Studying Human Cardiomyocytes in vitro

Primary cells and human embryonic stem cell sources


Traditionally, there have been difficulties in obtaining and culturing human cardiac cells of a high quality due to a scarcity of healthy donor material, culture issues associated with the non-dividing state of terminally differentiated cardiomyocytes. The development of human embryonic stem cell technology allowed major advancements to be made as protocols were developed to differentiate cardiomyocytes from a replenishable pluripotent source. A huge issue with the use of human embryonic stem cell-derived cells was the controversial ethical issues and strict regulations regarding their use. This meant that Yamanaka's breakthrough when his group produced the Nobel Prize winning technology in the form of  induced pluripotent stem cells had a gargantuan impact on the study of human cardiac cells. 

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Revolutionalizing research

The advent of iPS cell technology for human cells in 2007 opened up new possibilities in cardiovascular research as it brought with it the ability to attain unlimited numbers of cells collected in a less ethically controversial way than human embryonic stem cells. The differentiation of iPS cells into cardiomyocytes from not only healthy donors, but those with diseases meant that it was now possible to study cardiomyocytes in vitro in a disease and genetically relevant background. This field is rapidly expanding with >900 publications including the search term "induced pluripotent stem cell cardiomyocyte" already being published in 2015! The use of iPSC-derived cardiomyocytes for human disease modeling provides advantages over primary cells as they provide a continuous source with which to produce terminally differentiated cells. Furthermore, iPSC-cardiomyocytes have an advantage over those derived from human embryonic stem cells in that they avoid a field fraught with controversial ethical issues. While, like any model, cardiac cells derived from iPS cells have limitations, they provide a valuable tool for the cardiovascular research community. A major focus has been the study of cardiac diseases, beginning with those that have a known genetic cause.

Characteristics of iPSC-Cardiomyocytes

Differentiation to the cardiac lineage relies on many signalling pathways. Two important pathways are BMP signalling, an inducer and Wnt signalling, an inhibitor of cardiac specification. A number of different methods have been developed to differentiate cardiomyocytes from human embryonic stem cells and human induced pluripotent stem cells by modulation of the above mentioned signalling pathways. 

iPSC-derived cardiomyocytes cultures include spontaneously beating cells and while immature in culture the cells co-express a mixture of atrial, ventricular and nodal markers - a diffferent expression profile to adult cardiomyocytes. During the culture process the cells begin to mature - at around 16 days in culture the cells begin to segregate and by 30 days in culture become subtype specific i.e. ventricular, atrial or nodal. Unlike their adult counterparts, the cardiomyocytes derived from iPS cells maintain a functional phenotype in culture. 

In similarity to adult cardiomyocytes, iPSC-cardiomyocytes express major cardiac ion channel proteins and sarcoermeric proteins, exhibit action potentials and react to calcium. 

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Disease Modeling Using iPSC-Cardiomyocytes

Cardiac disease modeling using iPSC-derived cardiomyocytes takes advantage of a system that allows the assessment of the long term impacts of expression of mutated proteins for example and the impacts of various drug treatments on healthy and diseased backgrounds. The homogenous nature of the cells and the ability to go back to the same source repeatedly negates some of the drawbacks that come with using heterogeneous sources such as primary cells. An initial focus has been to examine cardiomyopathies and arrythmias using cells derived from patients with familal cardiac diseases with known genetic mutations. Let's examine the results from some of these studies. 

Cardiomyopathies

Familial Hypertrophic Cardiomyopathy (HCM)

  • Hereditary disorder associated with abnormal thickening of the left ventricular myocardium leading to arrhythmia and sudden cardiac death. 
  • Study by Lan et al., published in 2013 demonstrated abnormal phenotypes including cellular hypertrophy and  cardiac arrhythmia in iPSC-cardiomyocytes produced from patients with an Arg663His mutation in MYH7. 
  • Reversed the arrhythmia phenotype by pharmaceutically blocking Ca2+ and Na+ entry.
  • Provided new insights into disease mechanism and potential therapeutics.

Dilated Cardiomyopathy (DCM)

  • Hallmarks of DCM are ventricular dilation and impaired systolic function, which can lead to the need for transplantation.
  • Sun et al., is one example of familial iPSC-derived cardiomyocytes being utilized as a tool for studying the disease. 
  • They obtained cells from familial patients with an R173W mutation in the gene encoding Troponin T.
  • The cells recapitulated some disease characteristics such as decreased beating rates and abnormal calcium handling.
  • Demonstrated the use of iPSC-derived caridiomyocytes as a disease model for DCM.

Barth Syndrome 

  • Barth Syndrome is a mitochondrial cardiomyopathy that is caused by mutations in the TAZ gene.
  • As shown by Wang et al., in their Nature Medicine publication, cardiomyocytes generated from iPSCs from Barth Syndrome patients have impaired mitochondrial functionality, increased ROS production and defective sarcomere assembly.
  • Revealed new insights into the pathogenesis of Barth Syndrome and also provided insights into the role of mitochondrial defects in cardiac diseases.

Cardiac Arrhythmias

Long QT Syndromes

  • Long QT syndromes represent a group of hertiable disorders characterized by an extended QT interval with high incidences of sudden cardiac death. 
  • LQT1 is caused by mutations in KCNQ1, LQT2 is caused by hERG protein mutations while LQT3 is caused by gain-of-function mutations in SCN5A.
  • A number of studies have been carried out using iPSC-derived cardiomyocytes from LQT1-3 patient sources, which have shown disease-specific abnormalities in culture.
  • As an example, Moretti et al., showed that iPSC-derived cardiomyocytes from LQT1 patients exhibit prolonged action potentials.

Catecholaminergic Polymorphic
Ventricular Tachycardia (CPVT)

  • CPVT is an inherited ion channel disorder associated with sudden cardiac death in children and young adults. 
  • Mutations in RYR2 are the predominant cause of the disorder.
  • iPSC-derived cardiomyocytes from CPVT patients in a project completed by Kujal et al., in 2012 had aberrant Ca2+ cycling, thus replicating a key characteristic of the disorder.

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) 

  • ARVC is linked with sudden cardiac death and is associated with mutations in desmosomal proteins.
  • Ma et al., revealed that iPSC-derived cardiomyocytes from ARVC patients recapitulate some of the features of the disease including cellular lipid droplets akin to those observed in biopsies of abnormal tissue. 

Isogenic Disease Models

Harnessing The Power of Genome Editing Technology

As is evident by the numerous studies described above, the use of hiPSC-derived cardiomyocytes has revealed new insights into the pathology of a variety of cardiac diseases. They represent highly valuable disease models and can pave the way for the development of new therapeutics. One problem with the utilization of patient-derived cells for studying diseases has traditionally been the lack of a control with the same genetic background. This means that any subtle phenotypic changes would not be distinguished due to biological variation between donors even though there is a disease-relevant phenotype. The advent of genome editing technology including zinc finger nucleases, TALENs and CRISPR/Cas9 techniques have enabled the development of even more powerful tools. These technologies allow the correction of mutations in cells derived from patients with monogenetic diseases as well as the introduction of disease-relevant mutations into cells derived from healthy donors. 

One example of this in action is work completed by Joseph Wu's group, which is presented in their paper "Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing". They introduced disease-causing mutations for Long-QT Syndrome 1 and 2 into iPSCs and differentiated the cells into cardiomyocytes. The resultant cardiomyocytes displayed defects representative of Long-QT Syndrome in comparison to the control cells without the mutation. This phenotype could be rescued with the addition of appropriate drug treatments. Clearly, isogenic models of cardiovascular diseases generated from iPSCs are going to be increasingly valuable in a variety of applications, including high-throughput screening (HTS), as genome editing capacities progress.

Axol Custom Services

As part of our custom services, we can create isogenic lines for disease modeling and drug discovery using your mutations of interest. 

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In Summary 

  •  The ability to study the characteristics of human cardiomyocytes in vitro from both healthy and diseased backgrounds was greatly enhanced by the advent of iPS cell technology.

  • Human iPSC-derived cardiomyocytes provide a great research tool to complement studies performed in vivo using animal models and an easier to obtain/less heterogenous source of cells than primary and human embryonic stem cells.

  • In vitro models can recapitulate characteristics of cardiomyocytes seen in vivo, including successful recapitulation of cellular characteristics associated with cardiac diseases.