ABSTRACT
Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids, T lymphocytes, and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac, neurological, or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling, and variant-preferred healthy control lines were identified for specific disease settings.
Subject(s)
Cell Differentiation , Cell Lineage , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , CRISPR-Cas Systems , Cell Self Renewal , Cell Separation , Ectoderm/cytology , Ectoderm/metabolism , Gene Editing , Humans , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Neurons/cytology , Neurons/metabolism , Organoids , Phenotype , T-Lymphocytes/metabolism , Whole Genome SequencingABSTRACT
The lionfish (Pterois volitans) is a venomous invasive species found in the Caribbean and Northwestern Atlantic. It poses a growing health problem because of the increase in frequency of painful stings, for which no treatment or antidote exists, and the long-term disability caused by the pain. Understanding the venom's algogenic properties can help identify better treatment for these envenomations. In this study, we provide the first characterization of the pain and inflammation caused by lionfish venom and examine the mechanisms through which it causes pain using a combination of in vivo and in vitro approaches including behavioral, physiological, calcium imaging, and electrophysiological testing. Intraplantar injections of the venom produce a significant increase in pain behavior, as well as a marked increase in mechanical sensitivity for up to 24 hours after injection. The algogenic substance(s) are heat-labile peptides that cause neurogenic inflammation at the site of injection and induction of Fos and microglia activation in the superficial layers of the dorsal horn. Finally, calcium imaging and electrophysiology experiments show that the venom acts predominantly on nonpeptidergic, TRPV1-negative, nociceptors, a subset of neurons implicated in sensing mechanical pain. These data provide the first characterization of the pain and inflammation caused by lionfish venom, as well as the first insight into its possible cellular mechanism of action.
Subject(s)
Fish Venoms/toxicity , Gene Expression Regulation/drug effects , Pain Measurement/drug effects , Pain/chemically induced , Pain/metabolism , TRPV Cation Channels/metabolism , Acrylamides/therapeutic use , Analysis of Variance , Animals , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Calcium/metabolism , Calcium-Binding Proteins/metabolism , Capsaicin/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Exploratory Behavior/drug effects , Ganglia, Spinal/cytology , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Hyperalgesia/physiopathology , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/metabolism , Neurogenic Inflammation/chemically induced , Oncogene Proteins v-fos/metabolism , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , TRPV Cation Channels/genetics , Time Factors , TouchABSTRACT
BACKGROUND: Kilohertz-frequency electric field stimulation (kEFS) applied to the spinal cord can reduce chronic pain without causing the buzzing sensation (paresthesia) associated with activation of dorsal column fibers. This suggests that high-rate spinal cord stimulation (SCS) has a mode of action distinct from conventional, parasthesia-based SCS. A recent study reported that kEFS hyperpolarizes spinal neurons, yet this potentially transformative mode of action contradicts previous evidence that kEFS induces depolarization and was based on patch clamp recordings whose accuracy in the presence of kEFS has not been verified. OBJECTIVES: We sought to elucidate the basis for kEFS-induced hyperpolarization and to validate the effects of kEFS observed in patch clamp recordings by comparing with independent optical methods. METHODS: Using patch clamp electrophysiology and voltage-sensitive dye (VSD) imaging, we measured the response to kEFS applied in vitro to hippocampal and spinal neurons. RESULTS: The kEFS-induced hyperpolarization observed with current clamp recordings was corroborated by VSD imaging and rheobase measurements in patched neurons. However, no hyperpolarization was observed when imaging unpatched neurons or when recording with a voltage-follower amplifier rather than with a patch clamp amplifier (PCA). We found that EFS induced an artifactual current in PCAs that was injected back into current clamped neurons. The artifactual current induced by single, charge-balanced EFS pulses caused modest hyperpolarization, but these unitary hyperpolarizations accumulated when EFS pulses were repeated at kilohertz frequencies. CONCLUSION: Our results rule out hyperpolarization as the mechanism underlying kEFS-mediated analgesia and highlight the risk of recording artifacts caused by extracellular electrical stimulation.
