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

2018 ◽  
Vol 10 (6) ◽  
pp. 1991-2004 ◽  
Author(s):  
Evangelos Kiskinis ◽  
Joel M. Kralj ◽  
Peng Zou ◽  
Eli N. Weinstein ◽  
Hongkang Zhang ◽  
...  
Keyword(s):  
Motor Neuron ◽  
High Throughput ◽  
Neuron Model ◽  
Functional Characterization ◽  
All Optical ◽  
Human Ipsc

scholarly journals Transcriptomic Landscape and Functional Characterization of Induced Pluripotent Stem Cell–Derived Cerebral Organoids in Schizophrenia

2020 ◽  
Vol 77 (7) ◽  
pp. 745 ◽  
Author(s):  
Annie Kathuria ◽  
Kara Lopez-Lengowski ◽  
Smita S. Jagtap ◽  
Donna McPhie ◽  
Roy H. Perlis ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Functional Characterization ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

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 Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived—From the Same Induced Pluripotent Stem Cell Source

2020 ◽  
Vol 7 (4) ◽  
pp. 133
Author(s):  
Xiufang Guo ◽  
Agnes Badu-Mensah ◽  
Michael C. Thomas ◽  
Christopher W. McAleer ◽  
James J. Hickman
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Neuromuscular Junctions ◽  
Functional Characterization ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Type I ◽  
Induced Pluripotent

In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.

scholarly journals Microfluidics-enabled 96-well perfusion system for high-throughput tissue engineering and long-term all-optical electrophysiology

2020 ◽  
Author(s):  
Lai Wei ◽  
Weizhen Li ◽  
Emilia Entcheva ◽  
Zhenyu Li
Keyword(s):  
High Throughput ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Perfusion System ◽  
Excitable Cells ◽  
Hek Cells ◽  
All Optical ◽  
Electrophysiological Studies ◽  
Induced Pluripotent

ABSTRACTThis work demonstrates a novel high-throughput (HT) microfluidics-enabled uninterrupted perfusion system (HT-μUPS) and validates its use with chronic all-optical electrophysiology in human excitable cells. HT-μUPS consists of a soft multichannel microfluidic plate cover which could button on a commercial HT 96-well plate. Herein, we demonstrate the manufacturing process of the system and its usages in acute and chronic all-optical electrophysiological studies of human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CM) and engineered excitable (Spiking HEK) cells. HT-μUPS perfusion maintained functional voltage and calcium responses in iPSC-CM and Spiking HEK cells under spontaneous conditions and under optogenetic pacing. Long-term culture with HT-μUPS improved cell viability and optogenetically-tracked calcium responses in Spiking HEK cells. The scalability and simplicity of this design and its compatibility with HT all-optical electrophysiology can empower cell-based assays for personalized medicine using patient-derived cells.

scholarly journals All-optical electrophysiology refines populations of in silico human iPS-CMs for drug evaluation

2019 ◽  
Author(s):  
M Paci ◽  
E Passini ◽  
A Klimas ◽  
S Severi ◽  
J Hyttinen ◽  
...  
Keyword(s):  
High Throughput ◽  
In Silico ◽  
Pluripotent Stem Cell ◽  
Drug Effects ◽  
Induced Pluripotent Stem Cell ◽  
Drug Action ◽  
Drug Trials ◽  
All Optical ◽  
Induced Pluripotent

AbstractHigh-throughput in vitro drug assays have been impacted by recent advances in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) technology and by contact-free all-optical systems simultaneously measuring action potential (AP) and Ca2+ transient (CaTr). Parallel computational advances have shown that in silico models can predict drug effects with high accuracy. In this work, we combine these in vitro and in silico technologies and demonstrate the utility of high-throughput experimental data to refine in silico hiPS-CM populations, and to predict and explain drug action mechanisms. Optically-obtained hiPS-CM AP and CaTr were used from spontaneous activity and under pacing in control and drug conditions at multiple doses.An updated version of the Paci2018 model was developed to refine the description of hiPS-CM spontaneous electrical activity; a population of in silico hiPS-CMs was constructed and calibrated using the optically-recorded AP and CaTr. We tested five drugs (astemizole, dofetilide, ibutilide, bepridil and diltiazem), and compared simulations against in vitro optical recordings.Our simulations showed that physiologically-accurate population of models can be obtained by integrating AP and CaTr control records. Thus constructed population of models predicted correctly the drug effects and occurrence of adverse episodes, even though the population was optimized only based on control data and in vitro drug testing data were not deployed during its calibration. Furthermore, the in silico investigation yielded mechanistic insights, e.g. through simulations, bepridil’s more pro-arrhythmic action in adult cardiomyocytes compared to hiPS-CMs could be traced to the different expression of ion currents in the two.Therefore, our work: i) supports the utility of all-optical electrophysiology in providing high-content data to refine experimentally-calibrated populations of in silico hiPS-CMs, ii) offers insights into certain limitations when translating results obtained in hiPS-CMs to humans and shows the strength of combining high-throughput in vitro and population in silico approaches.SignificanceWe demonstrate the integration of human in silico drug trials and optically-recorded simultaneous action potential and calcium transient data from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) for prediction and mechanistic investigations of drug action. We propose a population of in silico models i) based on a new hiPS-CM model recapitulating the mechanisms underlying hiPS-CM automaticity and ii) calibrated with all-optical measurements. We used our in silico population to predict and evaluate the effects of 5 drugs and the underlying biophysical mechanisms, obtaining results in agreement with our experiments and one independent dataset. This work supports the use of high-content, high-quality all-optical electrophysiology data to develop, calibrate and validate computer models of hiPS-CM for in silico drug trials.

scholarly journals Multimodal on-axis platform for all-optical electrophysiology with near-infrared probes in human stem-cell-derived cardiomyocytes

2018 ◽  
Author(s):  
Aleksandra Klimas ◽  
Gloria Ortiz ◽  
Steven Boggess ◽  
Evan W. Miller ◽  
Emilia Entcheva
Keyword(s):  
Stem Cell ◽  
High Throughput ◽  
High Performance ◽  
Near Infrared ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Simultaneous Recording ◽  
Optogenetic Stimulation ◽  
All Optical ◽  
Induced Pluripotent

AbstractCombined optogenetic stimulation and optical imaging permits scalable, high-throughput probing of cellular electrophysiology and optimization of stem-cell derived excitable cells, such as neurons and muscle cells. We report a new “on-axis” configuration of OptoDyCE, our all-optical platform for studying human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and other cell types, optically driven by Channelrhodopsin2 (ChR2). This solid-state system integrates optogenetic stimulation with temporally-multiplexed simultaneous recording of membrane voltage (Vm) and intracellular calcium ([Ca2+]i) dynamics using a single photodetector. We demonstrate the capacity for combining multiple spectrally-compatible actuators and sensors, including newer high-performance near-infrared (NIR) voltage probes BeRST1 and Di-4-ANBDQBS, to record complex spatiotemporal responses of hiPSC-CMs to drugs in a high-throughput manner.

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