scholarly journals Robust Expression of Functional NMDA Receptors in Human Induced Pluripotent Stem Cell-Derived Neuronal Cultures Using an Accelerated Protocol

2021 ◽  
Vol 14 ◽  
Author(s):  
Jacob B. Ruden ◽  
Mrinalini Dixit ◽  
José C. Zepeda ◽  
Brad A. Grueter ◽  
Laura L. Dugan
Keyword(s):  
Nmda Receptors ◽  
Neural Progenitor Cells ◽  
Induced Pluripotent Stem Cell ◽  
Calcium Flux ◽  
Neuronal Cultures ◽  
Mature Neurons ◽  
Health And Disease ◽  
Nervous System Function ◽  
Induced Pluripotent ◽  
Human Ipsc

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease.

scholarly journals All-optical electrophysiology for high-throughput functional characterization of human iPSC-derived motor neuron model of ALS

2018 ◽  
Author(s):  
Evangelos Kiskinis ◽  
Joel M. Kralj ◽  
Peng Zou ◽  
Eli N. Weinstein ◽  
Hongkang Zhang ◽  
...  
Keyword(s):  
Motor Neuron ◽  
High Throughput ◽  
Neuron Model ◽  
Functional Characterization ◽  
Induced Pluripotent Stem Cell ◽  
Neuronal Cultures ◽  
All Optical ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractHuman 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 model of ALS. In a comparison of neurons 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 to numerical simulations of simple conductance-based neuronal models and to 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.

scholarly journals TREM2 knockout, but not the R47H Alzheimer’s variant, reduces neural phagocytosis and survival of human iPSC-derived macrophages

2020 ◽  
Author(s):  
Hazel Hall-Roberts ◽  
Juliane Obst ◽  
Thomas B. Smith ◽  
Elena Di Daniel ◽  
Emma Mead ◽  
...  
Keyword(s):  
Inflammatory Cytokine ◽  
Induced Pluripotent Stem Cell ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Calcium Flux ◽  
Loss Of Function ◽  
Human Microglia ◽  
Surface Receptor ◽  
Induced Pluripotent ◽  
Human Ipsc

Abstract BACKGROUND: TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer’s disease (AD), including R47H. Binding of ligands to TREM2 triggers Syk-dependent signalling through the DAP12 co-receptor, leading to phagocytosis, survival, and changes to microglial activation state. In biochemical assays, R47H impairs TREM2 binding to phosphatidylserine, a lipid “eat-me” signal exposed by apoptotic neurons. The effect of R47H TREM2 upon phagocytosis of apoptotic neurons by human microglia has not yet been reported. METHODS: We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines with homozygous R47H mutation or TREM2 knockout (KO). To assess microglial phenotypic function in the mutants, we measured: (1) pro-inflammatory cytokine responses by ELISA; (2) survival after growth factor-withdrawal; (3) phagocytosis by novel high-content imaging assays, using two neuron-derived cargoes that expose phosphatidylserine (fixed SH-SY5Ys and synaptosomes). Downstream signalling resulting from TREM2 activation was additionally assessed by assaying Syk phosphorylation and calcium flux. RESULTS: We demonstrated that TREM2 KO strongly diminished both pMac survival and neural phagocytosis, while having little impact on inflammatory cytokine response. R47H TREM2 modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, survival, or phagocytosis. CONCLUSIONS: Under healthy conditions in culture, the R47H mutation is not sufficient to cause defects in phagocytosis or survival of human pMac, unlike more severe T66M or W50C TREM2 loss-of-function mutations. We hypothesise that R47H TREM2 affects other microglia phenotypes yet to be explored, and/or that pathogenic manifestation requires other stressors relating to neurodegenerative disease.

scholarly journals High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chialin Cheng ◽  
Surya A. Reis ◽  
Emily T. Adams ◽  
Daniel M. Fass ◽  
Steven P. Angus ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Neural Progenitor Cells ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Clinical Investigation ◽  
Neuronal Model ◽  
Tau Phosphorylation ◽  
Neuronal Cultures ◽  
Proteomic Profiling ◽  
Induced Pluripotent

AbstractMutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.

scholarly journals Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes

2017 ◽  
Vol 312 (6) ◽  
pp. H1144-H1153 ◽  
Author(s):  
Sam Chai ◽  
Xiaoping Wan ◽  
Drew M. Nassal ◽  
Haiyan Liu ◽  
Christine S. Moravec ◽  
...  
Keyword(s):  
Action Potential ◽  
Expression Profile ◽  
Cardiac Electrophysiology ◽  
Human Heart ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Induced Pluripotent ◽  
Interfering Rna ◽  
Human Ipsc ◽  
Physiological Systems

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.

scholarly journals Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

2020 ◽  
Author(s):  
Elliot W. Swartz ◽  
Greg Shintani ◽  
Jijun Wan ◽  
Joseph S. Maffei ◽  
Sarah H. Wang ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Calcium Flux ◽  
Electrode Arrays ◽  
Spinal Motor Neurons ◽  
Culture Model ◽  
Skeletal Myotubes ◽  
Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

scholarly journals A multimodal 3D neuro-microphysiological system with neurite-trapping microelectrodes

2021 ◽  
Author(s):  
Beatriz Molina-Martinez ◽  
Laura V Jentsch ◽  
Fulya Ersoy ◽  
Matthijs van der Moolen ◽  
Stella Donato ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Electrophysiological Recording ◽  
Neuronal Cultures ◽  
Novel Approach ◽  
Clinical Models ◽  
Induced Pluripotent ◽  
Therapeutic Compounds ◽  
Automated Handling

Three-dimensional cell technologies as pre-clinical models are emerging tools mimicking the structural and functional complexity of the nervous system. The accurate exploration of phenotypes in engineered 3D neuronal cultures, however, demands morphological, molecular and especially functional measurements. Particularly crucial is measurement of electrical activity of individual neurons with millisecond resolution. Current techniques rely on customized electrophysiological recording set-ups, characterized by limited throughput and poor integration with other readout modalities. Here we describe a novel approach, using multiwell glass microfluidic microelectrode arrays, allowing non-invasive electrical recording from engineered 3D neural tissues. We demonstrate parallelized studies with reference compounds, calcium imaging and optogenetic stimulation. Additionally, we show how microplate compatibility allows automated handling and high-content analysis of human-induced pluripotent stem cell-derived neurons. This microphysiological platform opens up new avenues for high-throughput studies on the functional, morphological and molecular details of neurological diseases and their potential treatment by therapeutic compounds.

scholarly journals Population-scale proteome variation in human induced pluripotent stem cells

2018 ◽  
Author(s):  
Bogdan A Mirauta ◽  
Daniel D Seaton ◽  
Dalila Bensaddek ◽  
Alejandro Brenes ◽  
Marc J Bonder ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Induced Pluripotent Stem Cell ◽  
Protein Abundance ◽  
Protein Protein Interactions ◽  
Genetic Determinants ◽  
Ips Cell ◽  
Transcriptome Variation ◽  
Regulatory Effects ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractRealising the potential of human induced pluripotent stem cell (iPSC) technology for drug discovery, disease modelling and cell therapy requires an understanding of variability across iPSC lines. While previous studies have characterized iPS cell lines genetically and transcriptionally, little is known about the variability of the iPSC proteome. Here, we present the first comprehensive proteomic iPSC dataset, analysing 202 iPSC lines derived from 151 donors. We characterise the major genetic determinants affecting proteome and transcriptome variation across iPSC lines and identify key regulatory mechanisms affecting variation in protein abundance. Our data identified >700 human iPSC protein quantitative trait loci (pQTLs). We mapped trans regulatory effects, identifying an important role for protein-protein interactions. We discovered that pQTLs show increased enrichment in disease-linked GWAS variants, compared with RNA-based eQTLs.

Sign in / Sign up

Export Citation Format

Share Document