Ion channel profiling of stem cell-derived cardiomyocytes: Useful reagents for automated patch-clamp?

2018 ◽  
Vol 295 ◽  
pp. S49
Author(s):  
M. Morton
Keyword(s):  
Stem Cell ◽  
Patch Clamp ◽  
Ion Channel ◽  
Automated Patch Clamp

Automated patch clamp recordings of action potentials from stem cell derived cardiomyocytes

2010 ◽  
Vol 62 (2) ◽  
pp. e35
Author(s):  
Mohamed Kreir ◽  
Sonja Stoelzle ◽  
Alison Haythornthwaite ◽  
Claudia Haarmann ◽  
Cecilia Farre ◽  
...  
Keyword(s):  
Stem Cell ◽  
Patch Clamp ◽  
Action Potentials ◽  
Automated Patch Clamp

scholarly journals Characterizing Human Ion Channels in Induced Pluripotent Stem Cell–Derived Neurons

2012 ◽  
Vol 17 (9) ◽  
pp. 1264-1272 ◽  
Author(s):  
Alison Haythornthwaite ◽  
Sonja Stoelzle ◽  
Alexander Hasler ◽  
Andrea Kiss ◽  
Johannes Mosbacher ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ion Channels ◽  
Patch Clamp ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Excitable Cells ◽  
Automated Patch Clamp ◽  
Induced Pluripotent ◽  
Two Populations ◽  
A Current

Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na+ (Nav), Ca2+ (Cav), and K+ (Kv) channels as expected from excitable cells. The Nav current was TTX sensitive, IC50 = 12 ± 6 nM ( n = 5). About 50% of the Cav current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the Kv channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC50 = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABAA receptor antagonist bicuculline, IC50 = 632 ± 149 nM ( n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Nav currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching “excitable cells.” These results show high potential for pluripotent stem cell–derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.

Automated Patch Clamp Recordings of Human Stem Cell-Derived Cardiomyocytes

2016 ◽  
pp. 57-82 ◽  
Author(s):  
Alison Obergrussberger ◽  
Claudia Haarmann ◽  
Sonja Stölzle-Feix ◽  
Nadine Becker ◽  
Atsushi Ohtsuki ◽  
...  
Keyword(s):  
Stem Cell ◽  
Patch Clamp ◽  
Human Stem Cell ◽  
Automated Patch Clamp

scholarly journals A Yoda1-Based Approach to Investigate Piezo1 Channels in Red Blood Cells Using Automated Patch Clamp Technology

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1031-1031
Author(s):  
Maria Giustina Rotordam ◽  
Elisa Fermo ◽  
Nadine Becker ◽  
Wilma Barcellini ◽  
Andrea Brüggemann ◽  
...  
Keyword(s):  
Shear Stress ◽  
Patch Clamp ◽  
Ion Channel ◽  
Red Blood Cells ◽  
High Throughput ◽  
Blood Cells ◽  
Screening Assay ◽  
Whole Cell ◽  
Automated Patch Clamp ◽  
Induced Currents

Abstract Piezo1 is a mechanosensitive ion channel supposed to regulate the volume and maintain the structural integrity in Red Blood Cells (RBCs), as gain-of-function mutations in this channel are associated to the RBC disease Hereditary Xerocytosis (Zarychanski et al. Blood 2012; Bae et al. Proceedings of the National Academy of Sciences 2013). Piezo1 is activated by several mechanical forces, including stretching, poking and shear stress and allows Ca2+ and other cations to enter the cell generating an electrical response. In 2015, it has been discovered that Piezo1 is sensitive to a small molecule, Yoda1 (Syeda et al. Elife 2015), which keeps the channel open and affects its inactivation kinetics. This finding has created new possibilities to elucidate Piezo1 gating mechanism and explore its functional significance in physiological and pathophysiological conditions. Here, we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp-based high-throughput screening assay for Piezo1 activity. We established a protocol to detect functional Piezo1 mutations upon chemical stimulation by Yoda1, yet were not able to stimulate the channel via mechanical force, i.e. pressure steps and shear-stress. The assay was first developed on Neuro2A (N2A), a neuroblastoma cell-line endogenously expressing Piezo1 channels (kindly provided by Max-Delbrück Center, Berlin), due to larger abundance of Piezo1 channels in these cells. Initial experiments were performed on the Patchliner (Nanion Technologies GmbH, Munich), a medium-throughput automated patch clamp system able to record up to 8 cells at a time. Currents were elicited using a voltage ramp ranging from -100 to +80 mV for 300 ms, the holding potential was set to -60 mV. A significantly increased whole-cell current was observed upon 10 µM Yoda1 application in half of the recorded cells and the resulting Yoda1-induced currents were inhibited by 30 µM gadolinium chloride, a non-specific blocker of stretch-activated channels. The assay was then implemented on the SyncroPatch 384PE (Nanion Technologies GmbH, Munich), capable of recording up to 384 cells in parallel under identical experimental conditions, thus allowing for reliable statistical analysis. Yoda1 responding cells were selected based on strict quality control (QC) criteria, i.e. the seal resistance stability over time. In one example NPC-384 chip 140 out of 384 N2A cells (37%) passed the QC criteria and 85 cells (60% of the valid cells) were considered as Yoda1 responders. Finally, we investigated Piezo1 electrophysiological properties in healthy and patient RBCs carrying the novel PIEZO1 R2110W mutation. Similar to N2A cells, RBCs currents were analyzed and divided into Yoda1 responders and non-responders according to our QC criteria. The increase in whole-cell currents induced by Yoda1 application was significantly higher in patient compared to control RBCs, which was also reflected by a higher number of Yoda1 responders compared to control. Residue R2110W is structurally located in a gating sensitive area of the channel protein suggesting a gain-of-function. This would be in line with previously described mutations in PIEZO1 (Albuisson et al. Nature Communications 2013) and the mild form of anaemia observed in the patient. Furthermore, we excluded any involvement of Gardos channels in the Yoda1-induced currents by comparing measurements in the presence and absence of the specific Gardos channel inhibitor TRAM-34. Altogether, our work demonstrates that high-throughput patch clamping can provide a robust assay to study functional Piezo1 impairments in primary RBCs without expressing the mutated channel protein in a heterologous expression system. Our approach may be used to detect other channelopathies not only in RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Automated Patch Clamp on mESC-Derived Cardiomyocytes for Cardiotoxicity Prediction

2011 ◽  
Vol 16 (8) ◽  
pp. 910-916 ◽  
Author(s):  
Sonja Stoelzle ◽  
Alison Haythornthwaite ◽  
Ralf Kettenhofen ◽  
Eugen Kolossov ◽  
Heribert Bohlen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ion Channels ◽  
Patch Clamp ◽  
Cell Model ◽  
Single Type ◽  
Ion Currents ◽  
Drug Induced ◽  
Cardiovascular Side Effects ◽  
Automated Patch Clamp

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro–generated stem cell–derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell–derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell–derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Cardiac Ion Channel Safety Profiling on the IonWorks Quattro Automated Patch Clamp System

2010 ◽  
Vol 8 (6) ◽  
pp. 766-780 ◽  
Author(s):  
Xueying Cao ◽  
Yan Tony Lee ◽  
Mats Holmqvist ◽  
Yingxin Lin ◽  
Yucheng Ni ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Automated Patch Clamp

Minimized cell usage for stem cell-derived and primary cells on an automated patch clamp system

2013 ◽  
Vol 68 (1) ◽  
pp. 82-87 ◽  
Author(s):  
Nadine Becker ◽  
Sonja Stoelzle ◽  
Sven Göpel ◽  
David Guinot ◽  
Patrick Mumm ◽  
...  
Keyword(s):  
Stem Cell ◽  
Patch Clamp ◽  
Primary Cells ◽  
Automated Patch Clamp
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