Helping to End Addiction Long-term (HEAL): Induced Pluripotent Stem Cell (iPSC)-Derived Cell Types for Pain, Addiction and Overdose

National Center For Advancing Translational Sciences

Investigators

Abstract

Developing iPSC-derived Schwann cells for Disease Modeling and Treating Pain Schwann cells, the most abundant glial cells in the peripheral nervous system (PNS) wrap around large-diameter axons to produce a myelin sheath, while non-myelinating Schwann cells associate with smaller-diameter axons. These Schwann cell-axonal interactions are critical for controlling the normal function of both cell types and disruption of the interaction can lead to neurological symptoms and diseases, including pain. The project team intends to optimize a currently available Schwann cell differentiation protocol that is long-lasting and inefficient. These studies will include detailed cell characterization of iPSC-derived Schwann cells and identify conditions that can enhance myelination with the ultimate goal to utilize these cells for therapeutic development. Developing iPSC-derived Satellite Glial Cells for Drug Development Peripheral neuropathy (PN) is a common cause of neuropathic pain, often requiring opioid use in a subset of patients. Although causes of PN vary, it is relatively common in patients with certain common autoimmune conditions such as Sjogren syndrome (SS) and systemic lupus erythematosus (SLE). A novel autoantibody associated with neuropathic pain and PN in Sjogrens disease patients has been identified. Notably, this autoantibody was directed against a protein in satellite glial cells (SGC) in the dorsal root ganglia (DRG). This research project will develop SGCs from human iPSCs to be used in mechanistic studies as well as a tool for screening additional patients for novel autoantigens that may play a role in neuropathic pain. The project team intends to first generate scRNA-Seq data from rodent DRGs to identify additional developmental markers of SGCs, which overall represents an understudied cell type. This information will then be utilized in the development and optimization of a new protocol to generate human SGCs from human iPSCs. Development of iPSC-derived human A sensory neurons for the identification and characterization of novel neuropathic pain therapies Mechanical allodynia, the painful sensation generated by otherwise innocuous stimuli, is a hallmark of neuropathic pain. Allodynia develops, in part, due to aberrant activation of pain circuits within the dorsal horn of the spinal cord by A fibers following injury to the peripheral somatosensory system. For drug discovery programs targeting neuropathic pain the ability to evaluate novel therapeutics in human A neurons is of great value and would help to assess potential therapeutic benefits early in the discovery process. The project team intends to initially develop the differentiation protocol and expression analysis of the iPSC derived A neurons. Once the protocol is developed and validated, iPSC-derived A neurons will be comprehensively characterized using molecular and cellular methods and ultimately evaluated for functional modulation by chemogenetic approaches. Recent progress has been made by SCTL toward generation of sensory neurons that contain a population of cells expressing the desired A-fiber markers (SHOX2, PIEZO1, and TRKB) with the TRKB+/TRKC- group of sensory neurons showing highest expression of A-fiber markers. Testing a Reversible Gene Editing Method for Analgesia using iPSC-derived Sensory Neurons Human genetic studies have identified new targets that are important for nociception, with the voltage-gated sodium channel, NaV1.7 (SCN9A), being perhaps the most promising candidate for analgesic drug development. Specifically, a hereditary loss-of-function mutation in NaV1.7 leads to insensitivity to pain without other neurodevelopmental alterations. However, the similarity between various NaV sodium channels presents challenges for developing nociceptor selective inhibitors. Collaborators developed targeted epigenetic repression of NaV1.7 via epigenome engineering approaches as a potential treatment for chronic pain. The current goal is to optimize epigenetic repression reagents targeting the human NaV1.7 gene in a human iPSCs-derived nociceptor cell model to enable its translation into the clinic. In the past year, our collaborators have generated zinc finger constructs for targeted repression of NaV1.7. SCTL is currently working on increasing AAV/lentiviral transduction efficiencies in iPSC-derived nociceptors to ensure sufficient knockdown with the constructs is achievable. New iPSC lines from Phelan-McDermid Syndrome Patients to Identify Pain Mechanisms Patients with Phelan-McDermid syndrome show altered pain perception but little is known about the underlying mechanisms and molecular changes. Disease-in-a-dish models utilizing iPSCs from patients with pain or addictive disorders may advance understanding of different types of pain, and differences in individual pain responses or risk of developing chronic pain or addiction upon opioid exposure. This project is generating new iPSC lines from fibroblast cell lines, which in turn were derived from skin punches obtained from 22 Phelan-McDermid syndrome and 13 controls from family members. All fibroblast cell lines are already part of the NIMH Repository study 115 and managed by Rutgers University/RUCDR Infinite Biologics. The goal is to reprogram fibroblasts into iPSC lines and then make them available for distribution, serving as a valuable resource for the scientific community. Manufacturing and Functional Characterization of iPSC-derived Nociceptors and Astrocytes Existing protocols to differentiate human nociceptors from stem cells are inefficient, take a long time, produce limited numbers of neurons which are immature and do not have the full functional repertoire or gene expression profiles of primary nociceptors. NCATS has developed a new protocol for differentiating human nociceptors from iPSCs. The protocol is highly efficient and reproducible and based on the characterization performed to date by NCATS and external collaborators, these iPSC-derived nociceptors resemble primary sensory neurons much closer than neurons generated with previously published protocols. Hence, the advanced human nociceptor platform will be used in several projects for new target identification as well as high-content and high-throughput drug screening. In the past year, our collaborators have performed phenotypic screening with the Panacea Channel Modulator Library to screen for compounds that selectively decrease nociceptor excitability as measured by GCaMP6. Their preliminary data identify several compounds that selectively block nociceptor activity without blocking cortical/motor neuron activity, which will be investigated further. Our collaborators have also used the iPSC-derived nociceptors to screen for protective compounds using a paclitaxel-induced neuron degeneration model of chemotherapy-induced peripheral neuropathy. Human iPSCderived nociceptors for an in vitro model of pain signaling in the dorsal horn The goal of this project is to develop a microphysiological system to model the afferent nociceptive sensory circuit with coculture of human iPSC-derived dorsal spinal neurons and nociceptor cells in a micropatterned hydrogel model. The model consists of a 3D spheroid formed from peripheral sensory neurons that project axons through a micropatterned hydrogel toward a 3D spheroid formed from dorsal spinal cord neurons with an integrated microelectrode array (MEA) system for assessment of neural activity. The project teams intend to utilize iPSC-derived nociceptors and spinal cord neurons and define culture conditions for tissue-engineering of afferent nociceptive transmission and characterization of scalable MPS tissue model by assessing functional integration of spheroids with 3D MEAs and determination of circuit physiology. Ultimately, this pain-on-a-chip model will be used for