A central elicitor of ischemia/reperfusion (I/R) injury in the human heart is hypoxia/ischemia and yet there appears to be a lack of suitable in vitro models for the investigation of I/R-induced cellular mechanisms in human cardiac cells.
To examine whether induced human pluripotent stem cell (iPSC)-derived cardiomyocytes respond to physiologically defined transient hypoxic/ischemic conditions.
Materials and Methods
Enzymatically induced hypoxic/ischemic conditions (Zitta et al. Eur J Pharmacol 2010, Zitta et al. Mol Med 2012, Zitta et al. Exp Cell Res 2012, Hummitzsch et al. Exp Cell Res 2014) were imposed on spontaneously contracting human iPSC-derived cardiomyocytes (ax2505; generously provided by Axol Bioscience Ltd., Cambridge, UK) using glucose oxidase (GO, 2 U/ml) and catalase (CAT, 120 U/ml).
Hypoxia/ischemia-induced cytotoxicity was evaluated and quantified through morphological assessment and measurements of LDH activity released from damaged cells (Cytotoxicity Detection Kit; Roche, Mannheim, Germany) and Troponin T (TnT; Department of Clinical Chemistry, Kiel). A loop recorder attached to the bottom of the culture dish was used to assess lectrophysiological parameters (Medtronic, Dublin, Ireland).
Once GO/CAT had been added, hypoxic/ischemic conditions established rapidly, resulting in pO2 levels <10 mmHg after 60 minutes (lasting for at least 6 hours; A), a gradual decrease of glucose concentration (from 2 g/l to <1 g/l after 4 hours) was also observed as well as a decline of pH from 7.65 to 6.98.
Spontaneous synchronized contractions were seen in iPSC-derived cardiomyocytes after 10 days in culture with a beating frequency of 24.41±4.02 bpm under normoxia and 18.87±3.21 bpm under hypoxic/ischemic conditions.
The frequency of contractions immediately increased by 1.8-fold in the hypoxia/ischemia group (B) after replacing the hypoxic/ischemic medium by normoxic culture medium.
The electrophysiological signal of the cardiomyocytes was in line with a standard ventricle polarization with an amplitude of 0.73±0.26 mV and a QRS-interval of 0.11±0.02s.
A 10-fold increase in asystole episodes were detected during hypoxia (normoxia: 7%; hypoxia/ischemia: 70%; B). iPSC-derived cardiomyocytes showed clear morphological signs of cell damage (for example, cell rounding, swelling and detachment from the growth surface (C)) 24 hours after the 4-hour hypoxia period. This was associated with irregular and asynchronized beating of single cells.
Cultures subjected to 4 hours of hypoxia, displayed an LDH release as a marker of cell damage that increased 3-fold. iPSC-derived cardiomyocytes, cultured under normoxic conditions, revealed no morphological changes or increased LDH release after 24 hours (normoxia: 0.10±0.00au; hypoxia/ischemia: 0.29±0.02 au; P<0.05; D).
Furthermore, concentrations of Troponin T (TnT) were examined in cell culture supernatants and revealed increased levels in the hypoxia group (E).
Image Credit: Axol Bioscience
Human-induced pluripotent stem cell-derived cardiomyocytes respond to transient hypoxia/ischemia in vitro. Hypoxic/ischemic conditions in vivo closely resembled the described culture system. It may help in explaining cellular/molecular mechanisms of ischemia/reperfusion injury and support a search for cardioprotective strategies.
Produced from materials originally authored by Martin Albrecht PhD, Kerstin Parczany, Matthias Gruenewald MD, Ole Broch MD, Lars Hummitzsch MD, Rouven Berndt MD, Jochen Cremer MD, Markus Steinfath MD, and Karina Zitta PhD from the Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Kiel, Germany, and the Department of Cardiovascular Surgery, University Hospital Schleswig-Holstein, Kiel, Germany.
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
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