon degeneration within the epidermis of DPN patient skin and support the notion that diabetic skin induces degeneration by acting as an inhospitable environment for axonal sprouting. Currently, there is only one study modeling DPN using iPSC-derived sensory neurons, which outlines the bidirectional modulation of FBF13/Nav1.7 protein-protein interaction complex regarding type 2 diabetic neuropathy³¹.

Genetic neuropathies, with sensory deficits, have also been modeled on patient derived iPSCs. For example, three iPSC lines have been developed to model hereditary sensory neuropathy type 1 (HSN1)¹¹. The researchers who studied the mechanisms of HSN1 showed that many impairments in the HSN1 iPSC model were prevented by l-serine supplementation, supporting it as a potential therapy¹¹. Multiple groups have also modeled Frederich ataxia (FRDA), a rare disorder that causes progressive damage to the nervous system due to the breakdown of nerve fibers in the spinal cords and peripheral nerves^12,32–35. Patient derived samples were used to recapitulate the FRDA disease model (Table 1). Additionally, other rare genetic neuropathies, such as one caused by loss-of-function mutations in the mechanosensitive ion channel PIEZO2, have been similarly modeled using iPSC-derived sensory neurons³⁶.

Various methods have been used to model pain and nociception. To characterize nociceptors and assess their functionality, healthy iPSC lines have been used, whether it be created by the researchers themselves or purchased commercially^31,37–45. These include ones modeling inflammatory pain³⁸, peripheral analgesia⁴³, and chronic pain⁴⁵. As for inherited diseases such as inherited erythromelalgia, sickle cell disease, and Fabry disease, researchers derive patient lines from dermal fibroblasts or PBMC for effective in vitro modeling^10,46–50.

The basic mechanisms of itch have also been modeled using iPSC-derived sensory neurons. Both studies listed in Table 1 utilize fibroblast-derived iPSCs, whether it be studying atopic dermatitis⁵¹ or developing a histamine-dependent itch model⁵².

Many investigators have previously generated sensory neurons from iPSC. Human iPSC-derived sensory neurons and their respective differentiation protocols have been characterized in terms of gene expression profiles and functional assessments as summarized in Table 2. Twelve studies focusing on sensory neuron differentiation protocols were reviewed. There was a large range of protocol lengths to reach sensory neuron maturation, from 14 to 42 days. Most of the methodologies came from two types—a method employing a small molecule differentiation⁵³ and an accelerated method by Anatomic Incorporated. Published differentiation protocols often start with restricting stem cells to neural crest fate and proceed with steps towards the generation of mature sensory neurons, which are processes that can take weeks. Given the varied protocol lengths to reach sensory neuron maturity (up to 42 days), in this report, we described a fast (within 10 days) and robust generation of mature sensory neurons from human iPSC using mRNAs that don’t leave a genetic footprint. An efficient and standardized protocol to generate sensory neurons can be instrumental in advancing neuropathy modeling and aid in therapeutic development.

Table 2.

Summary of iPSC-derived sensory neuron differentiation protocols.

Study aims	iPSC source	Protocol length	Citation
Characterization; differentiation protocol	iPSC lines: C14, C72; passages 10–20.	15 – 30 days.	Chambers et al., 201253
Large-scale differentiation protocol	iPSC lines derived from Coriell fibroblasts: GM03814, GM03813, GM00232, GM22592, GM04603.	25 days.	Maury et al., 201567
Differentiation protocol	No iPSC source.	12 days for sensory precursors.	Chambers et al., 201669
Differentiation protocol; Schwann cell co-culture	No iPSC source.	14 days.	Cai et al., 201875
Characterization & functional assessment	Coriell Institute cell line: GM23279A.	33 days for immature peripheral sensory neurons.	Guimaraes et al., 201876
Characterization	107 fibroblast-derived iPSC lines from unrelated healthy individuals by the HIPSCI resource77	P1 protocol: 12 days for sensory precursors. P2 protocol: 14 days for sensory precursors.	Schwartzentruber et al., 201865
Functional assessment	iPSC line: UMN PCBC16iPS78.	14 days. Used Senso-DM Anatomic Incorporated 7007 & 7008 differentiation kits.	Li et al., 202179
Differentiation protocols for sensory neuron subtypes	Control and familial dysautonomia iPSCs80. Fibroblasts from Coriell, GM00316 & GM04899.	20 days.	Saito-Diaz et al., 202181
Characterization; passage number impact on differentiation	iPSC lines derived from PBMC	33 days.	Cantor et al., 202282
Differentiation protocol (small molecule-based)	iPSC lines derived from mesenchymal stromal cells, fibroblasts, and PBMC of healthy donors and patients.	17 days.	Neureiter et al., 202240
Differentiation protocols for neuronal subtypes	No iPSC source.	15 – 28 days.	Tay et al., 202268
Differentiation protocol optimization; iPSC into sensory-link neurons	Healthy iPSC cell line49.	42 days.	Schottmann et al., 202483
Differentiation protocol comparison (accelerated vs. small molecule)	iPSC lines derived from mesenchymal stromal cells, fibroblasts, and PBMC.	28 days.	Kalia et al., 202473

2. Method

2.1. iPSC Maintenance

Two healthy control iPSC lines (Ansari ALS Stem Cell Core at JHMI) were maintained using a protocol adapted from those suggested by Ricoh Biosciences and AMSBIO. Stem cells were cultured in iMatrix (Ricoh Biosciences Cat # NI511S)-coated 6-well plates split weekly using a TrypLE-Select (ThermoFisher Cat # 12563011) – EDTA (Sigma-Aldrich Cat # E8008–100ML) mix to maintain the lines. Media was changed Monday, Wednesday, and double- or triple-fed on Friday for weekend-free media changes. Differentiating colonies were only cleaned on passaging or differentiation days.

2.2. Differentiation of Sensory Neurons from iPSCs and Culture in Microfluidic Devices

We differentiated sensory neurons from iPSCs following the mRNA-based protocol outlined in Figure 1A. iPSCs were seeded into at least 4 wells of a 24-well plate on day 0. The wells with the best density on day 1 (50–70% confluency) were selected for differentiation. Two mRNA treatments were done on day 1 and day 2, and puromycin (Milipore Sigma Cat # P8833–25MG) was added to the last media change on days 1–3 to select for transfected cells and purify the neuronal population. All the mRNA treatments were supplied in the Ricoh Biosciences’ Quick Neuron^™ Sensory-mRNA kit (Cat # SS-mRNA) as QNS-mRNA-P. From days 0–1, the cells were grown in Stemfit (AMSBIO Cat # SFB-504-CT), then transitioned to N(S1) media from day 2–4. N(S1) media is made up of DMEM/F12 (Thermofisher Cat # 21331020; 49.6 mL), Neurobasal (ThermoFisher Cat # 21103049; 49.6 mL), GlutaMAX (ThermoFisher Cat # 35050061; 520 uL), Penicillin-Steptomycin (ThermoFisher Cat # 15140122; 1 mL), Component N (supplied in the kit; 3.2 mL), and Component S1 (supplied in the kit; 104 uL), and was prepared as outlined in the Ricoh Biosciences kit’s protocol.

Figure 1. mRNA-based differentiation protocol of sensory neurons.

A. This diagram depicts the Ricoh Biosciences mRNA sensory neuron differentiation protocol. iPSCs are treated 4 times with mRNA transcription factors to drive cells towards sensory neuron development. Cells reach the neural progenitor state by day 4, which is 3–5 times faster than other methods. The cells begin to develop into neurons by day 5. By day 7, the sensory neurons are considered mature, and they can be maintained for an additional 30+ days in culture. B. Brightfield images of cells at various timepoints throughout the differentiation process (Figure 1A) from day 1 to day 35. iPSC cells are seeded on day 0, and mRNA treatment begins on day 1. Cells reach the neural progenitor stage and are re-plated into devices on day 4. By day 7, neurons are considered mature. These neurons continue to survive in culture for weeks and will form large neuronal clusters with expansive axon networks as shown in day 28 and day 35. C. Sensory axons at day 29 in a microfluidic device, stained for neurofilament M (green). These neurons can generate axons that are over 3mm in length. Scale bar = 200um.

Cells were plated into Matrigel-coated (Corning Cat # 354230) devices on day 4. 100,000 neurons were plated in the open cell body compartment of eNuvio Omega-4 devices. For devices made in-house^54–56, cells were plated into the cell body compartment at a concentration of 50,000 cells/uL. 3 uL of cell suspension were added into one port, and cells were pulled through the compartment by removing 3 uL from the other end. Media was changed every MWF. Ricoh Biosciences’ N(S1P) media was used for the first week, then switched back to N(S1) media from the second week onwards. Half-volume media changes were performed to avoid damaging fragile axons. N(S1P) media was made up of the N(S1) media listed above with an added Component P (supplied in Ricoh Biosciences’ kit) in a 1:1000 ratio.

2.3. Immunofluorescence staining

Sensory neurons were fixed and stained after 21–28 days. Cell-culture media was washed from cells by three 1X-phosphate-buffered saline (PBS) washes. All the following steps were generally done with the cells on a rocker. Cells are fixed with 4% paraformaldehyde (PFA) in PBS chilled to 4°C for 20 min at room temperature (RT). Cells are permeabilized with PBST for 10 min at RT and then blocked with 5% goat serum in PBST for 15 min at RT. PBST is generally 0.3% Triton X-100 in PBS. However, when staining for membrane proteins, 0.1% Triton can be used or a gentler detergent, Tween, can be used at 0.1% or 0.3%.

Primary antibodies were diluted into 5% goat serum PBST and incubated overnight at 4°C. The following primary antibodies were used: β3-tubulin at 1:2000 (Biolegend), Brn3A at 1:100 (Sigma-Aldrich), Islet 1 (ISL1) at 1:250 (Abcam), neurofilament M (2H3) at 1:100 (DSHB), transient receptor potential vanilloid 1 at 1:100 (Invitrogen), voltage-gated sodium channel 1.7 (NaV1.7) at 1:100 (Abcam), and voltage-gated sodium channel 1.8 (NaV1.8) at 1:100 (Abcam). All the subsequent steps were done at RT (Supplementary Figure 1). Devices were washed four times with PBST for 5 min, then the appropriate Alexa Fluor-conjugated secondary antibodies added into 5% goat serum PBST (generally at either 1:1000 or 1:2000) for 45 min. Once secondary antibodies were added, cells were kept in the dark. Cells were then washed once with PBST, three times with PBS for 5 minutes, then stained with Hoechst (1:2000 in PBS) for 10 min. Devices are washed 3 final times with PBS for 5 min. Images were acquired by Zeiss confocal microscope.

2.4. Statistical Analysis

All data analysis was done in GraphPad Prism, and pairwise comparisons were done using a student’s t-test. A p value of < 0.05 was considered statistically significant. Plots of sample group data include mean ± standard error bars.

3. Results

3.1. Sensory neuron differentiation methodology

Many sensory neuronal differentiation protocols from iPSCs are described in Table 2. However, new mRNA-based differentiation methods with much shorter timelines have been developed for a variety of cell types, including sensory neurons. We tested a new method, an mRNA-based approach that produces sensory neurons without a genetic footprint, to differentiate iPSCs into sensory neurons. Specifically, we utilized Ricoh Biosciences’ Quick Neuron^™ Sensory-mRNA kit, which successfully yielded differentiated sensory neurons from human iPSCs. Following the protocol outlines in Figure 1A, we were able to obtain mature sensory neurons earlier than traditional protocols. Representative images of cells at various stages throughout the differentiation process are shown in Figure 1B. By day four post-differentiation, the iPSCs became neurons. Three days later, they clustered into mature neuronal bodies (Figure 1B). These sensory neurons survive weeks in culture and were able to successfully grow long axons in microfluidic devices.

3.2. Sensory neuron sub-types

We assessed differentiation efficiency by staining for sensory neuron markers between 21–30 days post-initiation of differentiation. We confirmed that we were able to efficiently generate human sensory neurons from iPSCs, with 65% of cells islet 1-positive and 75% Brn3A-positive (Figure 2). Most of the cells expressed the markers of functionality NaV1.7 and NaV1.8: 55% and 65% respectively (Figure 3A and B).

Figure 2. hiPSC-derived sensory neurons.

To generate sensory neurons from iPSCs, we used an mRNA-based sensory neuron differentiation protocol (Figure 1). A. Neurons stained for neurofilament M (green, left), islet 1 (violet, middle), a merged image of left and middle plus Hoechst counterstaining (blue, right). Scale bars = 50 mm. B. Quantification of islet 1-positive neurons (each dot representing one of ten independent clusters), showing that on average 65% of cells are islet 1-positive cells 21 days post-initiation of differentiation. C. Neurons stained for neurofilament M (violet, left), Brn3A (green, middle), a merged image of left and middle plus Hoechst counterstaining (blue, right). Scale bars = 50 mm. D. Quantification of Brn3A-positive neurons (each dot representing one of ten independent clusters), showing that on average 75% of cells are Brn3A-positive cells 21 days post-initiation of differentiation. Bars in B and D represent mean ± SEM.

Figure 3. hiPSC-derived sensory neurons positive for cell-type specific markers.

A. Hoechst counterstaining (blue, left), NaV1.7 (red, middle), and merged (right) staining. B. Quantification of NaV1.7-positive neurons (each dot representing one of ten independent clusters), showing that on average > 55% of cells are NaV1.7-positive cells 30 days post-initiation of differentiation. C. Hoechst counterstaining (blue) and NaV1.8 (green, middle) and merged (right) staining. D. Quantification of NaV1.8-positive neurons (each dot representing one of eleven independent clusters), showing that on average > 60% of cells are NaV1.8-positive cells 30 days post-initiation of differentiation. Scale bars = 50 mm in A and C. Bars in B and D represent mean ± SEM.

Small fiber neuropathies, such as DPN and painful neuropathies, all effect c-fibers. One of the markers that c-fibers express is TRPV-1, a nonselective cation channel found in neurons associated with nociception⁵⁷. To examine whether our sensory differentiation protocol generates small axons that are known to be involved in relevant neuropathies, we stained our cells with TRPV-1 and found that 80% of cells are TRPV-1 positive (Figure 4).

Figure 4. hiPSC-derived TRPV1-positive sensory neurons.

A. TRPV-1 (green), Hoechst counterstaining (blue, middle) and merged image (right). B. Quantification of TRPV1-positive neurons (each dot representing one of ten independent clusters), showing that on average > 80% of cells are TRPV1-positive after 21–30 days post-initiation of differentiation. Scale bars = 50 mm in A. Bars represent mean ± SEM.

Together, the characterizations and quantifications of sensory markers show that we can obtain efficient sensory neurons from human iPSCs. Furthermore, most of these cells expressed functional markers (for sodium channels) and TRPV-1, a marker for c fibers.

4. Discussion

In this report, we described a robust and efficient method to generate mature human sensory neurons from iPSCs in ten days—substantially faster than the protocol summarized in Table 2 (protocols of 14 – 42 days in length). This rapid differentiation protocol could accelerate our understanding of small fiber neuropathies and search for relevant therapeutics, especially regarding acquired forms with no known genetic causes such as DPN and CIPN.

This method of sensory neuron differentiation can also be helpful in modeling other neuropathies, such as those relating to itch, pain, genetic, and induced neuropathies. For induced conditions (such as CIPN or SARS-CoV-2-induced neuropathy) and various pain models, investigators typically use iPSC lines derived from healthy donors. As for inherited neuropathies, patient iPSC lines are derived from mesenchymal stromal cells, fibroblasts, and PBMC. A standardized, rapid protocol for generating sensory neurons could help elucidate disease mechanisms and accelerate therapeutic testing across these contexts.

Since small fiber neuropathies involve interactions between sensory axons and their target tissues, in vitro models should incorporate co-culture systems (for example, sensory axons with skin or intestinal tissues). An efficient differentiation protocol is therefore essential; our protocol described in this report generates mature sensory neurons within 10 days. Most of the sensory neurons generated in our protocol express TRPV-1, which is known to be involved with painful neuropathies^57,58. TRPV-1, a non-selective cation channel, plays a role in pain and body temperature regulation. Furthermore, these channels can be activated by vanilloid capsaicin, intense heat of over 43C, low acidity, electrical potential, and various lipid types. The involvement of TRPV-1 in pain perception has made it a primary target for pain management.

Similarly, this protocol can be paired with human skin explants to model innervation. In the field of dermatology, culturing of human skin explants has been used in research for decades before investigators were able to isolate specific skin cell types such as keratinocytes. This is due to the ease of obtaining skin biopsies and culturing of intact skin tissues⁵⁹. Conditions for culturing skin from most body parts have been developed, including from legs^60–62, which are where skin biopsies are normally obtained for diagnosis of DPN^26,28,63. Skin explants and sections are commonly cultured for 8–12 days, so a rapid neuronal differentiation protocol is needed for successful co-culturing—our protocol addresses this need.

The development of reproducible skin biopsy protocols has aided the accuracy of assessing DPN progression in human patients^26,28,63. This is because the typical features of DPN, such as nerve fiber swelling and tortuosity, are easily quantified via epidermal nerve fiber density of skin biopsies. Epidermal nerve fibers have been shown to be affected early during DPN development, acting as a “canary in the coal mine”. To what degree the target environment, in this case the skin of diabetic patients, contributes to sensory neuron degeneration is unknown. However, recent advances in human iPSC-derived neurons and availability of skin biopsies make addressing this question in the context of human cells feasible. The method in this report would be instrumental in co-cultures of diabetic skin and iPSC-derived sensory neurons because of its rapid differentiation protocol. Together, this rapid, reproducible method for generating mature human sensory neurons from iPSCs provides a practical platform for modeling target tissue–neuronal interactions, probing disease mechanisms, and accelerating the development and testing of therapies for small-fiber neuropathies.

Supplementary Material

1

Supplementary Figure 1. Expression of sensory differentiation markers.

A. Merged image of Nav1.8 (red) and Hoechst counterstaining (blue). B. Merged image of Islet 1 (green) and Hoechst counterstaining (blue). Differentiated sensory neurons cluster in a group once they express sensory markers. Scale bars = 20 mm in A and B.

Highlights.

• Efficient differentiation of human sensory neurons.

• Generation of c-fibers.

• Comparison of protocols for sensory neuron differentiation.

• Evaluation of neuropathy models using hiPSC-derived sensory neurons.