Human iPSC-derived Neuronal Cultures Research Articles

Screening for modulators of neural network activity in 3D human iPSC-derived cortical spheroids.

Human induced Pluripotent Stem Cells (iPSCs) are a powerful tool to dissect the biology of complex human cell types such as those of the central nervous system (CNS). However, robust, high-throughput platforms for reliably measuring activity in human iPSC-derived neuronal cultures are lacking. Here, we assessed 3D cultures of cortical neurons and astrocytes displaying spontaneous, rhythmic, and highly synchronized neural activity that can be visualized as calcium oscillations on standard high-throughput fluorescent readers as a platform for CNS-based discovery efforts. Spontaneous activity and spheroid structure were highly consistent from well-to-well, reference compounds such as TTX, 4-AP, AP5, and NBQX, had expected effects on neural spontaneous activity, demonstrating the presence of functionally integrated neuronal circuitry. Neurospheroid biology was challenged by screening the LOPAC®1280 library, a collection of 1280 pharmacologically active small molecules. The primary screen identified 111 compounds (8.7%) that modulated neural network activity across a wide range of neural and cellular processes and 16 of 17 compounds chosen for follow-up confirmed the primary screen results. Together, these data demonstrate the suitability and utility of human iPSC-derived neurospheroids as a screening platform for CNS-based drug discovery.

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS.

SummaryHuman induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, “Optopatch,” pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons?

Current neurotoxicity testing heavily relies on expensive, time consuming and ethically debated in vivo animal experiments that are unsuitable for screening large number of chemicals. Consequently, there is a clear need for (high-throughput) in vitro test strategies, preferably using human cells as this increases relevance and eliminates the need for interspecies translation. However, human stem cell-derived neurons used to date are not well characterised, require prolonged differentiation and are potentially subject to batch-to-batch variation, ethical concerns and country-specific legislations. Recently, a number of human induced pluripotent stem cell (iPSC)-derived neurons became commercially available that may circumvent these concerns. We therefore used immunofluorescent stainings to demonstrate that human iPSC-derived neurons from various suppliers form mixed neuronal cultures, consisting of different types of (excitatory and inhibitory) neurons. Using multi-well microelectrode array (mwMEA) recordings, we demonstrate that these human iPSC-derived cultures develop spontaneous neuronal activity over time, which can be modulated by different physiological, toxicological and pharmacological compounds. Additional single cell calcium imaging illustrates the presence of functional GABA, glutamate, and acetylcholine receptors as well as voltage-gated calcium channels. While human iPSC-derived neuronal cultures appear not yet suitable to fully replace the rat primary cortical model, our data indicate that these rapidly differentiating, commercially available human iPSC-derived neuronal cultures are already suitable for in vitro prioritisation and effect screening studies. Further characterisation and toxicological validation is now required to facilitate acceptance and large-scale implementation of these animal-free, physiologically-relevant human iPSC-based modelsfor future neurotoxicity testing.

Comparing drug-induced seizure-liability in human iPSC-derived and primary mouse neurons grown on micro-electrode arrays

Comparing drug-induced seizure-liability in human iPSC-derived and primary mouse neurons grown on micro-electrode arrays

Characterization of 4-Aminopyridine-induced network plasticity in rat cortical and human iPSC-derived neuronal cultures

Event Abstract Back to Event Characterization of 4-Aminopyridine-induced network plasticity in human iPSC-derived neuronal cultures Gaye Tanriöver1* and Katja Nieweg1 1 Phillips University, Institute of Pharmacology and Clinical Pharmacy, Germany Motivation Homeostatic network plasticity is a key regulatory mechanism for fine-tuning network activity. 4-aminopyridine (4-AP), which is commonly used to model epilepsy in vitro and in vivo has recently been described to trigger homeostatic plasticity in cultures of mouse hippocampal neurons [1]. The inhibition of several types of K+ channels (D-type and A-type K+ currents and a fraction of delayed rectifier currents) by 4-AP leads to prolonged action potentials and increased neurotransmitter release [2]. This induces hyperactivity, which is counteracted by mechanisms leading to decreased neuronal excitability. Human induced pluripotent stem cell (hiPSC)-derived neurons have been proposed to be a highly valuable in vitro model to be used in neurotoxicology, drug screening and regenerative medicine. Since spontaneous synchronous network activity develops in hiPSC-derived neuronal networks, our aim was to study the capacity of hiPSC-derived neuronal networks to exhibit homeostatic plasticity in response to 4-AP. Material and Methods hiPSCs were differentiated to cortical neurons as described previously [3]. Fifty thousand cells were plated after enrichment of neurons by immunopurification. After long-term (2-3 months) culture of the neurons for maturation, networks were transferred on the PO and laminin-coated 60-electrode multi-electrode arrays (Multichannel Systems, 200 µm electrode distance, 30µm electrode diameter) (MEAs) for recording. Calcium imaging was performed using Fura-2-AM ratiometric dye to identify single cell activity. Cells were loaded with 3µM Fura-2 AM for 20 min at 37°C in HEPES buffer. Fluorescence intensities at the two wavelengths (F340 and F380) were recorded separately and combined (fluorescence ratio: r=F340/F380) after background subtraction. Cells were imaged before and during 4-AP treatment as well as after removal of 4-AP. Neuronal networks with spontaneous network activity were incubated with 4-AP (100 µM) in order to trigger hyperactivity and measured during 4 days of 4-AP treatment as well as post-washout to monitor homeostatic plasticity. Results We modified our previously published [3] protocol for immunopanning of human iPSC derived neurons in order to permit differentiation of astrocytes from persisting neural precursor cells. Thus, we were able to generate mature, spontaneously active cortical like astrocyte-neuronal networks, shown by the presence of synchronous calcium transients around two months after immunoisolation. Analysis of network maturation was performed by culturing on 60-electrode MEAs. Single spikes and spike trains could be detected long before the occurrence of synchronous network bursts, which appeared two to three months after immunoisolation. To test, whether 4-AP triggered hyperactivity can induce homeostatic plasticity in human iPSC derived neuronal networks as it has been described for mouse hippocampal neurons [1], we treated human neurons of different maturational stages with 100µM 4-AP for 2-4 days and analyzed spontaneous network activity by MEA and calcium imaging. Mature, bursting human networks responded to 4-AP treatment with increased spiking and bursting rates (Fig. 1). Within the first 24h of treatment, mechanisms of homeostatic plasticity lead to downregulation of the activity close to baseline levels. One day after 4-AP removal, we observed that the overall network activity was strongly reduced even below baseline levels. The activity slowly recovered during the following days. Calcium imaging, which permits analyzing the activity of single cells within a network revealed similar results in mature human networks. However, immature human cultures without synchronous bursting activity did not respond to 4-AP and thus homeostatic plasticity could not be triggered in these cultures. Discussion 4-AP treatment of dissociated mouse hippocampal neurons cultured on MEAs was described, as a model to study homeostatic network plasticity by Pozzi et al. [1]. They showed that 4-AP induced hyperactivity lead to downregulation of neuronal excitability in mouse cultures. In our study, we applied this model to hiPSC derived neuronal networks and could show, that the maturational status of the network is a crucial parameter. Only spontaneously bursting cultures were able to respond in the expected manner. Whether downregulation of overall network activity occurs at the level of neuronal excitability or by synaptic downscaling will be analyzed by patch clamp recording. Conclusion In our study, we could show that mature hiPSC-derived neuronal networks cultured on MEAs are a valuable model to investigate mechanisms of homeostatic network plasticity in vitro. Since homeostatic plasticity is impaired in many psychiatric disorders and also at early stages of neurodegenerative diseases, a combination of patient derived iPSCs and the 4-AP plasticity model could serve as an innovative platform for drug screening.

High-content high-throughput assays for characterizing the viability and morphology of human iPSC-derived neuronal cultures.

Abstract Development of quantitative high-throughput in vitro assays that enable assessment of viability and morphological changes in neuronal cells is an active area of investigation in drug discovery and environmental chemical safety assessment. High-content imaging is an emerging and efficient tool for generating multidimensional quantitative cellular readouts; in addition, human induced pluripotent stem cell (iPSC)-derived neurons are a promising in vitro model system that emulates both the functionality and behavior of mature neurons, and they are available in quantities sufficient for screening workflows. The goal of this study was to develop high-content imaging and analysis methods to assess multiple phenotypes in human iPSC-derived neuronal cells. Specifically, we optimized cell culture, staining, and imaging protocols in a 384-well assay format and improved laboratory workflow by designing a one-step procedure to reduce assay time and minimize cell disturbance. Phenotypic readouts include quantitative characterization of neurite outgrowth and branching, cell number and viability, as well as measures of adverse effects on mitochondrial integrity and membrane potential. To verify the robustness of the workflow, we tested a series of compounds that are established toxicants. We report concentration-response effects of selected test compounds on human iPSC-derived neuronal cells and illustrate how the proposed methods may be used for high-content high-throughput compound toxicity screening and safety evaluation of drugs and environmental chemicals.