In this review, we provided an overview from the simplest to the most sophisticated human cellular models for cardiovascular research (Figure 1
The use of heterologous systems for the genetic manipulation of specific genes has dramatically fueled scientific research, allowing the comprehension of the functions of these genes in their healthy or pathologic version. Such observations occur in an isolated and highly reproducible context allowing the comparison of different mutations of the same protein in the same genetic setting. Nevertheless, these models lack the genetic and epigenetic backgrounds of the patients, which often influence the resulting phenotype, and are not useful for polygenic diseases or for acquired disorders. In general, any CVD in its entirety could not be studied on heterologous systems, which lack the molecular scenario and morphologic features of cardiovascular cells.
This is also true for patient-derived non-cardiovascular cells, useful when they express the same mutant proteins associated to CVDs. These cells can represent an additional model for mechanistic studies and drug discovery in large patient cohorts, thanks to their easy accessibility with non-invasive sampling techniques. However, despite the advantage of the direct derivation from patients, their non-cardiac origin restricts the range of investigation.
As previously mentioned, primary cell models are the most intuitive method for studying a CVD. However, adult cells carrying patient’ genetic background are poorly available. Moreover, samples are often obtained from subjects in advanced state of morbidity, making mechanistic studies hard to be finalized. Some primary cells, especially CMs, possess a short lifespan and cannot be amplified in vitro, limiting the number of experiments and the variety of assays.
Over the years, researchers tried to overcome these obstacles exploiting the expression of oncogenes to immortalize post-mitotic cells, forcing their cycle re-entry and the consequent proliferation. These attempts started from animal immortalized cardiac cells, such as mouse HL-1, to human with AC 16 line [50
]. The human ventricular AC 16 cells present the great benefit to be proliferative CMs, but, owing the SV40 transformation, they show some defects, such as expression of atrial markers, a pre-contractile stage, lack of many inward and outward currents, which do not start adequate action potentials, and they do not entirely recapitulate the cellular context of CMs [50
All these issues were partially solved with the introduction of hESCs. Their ability to proliferate for extended periods in culture and to differentiate towards tissue derivatives from the three germ layers, together with the possibility to generate recombinant hESC lines with different mutations by gene targeting techniques, promised a revolution in the comprehension of developmental processes, pathological molecular mechanisms, drug discovery and cell therapy. In the years following their isolation, numerous protocols for efficient differentiation were developed and refined, but ethical problems associated with the use of embryos for hESCs derivation and hiPSCs discovery restricted the use of hESCs as a disease model. hiPSCs made cardiac and vascular cells even more accessible, thanks to the possibility to generate pluripotent stem cells similar to hESCs, by reprogramming somatic cells from healthy donors and from patients affected by specific CVDs. hiPSCs allow to investigate genotype-phenotype relationships in monogenic, polygenic and genetically unknown conditions. A critical argument is the choice of controls: in order to avoid as much as possible the genetic variability between patients and control cells, the best option is the generation of an isogenic control, correcting the mutation in the patient hiPSC line through gene targeting approach [131
]. To date, cell types deriving form pluripotent stem cells show an immature phenotype. This is particularly true for CMs, whose maturation is now improved with various methods, including anisotropic signals, electromechanical conditionings, biochemical modulations, multiple cell type co-cultures [132
]. Co-cultures allow the generation of a more complex tissue, which better mimics the microenvironment of origin, thanks to the influence of extracellular matrix and cells that normally coexist in the tissue. New technologies have allowed to recreate this microenvironment in 3D constructs, mimicking the cell-cell and cell-matrix mechanics and structural organization observed in vivo like never before. In particular, OOC, together with 3D bioprinting technics, emerged as the most advanced methods to develop 3D tissue models in vitro with opportune anatomical and pathophysiological features [134
]. The resemblance to human organs reached with these novel strategies allows a more complete comprehension of pathogenic mechanisms and provides a more efficient tool for drug discovery. Furthermore, the recent concept of body-on-a-chip aims to reflect the interaction among organs in vivo, combining multiple OOC in a unique integrated system for various potential biomedical applications, such as disease modeling, drug discovery, biomarker detection [135
]. In this way, it could be possible to observe the role of other organs, such as nervous or respiratory systems, which are closely interconnected with heart and vessel physiology, and sometimes participate to pathogenesis of specific CVDs.
The generation of advanced and easily reproducible disease models, along with epigenetics, next generation sequencing and ‘omics’ studies, improves the comprehension of CVDs, leading to the development of personalized medicine in vitro, for both individuals and patients’ sub-populations [138
]. Different conditions, such as cardiac arrhythmias, cardiomyopathies and vascular hypertension, may show a similar phenotype despite different specific causes, with consequent distinct drug response. In these cases, differential diagnosis, risk stratification, and personalized treatments become necessary, in order to refine the understanding of genotype-phenotype relation and to implement the efficacy of clinical research [139
As previously mentioned, the fabrication of engineered 3D tissues, increasingly sophisticated and close to reality, provides flexible tools that also aims to replace various animal-based studies, reducing their use in laboratory to model CVDs. At the same time, in vivo models are necessary for the validation of 3D constructs before applying them on humans, since a future prospect is their application in regenerative medicine as biologically relevant patches for tissue repair [143
]. Moreover, pluripotent stem cells could become a tool for clinical applications: a lot of studies on cardiac regenerative therapy were carried out, showing improvements in cardiac function after transplantation in animals [146
]. Nevertheless, some factors must be considered, indeed, in vivo, hESCs cause an immune response and require immunosuppression, unlike hiPSCs, which can be derived from the specific patient. Moreover, because of the immature phenotype, the differentiation has to be improved, in order to obtain only terminally differentiated cells, avoiding teratoma formation [149
Obviously, further studies are needed before the application to the clinical practice will be a reality. Emerging technologies will allow in the future an increasingly precise understanding of the pathophysiology of CVDs and consequent therapeutic advancements.