Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.
Duchenne muscular dystrophy (DMD)–associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients’ somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient–derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.
Heart disease is the leading cause of mortality in western countries. Apart from congenital and anatomical alterations, ischemia is the most common agent causing myocardial damage. During ischemia, a sudden decrease in oxygen concentration alters cardiomyocyte function and compromises cell survival. The calcium handling machinery, which regulates the main functional features of a cardiomyocyte, is heavily compromised during acute hypoxic events. Alterations in calcium dynamics have been linked to both short- and long-term consequences of ischemia, ranging from arrhythmias to heart failure. In this perspective, we aimed at investigating the calcium dynamics in functional cardiomyocytes during the early phase of a hypoxic event. For this purpose, we developed a microfluidic system specifically designed for controlling fast oxygen concentration dynamics through a gas micro-exchanger allowing in line analysis of intracellular calcium concentration by confocal microscopy. Experimental results show that exposure of Fluo-4 loaded neonatal rat cardiomyocytes to hypoxic conditions induced changes in intracellular Ca(2+) transients. Such behavior was reversible and was detected for hypoxic levels below 5% of oxygen partial pressure. The observed changes in Ca(2+) dynamics were mimicked using specific L-type Ca(2+) channel antagonists, suggesting that alterations in calcium channel function occur at low oxygen levels. Reversible alteration in ion channel function, that takes place in response to changes in cellular oxygen, might represent an adaptive mechanism of cardiopreservation during ischemia.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.