Using Human iPSC Sensory Neurons Progenitors as a Model for Pain

Key Points

  • New developments in stem cell biology mean that researchers can now use sensory neurons, created from human induced pluripotent stem cells (hiPSCs), to produce physiologically-relevant human cell culture models. These models can be used to find and develop new drugs and to research pain mechanisms.
  • Sensory Neurons created using HiPSCs are more effective than using cell culture models derived from animals as they are more physiologically relevant to humans, whereas animal derived models are often non-translatable and can result in false conclusions.
  • Axol human Sensory neurons created using HiPSCs express sensory neural markers that are involved in the sensing of pain, displaying expected neural responses to thermal and chemical stimuli.
  • The research covered in this article demonstrates that Axol Sensory Neurons created using HiPSCs are a practical human cell culture model for cutting edge research into the physiology of pain disorders, which can be used to characterize new drug targets for research that has the potential to improve the quality of patients lives.

Aims and Objectives

  • This research looked into producing an in vitro model with Axol Sensory Neurons, created using HiPSCs, to emulate processes that occur during the human pain response.
  • The research used in vitro cultivated experimental hiPSC-Derived Sensory Neuron Progenitors (Axol Bioscience Ltd., UK) and rat-derived dorsal root ganglion (DRG) neurons as a control.
  • Once the cells had been fully cultivated the neural response of the cells to chemical and thermal stimuli was measured for both cell cultures using a multi-electrode array (MEA).
  • Immunofluorescent imaging was also used to determine the expression of associated pain receptors and sodium ion channels in the hiPSC- Derived Sensory Neurons.

Background

Diseases that impact the nervous system, for example inflammatory autoimmune disease and Mitchell’s Disease (erythromelalgia), can cause significant patient suffering and impact survival chances; meaning they represent a large challenge in public health provision and costs.

Determining the mechanisms by which sensory neurons function and their response to different drugs in development is a key research goal in the fields of healthcare and the life sciences. A better understanding of these mechanisms will allow the pathologies of sensory disorders to be better understood, facilitating the discovery of new drug targets for treatment of the disorders.

Conventionally research has used in vitro models of non-human mammalian nervous cells, most often collected from rats. The life sciences community are now realizing that these models are often not physiologically relevant to human models, meaning the results from the research cannot be transferred. The conclusions from this research can result in problems downstream in the drug development process such as false-positive results in drug screenings.

False-positives are problematic as they mean that the drug candidates will be cleared for human trials when they are doomed to fail. This puts trial participants under unnecessary risks, can result in losses for the drug developer and produces useless results.

Recent developments in the field of stem cell biology over the past 10 years have made it possible to produce human models that are physiologically-relevant. This has involved advances in human induced pluripotent stem cells (hiPSCs) development, which allows the creation of in vitro human sensory neurons.

hiPSC-Derived Sensory Neurons are the toolkit that researchers require to make groundbreaking research into the characterization of life-changing drug targets that are relevant to human models.

Materials and Methods

A culture of Human iPSC-Derived Sensory Neuron Progenitors (ax0055) (Axol Bioscience Ltd., UK) were the experimental culture. These cells were supported by a cell culture system, using a Sensory Neuron Maintenance Medium (ax0060), and coating reagents for plating on plastic, SureBond-XF (ax0053), and for painting on glass, SureBond-XF (ax0052), (all supplied by Axol Bioscience Ltd., UK).

A supplemental method (Human iPSC-Derived Sensory Neuron Progenitors) was followed to guarantee best practice for cell plating and thawing, non-neuronal cell growth arrest and maintenance of the cell culture.

The Axol hiPSC-Derived Sensory Neuron Progenitors were cultured to a cell density of 5.0 x 105 cells/cm2 on 64-channel MEA chips (MED-R515A; Alpha Med Scientific Inc.) coated with Axol SureBond+ReadySet Coating Solution at 37 °C in an atmosphere of 5% CO2/95% air.

A culture of rat dorsal root ganglion (DRG) neurons, collected from 10-year-old male Wister rats, were the experimental control. The cells were cultured to a cell density of 1.0 x 104 cells/cm2 on 64-channel MEA chips (MED-R515A; Alpha Med Scientific Inc.) that were coated with Laminin 511 at 37 °C in an atmosphere of 5% CO2/95% air.

Following two days in vitro (DIV) the culture medium was switched for a mitomycin C (Sigma-Aldrich Co. LLC) containing Sensory Neuron Maintenance Medium, for the removal of non-neuronal cells. At 5 DIV non-neuronal cells began to die with the full effects observed following 7 DIV.

The neuronal cells were maintained for at least six weeks using a Sensory Neuron Maintenance Medium that contained the growth factors NGF, BDNF, GDNF and NT-3. Every 3-4 days half of the medium volume was replaced. Following five weeks of cultivation researchers began to identify fully developed sensory neurons.

New: Achieve Faster Maturation, Quicker Results and Enhanced Marker Expression.

MATURATION MAXIMIZER is a fully defined media supplement to enhance the existing sensory neuron maintenance medium (ax0060) and  works by mimicking in-vitro, the cell to cell communication and interaction factors which occur when these cells are in their innate environment. The supplement is known to contain factors present in the peripheral nervous system and in particular the peripheral environment of sensory neurons."

Immunofluorescent Imaging

The matured hiPSC-Derived Sensory Neurons were stained using the immunostains TRPA1, TRPV1 and Nav 1.7, and counterstained using Hoechst and β-tubulin III. Confocal microscopy with the Leica TCS SP8 was used to collect immunofluorescent images of the neurons, in order to elucidate their morphology and the expression of pain receptors.

Stimuli Response Experiments

The culture’s evoked and spontaneous extracellular field potentials were measured in a 5% carbon dioxide atmosphere at a temperature of 37 °C using a 64-channel MEA system (MED64-Basic; Alpha Med Scientific Inc.) at a 20 kHz/channel sampling rate. The signals were low-pass filtered at 100 Hz and saved on a PC. Mobius software (Alpha Med Scientific Inc.) was used to analyze firings and sort spikes.

The response of the culture to thermal and chemical stimuli was first measured following 7 DIV. The measurements were carried out several times, with the time interval between measurements determined by the time it took for the cells to recover and display the same level of activity (which tended to be between two days and one week).

The long lifetime of the cells in the culture, which remained viable after 10 weeks in vitro, allowed the experiment to be carried out multiple times.

Chemical Response Experiments

The chemical response of the control and experimental cultures were assessed by the application of menthol, capsaicin and wasabi (Allyl isothiocyanate- AITC) using the MED64 probe.

The neural responses of both cell cultures to each chemical application was measured.

Thermal Response Experiments

The culture was exposed to temperatures between 37 °C and 47 °C, at 1 °C intervals, using an MED64 thermal controller. The sensory firing responses and the control and the experimental cultures were measured with respect to the temperature applied.

Five Expert Tips for the Culture of hiPSC-Derived Sensory Neuron Progenitors

Researchers at Axol have shared their top 5 pieces of advice for the culturing of Axol hiPSC-Derived Sensory Neuron Progenitors.

  1. About 24 hours after thawing vials of the Axol hiPSC- Derived Sensory Neuron Progenitors, you will see two types of cell under the microscope: fat, rounded neurons and darker, flatter cells (see image A). Sometimes you will see more of the flat cells, because the hiPSC-Derived Sensory Neuron Progenitors are embedded in these, but mitomycin C treatment will eliminate these flat cells.
  2. It is normal to observe significant cell death after mitomycin C treatment, which targets the flatter proliferating cells. Consequently, the population of hiPSC-Derived Sensory Neuron will be more homogeneous (i.e., almost 80-90% pure sensory neurons) post-mitomycin C treatment.
  3. For each addition of mitomycin C to the Sensory Neuron Maintenance Medium, always make fresh mitomycin C for best results.
  4. Under a phase contrast microscope, hiPSC-Derived Sensory Neuron appear slightly rounder, have larger somas, and are lighter in color than other neuronal subtypes (see image B); make sure you do not mistake them for dying cells!
  5. After longer culture periods (approximately five to six weeks in vitro), neurites will become thicker and longer, and somas will become more spaced out (see image C).

Pre-mitomycin C   Day 8 post-mitomycin C

A) Pre-mitomycin C    B) Day 8 post-mitomycin C

Day 35 post-mitomycin C

C) Day 35 post-mitomycin C

Results

Following 33 DIV of culturing the cells on 64-channel MEA chips the hiPSC-Derived Sensory Neurons were demonstrating spontaneous firing activities, mimicking in vivo behavior. Immunofluorescent imaging also showed that sensory neural markers associated with pain sensing (TRPA1, TRPV1 and Nav 1.7) had been expressed in the cells following 5 weeks of culturing (Figure 1).

After 5 weeks in culture, Axol hiPSC-Derived Sensory Neurons express TRPV1, TRPA1 and Nav 1.7

Figure 1. After 5 weeks in culture, Axol hiPSC-Derived Sensory Neurons express TRPV1, TRPA1 and Nav 1.7.

Concentration-dependent changes in the firing rates of the hiPSC-Derived Sensory Neurons in response to all three chemical stimuli: menthol, capsaicin and wasabi (Allyl- isothiocyanate- AITC), were observed following 14 DIV. In addition, increased firing rates at higher temperatures were also observed with the greatest increase observed at 43 °C (Figure 2 and Figure 3).

Number of spikes (%) in response to increasing temperature after 14 DIV and after 21 DIV

Figure 2. Number of spikes (%) in response to increasing temperature after 14 DIV and after 21 DIV.

Neuronal firing responses of hiPSC-derived sensory neurons to chemical stimuli: capsaicin, menthol and wasabi (AITC)

Figure 3. Neuronal firing responses of hiPSC-derived sensory neurons to chemical stimuli: capsaicin, menthol and wasabi (AITC).

It was possible to classify the hiPSC-Derived Sensory Neurons into 27 unique types on the basis of their physiological responses to the three different chemical stimuli: menthol, capsaicin and wasabi (Allyl- isothiocyanate- AITC) (Figure 4).

The 27 types of hiPSC-derived sensory neurons and rat DRG neurons defined by their firing response rates to capsaicin (Cap), menthol (Men) and wasabi (AIT). + = increase in firings; - = decrease in firings; and ± = no change.

Figure 4. The 27 types of hiPSC-derived sensory neurons and rat DRG neurons defined by their firing response rates to capsaicin (Cap), menthol (Men) and wasabi (AIT). + = increase in firings; - = decrease in firings; and ± = no change.

Conclusions

This research demonstrated that Axol human iPSC-Derived Sensory Neurons show the expected characteristics and firing behavior as human sensory neurons, showing that they are a viable model for research into pain disorder mechanisms and for the characterization of drug targets in humans.

The research also showed that electrophysiological measurements, using multi-electrode arrays (MEA) systems, of hiPSC-Derived Sensory Neurons can be used for toxicological research and that this could be used for screening the toxicity of drugs towards peripheral nerves.

The study has demonstrated that researchers do not have to use animal-derived cell models, which are limited in their scope with respect to human cell behavior, as they now have access to a useful and physiologically-sound human model.

Access to this model will allow researchers to make breakthroughs and therapeutic treatments in the area of pain relief, and could help to reduce human risk and the associated cost in Phase I and Phase II trials.

Using this novel and modern approach will allow researchers to gain ground breaking insight into the mechanisms of pain formation, allowing them to find new therapeutic ways of improving patient life quality.

Highlighted products used in this application note and where to find them

Product Name Product Code Supplier
Human iPSC-Derived Sensory Neuron Progenitors (Male) ax0055 Axol Bioscience
Sensory Neuron Maintenance Medium ax0060 Axol Bioscience
SureBond+ReadySet ax0052 Axol Bioscience
SureBond-XF ax0053 Axol Bioscience
64-channel MEA probe for MED64-Basic MED-R515A Alpha MED Scientific Inc
Mitomycin C M4287 Sigma
Glial-Derived Neurotrophic Factor (GDNF) 450-10 Peprotech
Nerve Growth Factor (NGF) 450-01 Peprotech
Brain-Derived Neurotrophic Factor (BDNF) 450-02 Peprotech
Neurotrophin-3 (NT-3) 450-03 Peprotech

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Jul 12, 2019 at 5:22 AM

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