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‘Pain in a Dish’ Models Peripheral Pain and Sets the Stage for Drug Screening

ARTICLE IN BRIEF

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HUMAN NOXIOUS STIMULUS-DETECTING sensory neurons produced by converting skin cells with a set of five genes — enabling study of “pain in a dish.”

In a plenary lecture at the AAN Annual Meeting, Brian J. Wainger, MD, PhD, described his research to advance the molecular understanding of the physiology of nociceptors, the sensory cells that stand at the head of the pain perception cascade.

WASHINGTON—New cell culture models of human nociceptors are poised to accelerate the search for new drugs to treat pain, Brian J. Wainger, MD, PhD, an assistant professor of neurology and anesthetics at Harvard Medical School in Boston, told attendees of a plenary lecture here in April at the AAN Annual Meeting.

In an overview of his research, Dr. Wainger said, “We think developing human pain sensory neurons from patients with specific pain disorders will allow for a tailored, personalized approach for deriving pain treatment for specific pain diseases.

“Animal models have provided instrumental insights into sensory physiology, but there are limits to the extent to which they can capture the complexity and heterogeneity of human pain conditions,” he explained. “Despite having successful treatments in animal models, these treatments have not translated as well as desired into human pain therapy,” providing the rationale for the new human cell-based models.

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DR. BRIAN J. WAINGER: “We think developing human pain sensory neurons from patients with specific pain disorders will allow for a tailored, personalized approach for deriving pain treatment for specific pain diseases.”

Molecular understanding of the physiology of nociceptors, the sensory cells that stand at the head of the pain perception cascade, has advanced markedly in the past two decades, he said, with the identification and characterization of ionotropic receptors and ion channels responsible for detection and transmission of specific pain stimuli, including heat, cold, and pH.

“Modulation of these receptors is responsible to a large extent for the transition to chronic pain disorders,” Dr. Wainger said. For example, prostaglandin E2 sensitizes the capsaicin receptor TRPV1 so that it is able to activate at warm, instead of just noxiously hot, stimuli. “This property, by which normally harmless stimuli can elicit a pain response, is one of the prime properties of chronic pain,” he explained, adding that mutations in the genes for pain receptors are implicated in a large fraction of common pain disorders, including small fiber neuropathy.

STUDY METHODOLOGY

To make valid cell-based models, Dr. Wainger wanted cells that displayed the same suite of responses to these same noxious stimuli as in vivo nociceptors. He began with human skin cells, which he reprogrammed into pluripotent stem cells. He then treated these stem cells with differentiation factors to induce them to become nociceptors.

He found that the cells responded to capsaicin and mustard oil, two agonists of TRPV1, with a response roughly the same as bona fide mouse nociceptors. The noxious chemicals not only caused calcium release, as the mouse cells do, but also triggered robust firing, with a pattern characteristic of nociceptors. In response to depolarization, the cells released calcitonin gene-related peptide, which is involved in inflammatory pain and neurogenic inflammation. In some cells, Dr. Wainger said, the neurons could actually fire in the presence of tetrodotoxin, the sodium channel-blocking poison from the puffer fish, which is a very specific property of nociceptors.

“One of our primary goals was to show that the neurons capture essential elements of the pathophysiological process,” including sensitization, Dr. Wainger said. He found that the initially low neuronal response to capsaicin could be increased by exposure to prostaglandin E2, just as occurs in nociceptors in vivo.

A similar mechanism of receptor sensitization is thought to underlie painful chemotherapeutic neuropathy, he said, “and this is a tremendous problem, as it is dose-limiting for on the order of half of chemotherapy regimens.” When the derived nociceptors were treated with the chemotherapy agent oxaliplatin, the firing rate jumped from below 200 spikes per minute to up to 1,000, recapitulating the sensitization seen in the human disorder.

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DR. RU-RONG JI said it has been difficult to develop new drugs with the traditional approach because, despite the basic similarities in pain physiology, the mouse is still not exactly the same as the human. With human cell lines, he said, it may be easier to see the potential of a putative treatment before spending the millions necessary for a clinical trial.

All these molecular pathways can be used for phenotypic drug screens, with reductions in the firing rate as the marker for a potentially useful analgesic, Dr. Wainger said. The ability to easily make millions of cells and monitor their responses in a dish holds the potential to accelerate drug discovery and development for pain conditions.

The cells may also be useful for developing a deeper understanding of, and new treatments for, genetically determined pain diseases, such as familial dysautonomia, he said. The disorder is caused by abnormal splicing of the IKBAP gene and causes a progressive degeneration of unmyelinated pain fibers and autonomic nerves, leading to severe pain and dysautonomia. Skin cells from affected family members, transformed into neurons, displayed the same abnormal splicing and a pattern of altered cell growth similar to that seen in patients. “What we hope is that, in the future, these phenotypes in a dish can be used to screen for novel treatments in a targeted, patient-specific manner,” Dr. Wainger said.

EXPERT COMMENTARY

“This is a very important approach for its translational potential,” Ru-Rong Ji, PhD, chief of pain research and a professor of neurology at Duke University Medical Center in Durham, NC, told Neurology Today. “It has been difficult to develop new drugs with the traditional approach because, despite the basic similarities in pain physiology, the mouse is still not exactly the same as the human.” Proteins may be expressed at different levels, or modified differently, and these small molecular differences may have large effects on the ability of a drug to block a receptor or ion channel. With human cell lines, Dr. Ji said, it may be easier to see the potential of a putative treatment before spending the millions necessary for a clinical trial. “This should help the drug discovery process,” he said.

However, he pointed out, the cell-based models are likely to be useful only for drug discovery aimed at primary nociception. Different pain modalities, such as central pain or emotionally or cognitively-modulated pain, “are more in the brain. This model will apply to the sensory component in the periphery,” and not to pain mediated by mechanisms elsewhere, he said.

Dr. Wainger agreed, noting that diverse pain conditions have too often been lumped together for the purposes of therapy development. “Different disease etiologies need to be evaluated and treated separately based on the different involved mechanisms,” he said. There may also be potential to examine more centrally-derived pain conditions using central neurons, perhaps in culture with astrocytes or other cells with which they interact.

“In the setting of the pain channelopathies, where the proteins are so locally expressed, I think there is good potential for successful modeling of those diseases” using the neuronal system he has developed, Dr. Wainger said. “But in cases where there is a much larger circuit responsible for the disease, we will likely need to address that with other types of models.”

LINK UP FOR MORE INFORMATION:

•. Wainger BJ, Buttermore ED, Oliveira JT, et al. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts http://www.nature.com/neuro/journal/v18/n1/full/nn.3886.html. Nat Neurosci 2015: 18(1): 17–24.
    •. Neurology Today archive on pain: http://bit.ly/paininadish-NT