Typically, it has been difficult to obtain and culture high quality human cardiac cells. This is usually a result of the scarcity of healthy donor material and culture issues associated with the non-dividing state of terminally differentiated cardiomyocytes.
The ascent of human embryonic stem cell technology permitted important progress, as protocols were advanced for differentiating cardiomyocytes from a replenishable pluripotent source. A major problem with the utilization of human embryonic stem cell-derived cells was the controversial ethical problems and stringent regulations governing their usage. Consequently, when Yamanaka's group produced the Nobel Prize winning induced pluripotent stem cells, their breakthrough was immensely beneficial for the study of human cardiac cells.
Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Revolutionizing Research
The introduction of iPS cell technology for human cells in 2007 exposed novel possibilities in cardiovascular research, bringing with it the capacity for attaining unlimited numbers of cells. Moreover, these would be obtained with less ethical controversy than human embryonic stem cells.
The differentiation of iPS cells into cardiomyocytes from both healthy donors and those with diseases enabled, for the first time, the study of cardiomyocytes in vitro in a disease and genetically relevant background. This area of interest is growing quickly, with over 900 publications containing the phrase "induced pluripotent stem cell cardiomyocyte" having been published in 2015 alone.
The utilization of iPSC-derived cardiomyocytes for human disease modeling offers advantages in comparison to primary cells, as they supply a continuous source with which to generate terminally differentiated cells. In addition, iPSC-cardiomyocytes possess advantages over those derived from human embryonic stem cells, as they bypass a field characterized by ethical controversy.
Although, as with any model, cardiac cells derived from iPS cells embody their own limitations, they also offer an efficacious tool for the cardiovascular research community. The study of cardiac diseases, starting with those that possess a perceived genetic cause, has been a key focus.
Characteristics of iPSC-Cardiomyocytes
Differentiation to the cardiac lineage depends on a number of signaling pathways. Two key pathways are BMP signaling, an inducer, and Wnt signaling, an inhibitor of cardiac specification. Numerous divergent approaches have been developed for differentiating cardiomyocytes from human embryonic stem cells and human induced pluripotent stem cells via modulation of the aforementioned signaling pathways.
iPSC-derived cardiomyocytes cultures comprise spontaneously beating cells and, despite being immature in culture, the cells co-express a combination of atrial, nodal and ventricular markers; a different expression profile to adult cardiomyocytes. During the culture process, the cells start to mature; at approximately sixteen days in culture the cells commence segregation and by thirty days in culture they become subtype specific i.e. atrial, nodal or ventricular.
In comparison to their adult equivalents, the cardiomyocytes derived from iPS cells embody a functional phenotype in culture. Much like adult cardiomyocytes, iPSC-cardiomyocytes exhibit action potentials, express major cardiac ion channel proteins and sarcoermeric proteins and react to calcium.
Disease Modeling Using iPSC-Cardiomyocytes
Cardiac disease modeling utilizing iPSC-derived cardiomyocytes capitalizes on a system that permits the evaluation of the long-term effects of expression of mutated proteins, in addition to the consequences of different drug therapies on healthy and diseased backgrounds. The homogeneous character of the cells and the ability to repeatedly return to an identical source compensates for some of the drawbacks associated with utilizing heterogeneous sources, such as primary cells.
An early focus has been the examination of arrythmias and cardiomyopathies utilizing cells derived from patients undergoing familial cardiac diseases with known genetic mutations. The results from some of these studies are presented below.
Familial Hypertrophic Cardiomyopathy (HCM)
Hereditary disorder associated with abnormal thickening of the left ventricular myocardium bringing about arrhythmia and sudden cardiac death
- Study by Lan et al. published in 2013 indicated abnormal phenotypes, comprising cellular hypertrophy and cardiac arrhythmia, in iPSC-cardiomyocytes generated from patients embodying an Arg663His mutation in MYH7
- Reversed the arrhythmia phenotype by pharmaceutically blocking Ca2+and Na+ entry
- Supplied novel insights into disease mechanism and possible therapeutics
Dilated Cardiomyopathy (DCM)
- Indicators of DCM are ventricular dilation and impaired systolic function, which can generate the requirement for transplantation
- Sun et al., represents one instance of familial iPSC-derived cardiomyocytes being used as a tool for examining the disease
- They acquired cells from familial patients embodying an R173W mutation in the gene encoding Troponin T
- The cells recapitulated certain disease properties, including decreased beating rates and abnormal calcium handling
- Demonstrated the utilization of iPSC-derived caridiomyocytes as a disease model for DCM
- Barth Syndrome is a mitochondrial cardiomyopathy that is brought about by mutations in the TAZ gene
- As indicated by Wang et al., in their Nature Medicine publication, cardiomyocytes produced from iPSCs from Barth Syndrome patients embody impaired mitochondrial functionality, augmented ROS production and defective sarcomere assembly
- Provided novel insights into the pathogenesis of Barth Syndrome and the significance of mitochondrial defects among cardiac diseases
Long QT Syndromes
- Long QT syndromes are a group of heritable disorders distinguished by an extended QT interval, with high incidences of sudden cardiac death
- LQT1 occurs as a result of mutations in KCNQ1, LQT2 is caused by hERG protein mutations and LQT3 is brought about by gain-of-function mutations in SCN5A
- Multiple studies have been undertaken utilizing iPSC-derived cardiomyocytes from LQT1-3 patient sources, which have indicated disease-specific abnormalities in culture
- For example, Moretti et al., demonstrated that iPSC-derived cardiomyocytes from LQT1 patients display prolonged action potentials
Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)
- CPVT is an inherited ion channel disorder linked to sudden cardiac death in children and young adults
- The predominant cause of the disorder are mutations in RYR2
- iPSC-derived cardiomyocytes from CPVT patients in a project undertaken by Kujal et al., in 2012 embodied aberrant Ca2+ cycling, therefore reproducing an important feature of the disorder
Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC)
- ARVC is associated with sudden cardiac death and mutations in desmosomal proteins
- Ma et al., indicated that iPSC-derived cardiomyocytes from ARVC patients recapitulate some of the characteristics of the disease, such as cellular lipid droplets similar to those found in biopsies of abnormal tissue
Isogenic Disease Models
Harnessing The Power of Genome Editing Technology
As is made clear by the multiple studies detailed above, the utilization of hiPSC-derived cardiomyocytes has indicated novel insights into the pathology of a multitude of cardiac diseases. They represent extremely valuable disease models and can assist in the development of novel therapeutics. One common downside with the use of patient-derived cells for investigating diseases has been the absence of a control with an identical genetic background.
This means that any subtle phenotypic alterations would not be distinguished as a result of biological variation between donors, even though there is a disease-relevant phenotype. The introduction of genome editing technologies, such as zinc finger nucleases, TALENs and CRISPR/Cas9 techniques, have permitted the development of even more robust strategies. Such technologies enable the correction of mutations in cells derived from patients with monogenetic diseases, in addition to the introduction of disease-relevant mutations into cells derived from healthy donors.
Research undertaken by Joseph Wu's group, outlined in the paper "Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing" is one example of this in action. Following introduction of disease-causing mutations for Long-QT Syndrome 1 and 2 into iPSCs, Wu’s group differentiated the cells into cardiomyocytes.
The resulting cardiomyocytes exhibited defects indicative of Long-QT Syndrome, in contrast to the control cells lacking this mutation. This phenotype could be saved with the addition of appropriate drug treatments. It is clear that, as genome editing capacities progress, isogenic models of cardiovascular diseases produced from iPSCs are going to be of increasing value in a variety of applications, such as high-throughput screening (HTS).
The capacity for studying the properties of human cardiomyocytes in vitro from both healthy and diseased backgrounds was significantly augmented by the introduction of iPS cell technology. Human iPSC-derived cardiomyocytes offer a great research opportunity for complementing research undertaken in vivo utilizing animal models. Moreover, they are an easier to acquire, and less heterogeneous source of cells than primary and human embryonic stem cells. In vitro models can recapitulate characteristics of cardiomyocytes observed in vivo, for example the successful recapitulation of cellular characteristics associated with cardiac diseases.
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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