Making Sense of Pain: Are Pluripotent Stem Cell–derived Sensory Neurons a New Tool for Studying Pain Mechanisms?
Authors: Kathrin Meyer, Brian K Kaspar
Journal: Molecular Therapy (2014)

Most current pain studies employ animal models or animal-derived cell lines, owing to the limited availability of neuronal tissues from humans. However, screening to isolate potential new drugs or to decipher or confirm molecular mechanisms requires human systems. Although reprogramming technologies used to generate induced pluripotent stem cells (iPSCs) from somatic cells could help to overcome the shortage of human material, their in vitro differentiation to sensory neurons has been inefficient. In this issue of Molecular Therapy, Young et al. report an optimized protocol for the generation of sensory neurons from human embryonic stem cells via small-molecule inhibition.1Young GT Gutteridge A Fox HDE Wilbrey AL Cao L Cho LT et al.Characterizing human stem cell–derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.Mol Ther. 2014; 22: 1530-1543Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar The authors were able to generate a highly enriched population of sensory neurons that expressed more than 80% of the ion channels found in adult human dorsal root ganglia (DRG). Indeed, the similarity between the expression profile of endogenous human DRG and the in vitro–differentiated sensory neurons was striking. Pain is essential to protect an organism from severe injury or harm. It induces responses such as reduced movement of an injured body part or retraction from a pain-causing stimulus such as heat, or mechanical or chemical irritation.2Basbaum AI Bautista DM Scherrer G Julius D Cellular and molecular mechanisms of pain.Cell. 2009; 139: 267-284Abstract Full Text Full Text PDF PubMed Scopus (2445) Google Scholar The sensory process producing the signals that lead to pain is called nociception. Information about the type, location, and intensity of a harmful stimulus is signaled via spinal cord to the thalamus and ultimately the cerebral cortex by sensory neurons in the DRG or the trigeminal ganglion.3Julius D Basbaum AI Molecular mechanisms of nociception.Nature. 2001; 413: 203-210Crossref PubMed Scopus (1918) Google Scholar The circuits and their biochemical and molecular interactions are very complex and plastic, and not fully understood. Alterations in the pathways of nociception can lead to hypersensitivity and chronic pain, a condition that affects up to 20% of adults and includes arthritis, shingles-induced neuralgia, and bone cancer. Understanding the signaling and modulation of pain is thus of great interest because it might help to develop new and better pain medication with fewer side effects. Young et al. grew the stem cells on a gelatinous mixture of proteins called Matrigel and treated them with medium containing a number of small-molecule drugs driving differentiation toward a neuronal fate for 10 days. The medium was subsequently switched to a neuronal growth medium containing a cocktail of growth factors (glial cell–derived neurotrophic factor, nerve growth factor, neurotrophin-3, brain-derived neurotrophic factor, and ascorbic acid) for 20–25 additional days to direct the cells to become nociceptive neurons. The authors carefully documented the course of their optimized differentiation process by comparing the expression profile of their cells to human DRG samples at 12 time points. They observed that the stem cells followed a differentiation pattern in vitro that was similar to in vivo development. After 5 days of differentiation, the cells expressed markers indicative of specification of the neural lineage in an early embryo (neuroectodermal markers). Between days 8 and 13, the expression of genes defining neural crest cells—the cell type responsible for DRG formation—was observed. Finally, markers for sensory neurons and nociceptors started to appear over days 9–16, and their expression gradually increased toward the end of the differentiation protocol. Next, the researchers used single-cell quantitative polymerase chain reaction to determine the heterogeneity of the generated cell populations at several time points during differentiation over 16 days. They used markers for a variety of cell types, including cochlear hair cells, photoreceptor cells, oligodendrocytes, astrocytes, and melanocytes, along with a wide variety of neuronal and sensory neuron markers. Many individual cells examined expressed markers indicative of DRG as early as 16 days of differentiation. However, it is also evident from this analysis that the cell population at day 16 remained heterogeneous. Analysis at later time points will be necessary to determine whether certain subtypes of sensory neurons are preferentially produced, as has been the case with other differentiation protocols for neurons.4Hester ME Murtha MJ Song SW Rao M Miranda CJ Meyer K et al.Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes.Mol Ther. 2011; 19: 1905-1912Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar More interestingly, Young et al. confirmed the functionality of a subset of channels that are known to be involved in pain signaling in rodents and humans via electrophysiology (for a review, see ref. 5Waxman SG Zamponi GW Regulating excitability of peripheral afferents: emerging ion channel targets.Nat Neurosci. 2014; 17: 153-163Crossref PubMed Scopus (263) Google Scholar). Inhibitory GABAA receptors are involved in enhancing the contrast between signal and noise by permitting the transmission only of more potent sensory inputs to the neurons in the spinal cord.6Hochman S Shreckengost J Kimura H Quevedo J Presynaptic inhibition of primary afferents by depolarization: observations supporting nontraditional mechanisms.Ann NY Acad Sci. 2010; 1198: 140-152Crossref PubMed Scopus (32) Google Scholar The team found active GABAA receptors on their differentiated cells that could be blocked by application of antagonists such as picrotoxin or bicuculline, and potentiated by diazepam. Subunit-specific modulators demonstrated the similarity of the channel composition to that of human DRG. The same was true for the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, where the authors demonstrated the presence of HCN1 and 3 but not HCN2 based on cyclic adenosine monophosphate insensitivity. These channels increase neuronal excitability, and their expression has been found to be enhanced during inflammation and chemotherapy.5Waxman SG Zamponi GW Regulating excitability of peripheral afferents: emerging ion channel targets.Nat Neurosci. 2014; 17: 153-163Crossref PubMed Scopus (263) Google Scholar DRG neurons express a diverse class of potassium channels of different families, and Young and colleagues were able to confirm the functionality of the voltage-gated potassium channels Kv7.2 and Kv7.3 by using a selective drug to activate them in a reversible manner. These voltage-gated potassium channels are involved in the control of the resting membrane potential and determination of firing frequency and duration.5Waxman SG Zamponi GW Regulating excitability of peripheral afferents: emerging ion channel targets.Nat Neurosci. 2014; 17: 153-163Crossref PubMed Scopus (263) Google Scholar Their opening inhibits spontaneous or prolonged firing, which is important for the suppression of hyperexcitability. In persistent and chronic pain conditions, sensory neurons become overexcitable.2Basbaum AI Bautista DM Scherrer G Julius D Cellular and molecular mechanisms of pain.Cell. 2009; 139: 267-284Abstract Full Text Full Text PDF PubMed Scopus (2445) Google Scholar The opening of Kv7 channels with retigabine has been shown in rats to be beneficial in the treatment of bone cancer as well as for inflammatory joint pain.7Zheng Q Fang D Liu M Cai J Wan Y Han JS et al.Suppression of KCNQ/M (Kv7) potassium channels in dorsal root ganglion neurons contributes to the development of bone cancer pain in a rat model.Pain. 2013; 154: 434-448Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,8Xu W Wu Y Bi Y Tan L Gan Y Wang K Activation of voltage-gated KCNQ/Kv7 channels by anticonvulsant retigabine attenuates mechanical allodynia of inflammatory temporomandibular joint in rats.Mol Pain. 2010; 6: 49Crossref PubMed Scopus (45) Google Scholar Finally, Young et al. also confirmed the presence and function of the acid-sensing ion channels 1, 2, and 3. These channels respond to a reduction in extracellular pH that can be caused by a variety of pain-causing stimuli.9Wemmie JA Taugher RJ Kreple CJ Acid-sensing ion channels in pain and disease.Nat Rev Neurosci. 2013; 14: 461-471Crossref PubMed Scopus (413) Google Scholar Overall, nearly 400 ion-channel genes have been identified in the human genome, and Young et al. detected expression of 168 of these in the adult human DRG samples. This illustrates the complexity of the signaling pathways involved in the perception and transmission of sensory inputs, including pain. Therefore, a human in vitro system to identify novel targets for treating acute and chronic pain through modulation of ion-channel activity is of great interest. The in vitro–differentiated cells expressed 141 of these 168 channels, implying the potential of the cells for drug screening and in deciphering of the molecular mechanisms of pain signaling. Despite the elegance of the study and the detailed electrophysiology, it will be crucial to evaluate the efficiency of the protocol as well as the purity of the generated cells using other stem cell lines, as differences in differentiation potential are frequently reported.10Yin D Tavakoli T Gao WQ Ma W Comparison of neural differentiation potential of human pluripotent stem cell lines using a quantitative neural differentiation protocol.Methods Mol Biol. 2012; 873: 247-259Crossref PubMed Scopus (4) Google Scholar Furthermore, the efficiency of differentiation might differ when using iPSCs generated from terminally differentiated somatic cells.10Yin D Tavakoli T Gao WQ Ma W Comparison of neural differentiation potential of human pluripotent stem cell lines using a quantitative neural differentiation protocol.Methods Mol Biol. 2012; 873: 247-259Crossref PubMed Scopus (4) Google Scholar The latter is especially important in the context of cell-replacement therapies, which aim to avoid immune reactions by using a patient's own fibroblasts to generate stem cells for differentiation. In fact, the previously published protocol showed substantial differences between the efficiency of differentiation of iPSCs and an embryonic stem cell line.11Chambers SM Qi Y Mica Y Lee G Zhang XJ Niu L et al.Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors.Nat Biotechnol. 2012; 30: 715-720Crossref PubMed Scopus (392) Google Scholar Similarly, a recent study showed that only 15% of cells differentiated from iPSCs generated from human embryonic fibroblasts displayed sensory characteristics.12Lee KS Zhou W Scott-McKean JJ Emmerling KL Cai GY Krah DL et al.Human sensory neurons derived from induced pluripotent stem cells support varicella-zoster virus infection.PLoS ONE. 2012; 7: e53010Crossref PubMed Scopus (60) Google Scholar It is therefore likely that further purification of the generated sensory neurons would be needed before their use in drug screens or for transplantation. An additional potential problem when using iPSC lines is phenotypic variation between different clones from the same patient.13Thatava T Kudva YC Edukulla R Squillace K De Lamo JG Khan YK et al.Intrapatient variations in type 1 diabetes–specific iPS cell differentiation into insulin-producing cells.Mol Ther. 2013; 21: 228-239Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar This variation sometimes makes it difficult to discern real disease-related observations versus randomly occurring phenomena. On the other hand, the testing of various different clones per line is labor-intensive and expensive. The emerging field of direct reprogramming may facilitate generation of neuronal progenitor cells or neuronal subtypes in a more direct way so as to avoid the need for clonal selection.14Meyer K Ferraiuolo L Miranda CJ Likhite S McElroy S Renusch S et al.Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS.Proc Natl Acad Sci USA. 2014; 111: 829-832Crossref PubMed Scopus (246) Google Scholar A key question is whether iPSC-derived sensory neurons from patients suffering from chronic pain will reproduce the hyperexcitability phenotype in vitro. This would shed light on the relative importance of effects of the microenvironment versus those affecting the whole organism (e.g., stress, altered metabolism, preconditions). Interestingly, for the neurodegenerative disorder amyotrophic lateral sclerosis, fibroblast-derived astrocytes seem to reproduce the phenotype observed in spinal cord–derived astrocytes.14Meyer K Ferraiuolo L Miranda CJ Likhite S McElroy S Renusch S et al.Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS.Proc Natl Acad Sci USA. 2014; 111: 829-832Crossref PubMed Scopus (246) Google Scholar,15Haidet-Phillips AM Hester ME Miranda CJ Meyer K Braun L Frakes A et al.Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.Nat Biotechnol. 2011; 29: 824-828Crossref PubMed Scopus (571) Google Scholar Because nociception is very complex and involves many different cell types interacting with one another at various positions in the peripheral and central nervous systems, the study of any single cell type might be insufficient. On the other hand, many receptors involved in pain transmission in sensory neurons are also expressed by other cell types. For these reasons, most potential new therapeutics fail in the clinic, and new methods for testing of drugs are urgently needed.5Waxman SG Zamponi GW Regulating excitability of peripheral afferents: emerging ion channel targets.Nat Neurosci. 2014; 17: 153-163Crossref PubMed Scopus (263) Google Scholar Electrophysiology can be a powerful tool to test the effect of potential therapeutics in vitro, although the scaling of this method to high-throughput screens remains a challenge. However, studying differentiated cells in vitro might lead to the discovery of new biomarkers that, along with the electrophysiological properties, could then facilitate initial drug screens. Alternatively, the emerging field of adeno-associated virus–based gene therapy might provide a more specific way of targeting individual cell types if cell type–specific promoters are used. Adeno-associated viral–mediated targeting of different pain-related ion channels recently proved successful in reducing pain in rodent models.16Hirai T Enomoto M Kaburagi S Yoshida-Tanaka K Ukegawa M et al.Intrathecal AAV serotype 9–mediated delivery of shRNA against TRPV1 attenuates thermal hyperalgesia in a mouse model of peripheral nerve injury.Mol Ther. 2014; 22: 409-419Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,17Samad OA Tan AM Cheng X Foster E Dib-Hajj SD Waxman SG et al.Virus-mediated shRNA knockdown of Na(v)1.3 in rat dorsal root ganglion attenuates nerve injury-induced neuropathic pain.Mol Ther. 2013; 21: 49-56Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar In conclusion, the in vitro–differentiated sensory neurons reported here have the potential to improve our basic understanding of signaling pathways as well as of receptor properties and interactions, which could have major benefits for pain research and might help to accelerate the development of new and more cell type–specific therapeutics.