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Opioid overdose is the leading cause of drug overdose lethality, posing an urgent need for investigation. The key brain region for inspiratory rhythm regulation and opioid-induced respiratory depression (OIRD) is the preBötzinger Complex (preBötC) and current knowledge has mainly been obtained from animal systems. This study aims to establish a protocol to generate human preBötC neurons from induced pluripotent cells (iPSCs) and develop an opioid overdose and recovery model utilizing these iPSC-preBötC neurons. A de novo protocol to differentiate preBötC-like neurons from human iPSCs is established. These neurons express essential preBötC markers analyzed by immunocytochemistry and demonstrate expected electrophysiological responses to preBötC modulators analyzed by patch clamp electrophysiology. The correlation of the specific biomarkers and function analysis strongly suggests a preBötC-like phenotype. Moreover, the dose-dependent inhibition of these neurons' activity is demonstrated for four different opioids with identified IC50's comparable to the literature. Inhibition is rescued by naloxone in a concentration-dependent manner. This iPSC-preBötC mimic is crucial for investigating OIRD and combating the overdose crisis and a first step for the integration of a functional overdose model into microphysiological systems., (© 2023 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)

The maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicular glutamate transporter 1 [VGlut1]) and inhibitory (GABA) markers, while late-stage cultures (day 40) expressed solely VGlut1, indicating a purely excitatory phenotype without containing glial cells. This maturation process was further validated utilizing patch clamp and MEA analysis. Particularly, induced long-term potentiation (LTP) successfully persisted for 1 h in day 40 cultures, but only achieved LTP in the presence of the GABA A receptor antagonist picrotoxin in day 21 cultures. This system was also applied to epilepsy modeling utilizing bicuculline and its correction utilizing the anti-epileptic drug valproic acid., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We report on a functional human model to evaluate multi-organ toxicity in a 4-organ system under continuous flow conditions in a serum-free defined medium utilizing a pumpless platform for 14 days. Computer simulations of the platform established flow rates and resultant shear stress within accepted ranges. Viability of the system was demonstrated for 14 days as well as functional activity of cardiac, muscle, neuronal and liver modules. The pharmacological relevance of the integrated modules were evaluated for their response at 7 days to 5 drugs with known side effects after a 48 hour drug treatment regime. The results of all drug treatments were in general agreement with published toxicity results from human and animal data. The presented phenotypic culture model exhibits a multi-organ toxicity response, representing the next generation of in vitro systems, and constitutes a step towards an in vitro "human-on-a-chip" assay for systemic toxicity screening.

Pre-clinical testing of drug candidates in animal models is expensive, time-consuming, and often fails to predict drug effects in humans. Industry and academia alike are working to build human-based in vitro test beds and advanced high throughput screening systems to improve the translation of preclinical results to human drug trials. Human neurons derived from induced pluripotent stems cells (hiPSCs) are readily available for use within these test-beds and high throughput screens, but there remains a need to robustly evaluate cellular behavior prior to their incorporation in such systems. This study reports on the characterization of one source of commercially available hiPSC-derived neurons, iCell(®) Neurons, for their long-term viability and functional performance to assess their suitability for integration within advanced in vitro platforms. The purity, morphology, survival, identity, and functional maturation of the cells utilizing different culture substrates and medium combinations were evaluated over 28 days in vitro (DIV). Patch-clamp electrophysiological data demonstrated increased capacity for repetitive firing of action potentials across all culture conditions. Significant differences in cellular maturity, morphology, and functional performance were observed in the different conditions, highlighting the importance of evaluating different surface types and growth medium compositions for application in specific in vitro protocols., (© 2015 American Institute of Chemical Engineers.)

One of the earliest morphological changes occurring in apoptosis is cell shrinkage associated with an increased efflux of K(+) and Cl(-) ions. Block of K(+) or Cl(-) channels prevents cell shrinkage and death. Recently, we found evidences for the activation of a voltage-dependent anion channel in the plasma membrane (pl-VDAC) of a hippocampal cell line undergoing apoptosis. Nothing is known on pl-VDAC in apoptotic cell death of neural cells at different stages of differentiation. We have addressed this issue in primary cultures of differentiated hippocampal neurons and embryonic neural stem cells (NSCs). In control hippocampal neurons, pl-VDAC is closed but acts as an NADH-ferricyanide reductase, while in apoptotic neurons, pl-VDAC is opened and the enzymatic activity is increased. Anti-VDAC antibodies block pl-VDAC and prevent apoptosis, as well as the increase in enzymatic activity. Conversely, in NSCs, pl-VDAC is scarcely seen and there is no NADH-ferricyanide reductase activity. In agreement, anti-VDAC antibodies do not affect the apoptotic process. Instead, we find activation of a Na(+) channel that has low voltage dependency, a conductance of 26 pS, and is blocked by amiloride, which also prevents apoptosis. Thus, it appears that activation of pl-VDAC during apoptosis is a critical event in differentiated neurons, but not in NSCs.

Ion channels in the plasma membrane play critical roles in apoptosis. In a recent study we found that a voltage-dependent anion channel in the plasma membrane (VDACpl) of neuronal hippocampal cell line (HT22) cells was activated during apoptosis and that channel block prevented apoptosis. Whether or not VDACpl is identical to the mitochondrial VDACmt has been debated. Here, we biophysically characterize the apoptosis-inducing VDACpl and compare it with other reports of VDACpls and VDACmt. Excised membrane patches of apoptotic HT22 cells were studied with the patch-clamp technique. VDACpl has a large main-conductance state (400 pS) and occasionally subconductance states of approximately 28 pS and 220 pS. The small subconductance state is associated with long-lived inactivated states, and the large subconductance state is associated with excision of the membrane patch and subsequent activation of the channel. The open-probability curve is bell shaped with its peak around 0 mV and is blocked by 30 microM Gd3+. The gating can be described by a symmetrical seven-state model with one open state and six closed or inactivated states. These channel properties are similar to those of VDACmt and other VDACpls and are discussed in relation to apoptosis.

Apoptotic cell death is an essential process in the development of the central nervous system and in the pathogenesis of its degenerative diseases. Efflux of K(+) and Cl(-) ions leads to the shrinkage of the apoptotic cell and facilitates the activation of caspases. Here, we present electrophysiological and immunocytochemical evidences for the activation of a voltage-dependent anion channel (VDAC) in the plasma membrane of neurons undergoing apoptosis. Anti-VDAC antibodies blocked the channel and inhibited the apoptotic process. In nonapoptotic cells, plasma membrane VDAC1 protein can function as a NADH (-ferricyanide) reductase. Opening of VDAC channels in apoptotic cells was associated with an increase in this activity, which was partly blocked by VDAC antibodies. Hence, it appears that there might be a dual role for this protein in the plasma membrane: (1) maintenance of redox homeostasis in normal cells and (2) promotion of anion efflux in apoptotic cells.