Future Directions in Cardiovascular Disease Modeling

Cardiovascular disease remains the leading cause of death in the United States and worldwide, indicating the continued need for basic science research into disease mechanisms and the development of therapeutics. In the statement “Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine,” Musunuru et al. nicely present an overview of induced pluripotent stem cell (iPSC) technology and its current and future application to the field of cardiovascular medicine.¹ This commentary will highlight several essential features of the statement while detailing additional approaches and concerns as the field progresses.

iPSC technology is a valuable platform for modeling cardiovascular disease because the cellular source is human in origin, often reprogrammed directly from the somatic cells of an affected individual, the cells can be differentiated into any somatic cell type following reprogramming, meaning cardiomyocytes, vascular smooth muscle cells, and endothelial cells can each be investigated, and the cellular population can self-renew, allowing for an essentially endless supply of cells. In this vein, iPSCs have been used to model an array of cardiovascular diseases, including cardiomyopathies, rhythmic disorders, vascular disease, and metabolic factors associated with heart disease.¹ Cardiovascular disease modeling using iPSCs has thus shed light on a multitude of disease mechanisms, and provided readily translatable insights into therapeutic resolution to disease progression.

The two principal approaches to studying cardiovascular disease with iPSCs are to reprogram the somatic cells from an individual affected by the disease, thus creating disease-specific patient-derived iPSCs, or to employ genetic editing technologies on wild type control iPSCs to introduce disease phenotypes. Both approaches are valid and each has its limitations. Though patient-derived iPSCs hold more promise to faithfully recapitulate the disease phenotype, it is not always possible to acquire sufficient sources of these cells, particularly for rarer conditions. Thus, genetic editing technologies can be employed to systematically introduce mutations into wild type iPSCs to create mutant cell lines. This approach is excellent for monogenic disorders, allows for the assessment of the sufficiency of a mutation to cause the disease phenotype, and can readily be applied as a pharmacogenomic platform for precision medicine. However, with complex, polygenic disorders, such an approach may be limited as a single mutation could lead to a minimal effect and the disease phenotype may not be faithfully reproduced. An ideal situation would therefore be to use the two approaches in concert, using the patient-derived iPSCs as a positive control and genetically modified wild type iPSCs for biological replicates. Additionally, employing genetic editing on a patient-derived mutant cell line as an attempt to correct the disease phenotype would assess the necessity of the mutation to cause the disease, adding confidence to using that mutation for genetically editing biological replicates.

Another current limitation in the field is the state of iPSC-derived cardiomyocyte maturity. As acknowledged in the statement, these iPSC-derived cardiomyocytes appear to be more similar to fetal cardiomyocytes than to adult cardiomyocytes, showing a lack of transverse tubules, fewer mitochondria, and higher resting membrane potentials than primary cells.¹ This is problematic, as the fetal and juvenile heart differs in many respects from the adult heart, and to model adult cardiovascular disease effectively, the iPSC-derived cardiomyocytes need to be more mature. Current methodology to overcome this obstacle has involved simply maintaining the cells for a longer time in culture, allowing them to mature on their own, though this approach is far from ideal, or modifying the growth conditions to force a state of maturation. Approaches to enhance cardiomyocyte maturity include increasing the stiffness of the growth substrate,² providing electrical stimuli to the developing cells,^3,4 or over- or underexpressing certain factors. Such factors have previously included microRNA overexpression⁵ and could plausibly include inhibition of factors which promote cardiac regeneration.⁶ Additionally, engineered heart tissue (EHT) approaches have been used to facilitate cardiomyocyte maturation. EHTs are three dimensional constructs produced through seeding cardiomyocytes onto a biomaterial scaffold,⁷ through the stacking of cell sheet monolayers,⁸ or through incorporation into a collagen gel.^9,10,11 Optimizing the culture conditions of EHTs, such as through the coculturing of cardiomyocytes with human foreskin fibroblasts or employing a serum free media under dynamic conditions which allow for enhanced nutrient uptake, has shown to increase the contractile force of the cardiomyocytes to approaching adult levels.^9,10,11,12

Similarly, in the field of vascular disease, the topic of lineage specificity has become of growing importance in the faithful modeling of disease phenotypes. Vascular smooth muscle cells come from a variety of different cellular lineages, and it has been established that populations of these cells behave differently upon stimulation by various factors depending upon the lineage.¹³ In particular, vascular smooth muscle cells originating from the neural crest lineage form the medial lining of the ascending aorta and have been implicated in the pathology of thoracic aortic aneurysm and dissection¹⁴ as well as supravalvular aortic stenosis.¹⁵ Additionally, cerebrovascular smooth muscle cells of neural crest origin are thought to be involved in the pathology of Alzheimer’s disease in the Down Syndrome brain.¹⁶ Therefore, techniques to take iPSCs and differentiate them into specific lineages of vascular smooth muscle has become of great importance in the field, and will enhance our knowledge of cardiovascular disease pathology.

Touched on briefly in the statement is the development of three dimensional platforms to assess cardiovascular disease biology. Formation of vascular tissue rings¹⁷ and tissue engineered blood vessels¹⁸ has been established in the field, and provides an opportunity to study vascular disease mechanisms while EHTs can be used as a platform to model cardiac diseases. These three dimensional approaches are useful as the cells are arranged in a network so as to more closely represent in vivo models through an in vitro human cell based approach. Engineered blood vessels could additionally be used therapeutically to replace current cardiovascular surgeries involving vessel bypass or stents, while EHTs could be used as beating conduits in the treatment of single ventricle anomalies or to replace damaged myocardium. Such therapeutic approaches could feasibly be done on either a patient-specific level or with a decellularized allogeneic scaffold for universal use. Both approaches would take advantage of the body’s own mechanisms of remodeling while neither should face overt immune rejection.

iPSCs continue to be an exciting and expanding field for cardiovascular disease modeling, and as our knowledge base grows, clinically relevant findings will reduce the health burden of these diseases. The ability for iPSCs to be derived and assess cardiovascular disease from multiple angles, and the separation from the confounding variables due to across species variation, will allow for effective and efficient analysis of disease mechanisms and streamline precision medicine therapies.