This research aimed to assess the potential of embryonic stem cell-derived cardiac cells to replace large numbers of animals currently used in heart disease research. Cardiac myocytes, from human and mouse embryonic stem cells, were compared to adult human and animal myocytes to determine their suitability for investigation of normal physiology, disease and drug research.
Heart disease has a huge impact on human health; because of this, a large number of animal studies are performed for the investigation of disease hypotheses, drug discovery and toxicology testing. Animals are used due to a lack of suitable in vitro models, and because human tissue is difficult to obtain.
Cardiac myocytes, derived from embryonic stem cells (ESCs) offer a number advantages, including the potential to replace a large number of animal studies. ESCs may be obtained from human sources and may be more easily manipulated physically and genetically than animal models.
Research details and methods
Human and mouse ESC-derived cardiac myocytes were validated by comparison with adult myocytes from both species. Their contractile responses to a bank of agents under investigation for their roles in heart failure (for example, neurohormones and cytokines) were determined, and subtypes of cellular receptors which mediate responses investigated using specific antagonists.
Comparisons were also made to the responses of normal adult human or mouse myocytes, either from past experiments or studies done in parallel. Myocytes were generated from a mouse mutant ESC line which lacks an inhibitory G-protein, to test hypotheses related to beta1- and beta2-adrenoceptor coupling.
Key impacts and findings
A range of acute and long term ESC cardiac myocyte assays were established as model systems for cardiac physiology and pathophysiology. Specific instances have been found where the ESC-derived myocytes are equal or superior to animal myocytes for modelling adult human ventricular myocyte responses.
For basic cardiac investigations human ESC cardiac myocyte models have the potential to replace approximately 180 animals per year within the Harding laboratory alone, and in the region of 1,500 studies per year worldwide. The potential for reducing animal use in drug discovery and development is even greater. Human ESC cardiac myocyte, and similar cells derived from skin rather than embryonic tissue, are now commercially available to researchers.
Because of the huge impact of heart disease on human health, a large number of animal studies are performed for the investigation of disease hypotheses, drug discovery and toxicology testing. One of the reasons for the predominance of in vivo work in this area is the difficulty associated with finding a suitable in vitro model. Human tissue is difficult to obtain, and there are scientific and ethical problems associated with its use. Primary animal cardiac myocytes have been a successful preparation because of the preservation of their structure and function after isolation, and a great deal of work has been done in our laboratory to validate the use of primary animal (and human) myocytes as a model for in vivo cardiac responses. However, they have insurmountable limitations in terms of survival in culture (1-2 days), and meaningful transfection is only possible using viral vectors. Embryonic stem cells have the potential to produce myocytes representing all phenotypes in the heart (atrial, nodal and ventricular). Since they may be obtained from human sources, they may act as a more relevant preparation than animal tissue. They can continue to beat regularly in culture for 70 days (the longest time tested) and can be transfected using plasmid DNA. They may also be created from mutant ES lines, raising the potential for creation of transgenic myocytes without the mouse. Large banks of these mutant ES lines exist, and are available for purchase.
In the present project we aim to validate the ES cell derived cardiac myocyte (ESCM) by comparison with adult myocytes for both human and mouse lines. Contractile responses to a bank of agents under investigation for their roles in heart failure (neurohormones, cytokines etc.) will be determined, and subtypes of receptors mediating responses will be investigated using specific antagonists. Comparisons will be made to responses of normal adult human or mouse myocytes, either from past experiments or studies done in parallel. Myocytes will also be generated from a mouse mutant ES line lack inhibitory G-protein to test hypotheses related to beta1- and beta2-adrenoceptor coupling. Human and mouse ESCM are established in our laboratory and we have developed methods for measuring their contraction. For basic cardiac investigations the ESCM preparation has the potential to replace approximately 180 animals per year in my own laboratory and 1500 studies per year worldwide. The long-term potential in terms of pharmaceutical drug discovery is much larger.
Földes G, Mioulane M, Kodagoda T, Lendvai Z, Iqbal A, Ali NN, Schneider MD, Harding SE (2014). Immunosuppressive agents modulate function, growth, and survival of cardiomyocytes and endothelial cells derived from human embryonic stem cells. Stem Cells Dev. 23(5): 467-76 doi:10.1089/scd.2013.0229
Mioulane M, Földes G, Ali NN, Schneider MD, Harding SE (2012) Development of high content imaging methods for cell death detection in human pluripotent stem cell-derived cardiomyocytes. Journal of cardiovascular translational research 5: 593-604 doi:10.1007/s12265-012-9396-1
Földes G, Mioulane M, Wright JS, Liu AQ, Novak P, Merkely B, Gorelik J, Schneider MD, Ali NN, Harding SE (2011) Modulation of human embryonic stem cell-derived cardiomyocyte growth: a testbed for studying human cardiac hypertrophy? Journal of molecular and cellular cardiology 50: 367-76 doi:10.1016/j.yjmcc.2010.10.029
Földes G, Liu A, Badiger R, Paul-Clark M, Moreno L, Lendvai Z, Wright JS, Ali NN, Harding SE, Mitchell JA (2010) Innate immunity in human embryonic stem cells: comparison with adult human endothelial cells. PLoS ONE 5(5): e10501 doi:10.1371/journal.pone.0010501
Abdul Kadir SH, Ali NN, Mioulane M, Brito-Martins M, Abu-Hayyeh S, Foldes G, Moshkov AV, Williamson C, Harding SE, Gorelik J (2009) Embryonic stem cell-derived cardiomyocytes as a model to study fetal arrhythmia related to maternal disease. Journal of Cellular and Molecular Medicine 13 (9b): 3730-3741 doi: 10.1111/j.1582-4934.2009.00741.x
Brito-Martins M, Harding SE, Ali NN (2008) Beta (1)- and beta (2) adrenoreceptor responses in cardiomyocytes derived from human embryonic stem cells: comparison with failing and non-failing adult human heart. British Journal of Pharmacology 153: 751-759 doi: 10.1038/sj.bjp.0707619
Harding SE, Ali NN, Brito-Martins M, Gorelik J (2007) The human embryonic stem cell-derived cardiomyocyte as a pharmacological model. Pharmacology & Therapeutics 113: 341-353 doi:10.1016/j.pharmthera.2006.08.008