Primary Cultures Research Articles

Repurposing Nitazoxanide for Potential Treatment of Rare Disease Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare genetic lung disease. Unfortunately, treatment with the mTORC1 inhibitor Rapamycin only slows disease progression, and incomplete responses are common. Thus, there remains an urgent need to identify new targets for the development of curative LAM treatments. Nitazoxanide (NTZ) is an orally bioavailable antiprotozoal small molecule drug approved for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum in children and adults, with a demonstrated mTORC1 inhibitory effect in several human cell lines. NTZ’s excellent safety profile characterized by its more than 20 years of clinical use makes it a promising candidate for repurposing. Our rationale for this study was to further investigate NTZ’s effect using in vitro and in vivo LAM models and to elucidate the underlying molecular mechanism beyond mTORC1 inhibition. For this purpose, we investigated cell proliferation, cell viability, and changes in protein phosphorylation and expression in primary human cell cultures derived from LAM lung samples before translating our results into a syngeneic mouse model utilizing Tsc2-null cells. NTZ reduced cell growth for all tested cell lines at a dose of about 30 µM. Lower doses than that had no effect on cell viability, but doses above 45 µM lowered the viability by about 10 to 15% compared to control. Interestingly, our western blot revealed no inhibition of mTORC1 and only a mild effect on active ß-Catenin. Instead, NTZ had a pronounced effect on reducing pAkt. In the mouse model, prophylactic NTZ treatment via the intraperitoneal and oral routes had some effects on reducing lung lesions and improving body weight retention, but the results remain inconclusive.

Comparative Study of Single and Coaxial Electrospun Antimicrobial Cross-Linked Scaffolds Enriched with Aloe Vera: Characterization, Antimicrobial Activity, Drug Delivery, Cytotoxicity, and Cell Proliferation on Adipose Stem Cells and Human Skin Fibroblast.

The preparation of materials with application in the biomedical field needs to attend some characteristics such as biocompatibility, nontoxicity, adequate mechanical properties, and the ability to mimic the extracellular matrix. Scaffolds for use in cell culture were prepared based on gelatin, polylactic acid (PLA), aloe vera mucilage, and tetracycline. Fibers were prepared in single and coaxial configuration and then cross-linked with glutaraldehyde saturated vapor. The fibers were obtained with cylindric morphology and changed to ribbon morphology and porous membranes, similarly to the extracellular matrix, when cross-linked. Membranes prepared by coaxial electrospinning showed core-shell structures when observed by transversal images, which is beneficial for controlled drug release. Characterization techniques such as scanning electron microscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy demonstrated the cross-linking due to the increase in diameter, formation of imine groups, and improvement of thermal stability. Antibiotic release tests showed that the prevalent release mechanism is diffusion and can be controlled considering the encapsulation effect, when fibers are prepared with a coaxial configuration, increasing the drug release time, making it a suitable material for controlled release. The biological evaluation of the scaffolds was carried out in two cell lines: mammalian adipose stem cells (ASCs), used as a primary cell culture, and Detroit 548 human skin fibroblasts as a dermal cell model. Aloe vera enriched scaffolds showed better activity in contact with both cell lines, exhibiting cell viability values greater than 90% and favorable results in live-dead assays when no damaged cells were observed. Cell proliferation was evaluated using Detroit 548 human skin fibroblast on gelatin-based scaffolds by the staining of the adhered cells; the images showed good confluence and morphology of the cells on the aloe vera and antibiotic loaded membranes for both of the studied configurations. Antibiotic loaded membranes presented antimicrobial activity against S. aureus, and this behavior increased when aloe vera is included. According to the results, the scaffolds prepared on single configuration enriched with aloe vera and tetracycline could be used in dermal tissue engineering as burn dressings, diabetic foot apposite, and skin substitutes, and the scaffolds prepared with a coaxial configuration are recommended for controlled release systems of antibiotics as treatments for chronic wounds such as diabetic foot and burn healing.

Metabolic preferences of astrocytes: Functional metabolic mapping reveals butyrate outcompetes acetate.

Disruptions to the gut-brain-axis have been linked to neurodegenerative disorders. Of these disruptions, reductions in the levels of short-chain fatty acids (SCFAs), like butyrate, have been observed in mouse models of Alzheimer's disease (AD). Butyrate supplementation in mice has shown promise in reducing neuroinflammation, amyloid-β accumulation, and enhancing memory. However, the underlying mechanisms remain unclear. To address this, we investigated the impact of butyrate on energy metabolism in mouse brain slices, primary cultures of astrocytes and neurons and in-vivo by dynamic isotope labelling with [U-13C]butyrate and [1,2-13C]acetate to map metabolism via mass spectrometry. Metabolic competition assays in cerebral cortical slices revealed no competition between butyrate and the ketone body, β-hydroxybutyrate, but competition with acetate. Astrocytes favoured butyrate metabolism compared to neurons, suggesting that the astrocytic compartment is the primary site of butyrate metabolism. In-vivo metabolism investigated in the 5xFAD mouse, an AD pathology model, showed no difference in 13C-labelling of TCA cycle metabolites between wild-type and 5xFAD brains, but butyrate metabolism remained elevated compared to acetate in both groups, indicating sustained uptake and metabolism in 5xFAD mice. Overall, these findings highlight the role of astrocytes in butyrate metabolism and the potential use of butyrate as an alternative brain fuel source.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Liver mechanosignaling as a natural anti-hepatitis B virus mechanism

The mechanisms underlying the natural control of hepatitis B virus (HBV) infection have long been an intriguing question. Given the wide physiological range of liver stiffness and the growing attention to the role of mechanical microenvironment in homeostasis and diseases, we investigated how physical matrix cues impact HBV replication. High matrix stiffness significantly inhibited HBV replication and activated YAP in primary hepatocyte culture system, a key molecule in mechanosignaling. YAP activation notably suppressed HBV transcription and antigen expression. Several YAP-induced genes exhibited strong anti-HBV effects. Single-cell analysis of liver tissue from male individuals with active HBV replication revealed a strong significant negative correlation between YAP signature activation and HBV transcript levels. Intraperitoneal administration of YAP small molecule agonist potently controls HBV in male mouse models. These findings unveil a mechanism that involves the mechanical environment of hepatocytes and YAP to clear hepatotropic viral infection in the liver, providing new perspectives for HBV cure studies and antiviral development.

Long-term 3D primary epithelioid cultures reveal genes that regulate esophageal cell fitness.

Long-term 3D primary epithelioid cultures reveal genes that regulate esophageal cell fitness.

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

14-3-3 phosphorylation inhibits 14-3-3θ's ability to regulate LRRK2 kinase activity and toxicity.

LRRK2 mutations are among the most common genetic causes for Parkinson's disease (PD), and toxicity is associated with increased kinase activity. 14-3-3 proteins are key interactors that regulate LRRK2 kinase activity. Phosphorylation of the 14-3-3θ isoform at S232 is dramatically increased in human PD brains. Here we investigate the impact of 14-3-3θ phosphorylation on its ability to regulate LRRK2 kinase activity. Both wildtype and the non-phosphorylatable S232A 14-3-3θ mutant reduced the kinase activity of wildtype and G2019S LRRK2, whereas the phosphomimetic S232D 14-3-3θ mutant had minimal effects on LRRK2 kinase activity, as determined by measuring autophosphorylation at S1292 and T1503 and Rab10 phosphorylation. However, wildtype and both 14-3-3θ mutants similarly reduced the kinase activity of the R1441G LRRK2 mutant. 14-3-3θ phosphorylation did not promote global dissociation with LRRK2, as determined by co-immunoprecipitation and proximal ligation assays. 14-3-3s interact with LRRK2 at several phosphorylated serine/threonine sites, including T2524 in the C-terminal helix, which can fold back to regulate the kinase domain. Interaction between 14-3-3θ and phosphorylated T2524 LRRK2 was important for 14-3-3θ's ability to regulate kinase activity, as wildtype and S232A 14-3-3θ failed to reduce the kinase activity of G2019S/T2524A LRRK2. Finally, we found that the S232D mutation failed to protect against G2019S LRRK2-induced neurite shortening in primary cultures, while the S232A mutation was protective. We conclude that 14-3-3θ phosphorylation destabilizes the interaction of 14-3-3θ with LRRK2 at T2524, which consequently promotes LRRK2 kinase activity and toxicity.

Co-cultivation of mastocytes and cumulus cells in polycystic ovary syndrome

The purpose of the study was to study the cytokine-producing function of cumulus cells of the ovaries in PCOS during co-cultivation of cumulus cells with mast cells to identify their mutual influence in the immunopathogenesis of PCOS. The study was approved by the Interdisciplinary Ethics Committee of the FSBEI of HE of the Ministry of Health of the Russian Federation. A permanent human mast cell line HMC-1 (Human mast cell, ATCC, USA) and a primary culture of cumulus cells were used. The condition of cumulus cells and mastocytes was determined using fluorescent dyes followed by flow cytometry before co-cultivation and after 7 days co-cultivation. The levels of IL-6, IL-10, and IFNã were studied on days 1, 3, and 7 of the experiment. In monoculture and during co-culture of cells, the synthesis of cytokines IL-6, IL-10, and IFNã continues, but to varying degrees of severity. In the permanent mast cell line, an increase in IL-6, IL-10, and IFNã is observed on the 1st, 3rd, and 7th days of cultivation. On the first day, cytokine levels of donor cumulus cells and mastocytes did not differ significantly (p 0.05). In the culture of donor cumulus cells, the synthesis of IL-6, IL-10, and IFNã progressively increases by the 7th day of the experiment (p 0.05). On the first day of the experiment, the balance of Th1/Th2 cytokines in PCOS was twice as high as in healthy women. The ratio increased, and on the 7th day reached 3.83 times higher than the ratio of Th1/Th2 cytokines in the control group. Hyperproduction of IFNã by mastocytes was most significant when they were co-cultured with cumulus cells, in monoculture mast cells synthesized excessively cytokine twice from the initial values of IFNã, and the monoculture cumulus cells in PCOS it practically did not contain. The studied cytokines are regulators of the function of ovarian cumulus cells and factors that increase the competence of the oocyte, indicating the need for their correction, which will allow a real influence on the links of pathogenesis in PCOS in the future.later on.

The time dependent influence of curvature and topography of biomaterials in the behavior of corneal endothelial cells.

Among the different layers of the cornea, the corneal endothelium, which is composed of corneal endothelial cells (CEC), plays a key role in the maintenance of cornea transparency. Based on the donor shortages and the limitations associated with transplantation, in this work we have developed collagen hydrogels with different patterned structures on the surface to provide topographies in ranges similar to the natural environment that CEC sense. This aimed at stimulating cells to maintain a typical CEC phenotype and provide alternatives to corneal transplantation. In this sense, we have elaborated curved collagen hydrogels (concave and convex) with three different topographies (50, 200 and 300µm grooves), with the help of 3D printed mold and replicating the mold with the collagen hydrogel, onto which CEC were cultured in order to analyze its behavior. Flat hydrogels were used as controls. Cell morphology, cell circularity and gene expression of ATP1A1 and ZO-1 genes were analyzed after 3 and 6days. Results showed an effect of the curvature and the topography compared to flat collagen hydrogels, showing higher expression of ZO-1 and ATP1A1 in curved non-patterned hydrogels at day 3. The patterned hydrogels did not have such a significant effect on gene expression compared to flat hydrogels, showing stronger effect of the curvature compared to the topography. The effect was opposite at day 6, showing higher gene expression at days 6 on the patterned hydrogels, especially for the ZO-1 gene. The gene expression results were in accordance with the cell morphology observed at the different time points, showing circularities closer to hexagon like morphology at shorter time points. Overall, this platform can serve as a system to culture cell under proper environment to further be able to transplant a CEC monolayer or to allow transplantation of thin collagen layers with cultured CEC.

Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

Blockage of ATGL-mediated breakdown of lipid droplets in microglia alleviates neuroinflammatory and behavioural responses to lipopolysaccharides

Lipid droplets (LD) are triglyceride storing organelles that have emerged as an important component of cellular inflammatory responses. LD lipolysis via adipose triglyceride lipase (ATGL), the enzyme that catalyses the rate-limiting step of triglyceride lipolysis, regulates inflammation in peripheral immune and non-immune cells. ATGL elicits both pro- and anti-inflammatory responses in the periphery in a cell-type dependent manner. The present study determined the impact of ATGL inhibition and microglia-specific ATGL genetic loss-of-function on acute inflammatory and behavioural responses to pro-inflammatory insult. First, we evaluated the impact of lipolysis inhibition on lipopolysaccharide (LPS)-induced expression and secretion of cytokines and phagocytosis in mouse primary microglia cultures. Lipase inhibitors (ORlistat and ATGListatin) and LPS led to LD accumulation in microglia. Pan-lipase inhibition with ORlistat alleviated LPS-induced expression of IL-1β and IL-6. Specific inhibition of ATGL had a similar action on CCL2, IL-1β and IL-6 expression in both neonatal and adult microglia cultures. CCL2 and IL-6 secretion were also reduced by ATGListatin or knockdown of ATGL. ATGListatin increased phagocytosis in neonatal cultures independently from LPS treatment. Second, targeted and untargeted lipid profiling revealed that ATGListatin reduced LPS-induced generation of pro-inflammatory prostanoids and modulated ceramide species in neonatal microglia. Finally, the role of microglial ATGL in neuroinflammation was assessed using a novel microglia-specific and inducible ATGL knockout mouse model. Loss of microglial ATGL in adult male mice dampened LPS-induced expression of IL-6 and IL-1β and microglial density. LPS-induced sickness- and anxiety-like behaviours were also reduced in male mice with loss of ATGL in microglia. Together, our results demonstrate potent anti-inflammatory effects produced by pharmacological or genetic inhibition of ATGL-mediated triglyceride lipolysis and thereby propose that supressing microglial LD lipolysis has beneficial actions in acute neuroinflammatory conditions.

P300/CBP KATs Are Critical for Maturation and Differentiation of Adult Neural Progenitors.

Epigenetic modifications play a pivotal role in the process of neurogenesis. Among these modifications, reversible acetylation fine-tunes gene expression for both embryonic and adult neurogenesis. The CBP/KAT3A and its paralogue p300/KAT3B are well-known lysine acetyltransferases with transcriptional coactivation ability that engage in neural plasticity and memory. The exclusive role of their KAT activity in neurogenesis and memory could not be addressed due to the absence of a p300/CBP modulator, which can cross the blood-brain barrier. Previous work from our laboratory has shown that a small molecule activator, TTK21, specific to CBP/p300, when conjugated to glucose-derived carbon nanospheres (CSP), is efficiently delivered to the mouse brain and could induce dendritic branching and extend long-term memory. However, the molecular mechanisms of p300 acetyltransferase activity-dependent enhanced dendritogenesis are yet to be understood. Here, we report that CSP-TTK21 treatment to primary neuronal culture derived from mouse embryo enhances the expression of five critical genes: Neurod1 (central nervous system development), Tubb3 (immature neural marker), Camk2a (synaptic plasticity and LTP), Snap25 (spine morphogenesis plasticity), and Scn2a (propagation of the action potential). Activation of these genes by inducing the p300/CBP KAT activity presumably promotes the maturation and differentiation of adult neuronal progenitors and thereby the formation of long and highly branched doublecortin-positive functional neurons in the subgranular zone of the dentate gyrus.

L-Glutamine mitigates bile acid-induced inhibition of growth factor activity in rat hepatocyte cultures

Bile acid-induced hepatotoxicity is inevitable in Cholestasis pathogenesis and L-Glutamine (L-Gln) has been reported to prevent total parenteral nutrition (TPN)-induced cholestasis in premature neonates. While mechanisms remain unknown, we hypothesize that bile acids impair growth factor (GF) function in hepatocytes which L-glutamine prevents through NAPDH oxidase (NOX) modulation. Glycochenodeoxycholic acid (GCDC, 0–100 µM) when added to primary hepatocyte cultures significantly (p < 0.01) decreased the FBS-induced BrdU incorporation, however inhibition of Fibroblast Growth factor (FGF)- or Hepatocyte growth factor (HGF)-induced DNA synthesis was more pronounced (p < 0.001). L-Gln markedly attenuated GCDC-mediated inhibition of DNA synthesis in both FBS and GF-treated cells. GCDC significantly increased the NADPH oxidase activity and NOX-1 protein expression that were markedly reduced by L-Gln and protein kinase c (PKC) inhibitor, LY-333531. Apocynin (APCN) and diphenyliodonium (DPI) significantly blocked the GCDC-mediated inhibition of GF-induced DNA synthesis. This study demonstrates that bile acid-induced hepatotoxicity involves dysfunction of certain growth factors via protein kinase c (PKC)- mediated NOX modulation which can be corrected, at least partly, by L-glutamine.

Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures

ObjectivesNontuberculous mycobacteria (NTM) infections present a global health challenge. This study describes unidentified mycobacteria species from extrapulmonary sites, using advanced identification and sequencing techniques. MethodsExtrapulmonary mycobacteria growth indicator tube primary cultures collected retrospectively between 2019 and 2023, featuring unidentified mycobacteria species detected by GenoType Mycobacterium line probe assays, underwent multilocus targeted next-generation sequencing using Oxford Nanopore Technology, polymerase chain reaction amplicon Sanger sequencing, and Deeplex Myc-TB analysis. Previously collected clinical and laboratory data were reported. ResultsA total of 28 cultures, collected from extrapulmonary sites, each from different patients, were included. Mycobacterial mixtures were identified in 19 of 28 (68%) cultures, with four of 28 (14%) showing unidentified species based on sequencing of rpoB and hsp65 targets. Mycobacterium monacense was present in 13 of 28 (46%) of the cultures. Culturable Mycobacterium tuberculosis complex (MTBC) was identified in five extrapulmonary specimens that previously tested negative for MTBC using Xpert MTB/RIF Ultra. The comparative analysis between Sanger and targeted next-generation sequencing using Oxford Nanopore Technology sequencing (for hsp65) demonstrated 27 of 28 (96%) agreement on the predominant strain. Deeplex Myc-TB could not identify NTM-MTBC co-infections in minor subpopulations. ConclusionsThis study highlights the role of advanced sequencing in identifying NTM mixtures and mycobacterial co-infections. It calls for ongoing efforts to integrate next-generation sequencing into mycobacteria testing algorithms.

Development and Characterization of Primary Brain Cultures from Japanese Quail Embryos.

Cell cultures play a crucial role in neuroscience research, facilitating the elucidation of the complexities of cellular physiology and pathology. The relative simplicity in producing cultures and the accessibility to cells that the cultures provide, in contrast to in vivo settings, allow users to manipulate and monitor cells more easily at higher throughputs and lower costs. These are ideal for screening purposes and electrophysiological characterizations. Despite the prevalence of methodologies for producing brain cultures from various animal models, rodents in particular, approaches for culturing neurons (and glia) from birds are less established or completely absent as in the case of the Japanese quail model. Here, we present a unique culturing protocol for brain cells (e.g., neurons at different maturation levels, such as progenitor cells, excitatory and inhibitory neurons, microglia, and endothelial cells) from entire forebrains of Japanese quail embryos for high-throughput screening of viral vectors in vitro and other various purposes. Following dissection and digestion methods uniquely suited for avian brains, we tailored the growth media and culturing surface to allow the survival of quail brain cultures for more than three weeks in vitro. Key features • We introduce a detailed protocol for producing primary brain cultures from quail embryos' forebrains for up to 30 days. • We show that the cultures support in vitro viral transfections effectively. • We demonstrate the use of the cultures for rapid (days) screening for suitable viruses for quail brain cells, electrophysiological characterizations, and single mRNA sequencing.

Generation of footprint-free human induced pluripotent stem cell line (SCIKFi001-B) from cGMP grade umbilical cord-derived mesenchymal stem cells (UC-MSCs) using episomal-plasmid based reprogramming approach

UCMSCs were reprogrammed to iPSCs using Yamanaka factor bearing episomal plasmids. SCIKFi001-B exhibited pluripotency, had typical iPSC morphology and didn’t retain any residual episomal plasmid. Although karyotyping showed chromosomal translocation, this abnormality seemed to have little impact on the functionality of SCIKFi001-B since it retained its ability to differentiate to three-germ layer. While karyotypic abnormality might negate use in therapeutic and clinical settings, this line remained a valuable educational tool for iPS cell culture techniques. Finally, our study highlighted the importance of routine karyotyping on iPSC lines as abnormal karyotypes oftentimes bear no discernible effect on cell morphology nor functionality.

Rat Sympathetic Neuron Calcium Channels Are Insensitive to Gabapentin.

The gabapentenoids such as gabapentin (GP) and pregabalin are approved for the treatment of chronic pain, but their utility is limited by persistent side effects. These adverse effects result from GPs affecting many types of neurons and muscle cells, not just the pain-sensing neurons that are the intended targets. We have recently discovered a type of peripheral neuron, rat sympathetic neurons from the superior cervical ganglion (SCG), that is uniquely insensitive to GP effects. Currents were measured using whole-cell patch-clamp electrophysiology from cells in primary culture from either the SCG or the Nodose Ganglion (NDG) as a positive control for the effects of GP. We find that the calcium current density was dramatically reduced by GP pretreatment in NDG neurons, but that neurons from the SCG were resistant. Further, when GP was cytoplasmically injected into these neurons, the resistance of SCG neurons to GP treatment persisted. These data demonstrate that rat sympathetic neurons appear to be uniquely resistant to GP treatment. These results may help us to better understand the mechanism of action of, and resistance to, GP in altering calcium channel current density, which may help to develop future treatments with fewer side effects.