Cardiovascular Disease: an alarming health problem
Cardiovascular disease remains one of the main causes of morbidity and mortality worldwide, leading to more than 17.5 million deaths worldwide per annum [
As the disease remains one of the most prevalent medical conditions throughout the UK, the demand for new pharmaceutical drugs is soaring. Research in the realm of cardiovascular drug discovery and development is ongoing, however many challenges are yet to be overcome. The lack of appropriate cardiac disease models for toxicology testing has provoked the need for a novel system that is scalable, reproducible and from an inexhaustible source.
The drug discovery and development process is often long, complex and expensive; however innovative research in the field of medical and pharmaceutical biotechnology has led to new advances in the use of human pluripotent stem cells. This review aims to evaluate the promising opportunities that pluripotent stem cells give rise to in the new era of cardiovascular medicine.
Cardiovascular Disease: the need for change in the drug industry
The cardiovascular drug industry has been plagued, over the past decades, with a large volume of molecular targets thanks to the sequencing of the human genome and the reliance on high-throughput screening (HTS) in order to identify and optimise drug candidates. However this reductionist approach, of one-target one-drug one-effect paradigm, is associated with numerous flaws [
Presently the reliance on immortalized cell lines - animal models of human disease and clinical trials - are consistent with the drug discovery process. This process has often been portrayed as a long, expensive and complex journey. Together with disappointing advances, the drug industry is facing increasing and growing concerns over the efficacy and safety of drug discovery. This has prompted and encouraged the drug industry to re-consider how new medicines are developed. One highly promising offering is the development of a more physiologically relevant in vitro model, which harbours the potential for enhancing the likelihood for successful translation of preclinical discoveries into clinical treatment. By using an improved testing platform the incorporation of the complexity of the clinical human disease phenotype can be achieved, addressing and potentially overcoming the limitations of current testing platforms [
Drug discovery and toxicology: seeking a reliable test system
It would be immensely valuable if a reliable test system, which would allow for the identification and characterisation of potential drug targets; the screening of compound libraries for the selection of drugs with desired effects; the detection of drug candidates; and the identification of potential associated adverse effects, could be developed for CVD [
The limited regenerative capability of human cardiac tissue, and difficulties in both obtaining and propagating cardiomyocyte cells out with the human body for drug testing, has provoked the need for new methodological developments for cultivating cardiomyocytes in vitro. Currently, studies investigating novel therapeutics for CVD involve the use of animal models. There are, however, both scientific and ethical problems associated with their use. In addition, primary myocytes derived from animal models, including rodents, have been used as models of cardiac response. However, as with human cardiac cells, they are limited in their ability to survive in culture media out with the body. The use of animal models in drug testing is also limited due to the physiological differences between animals and man. For instance, when drugs are administered orally, the main site for absorption is the small intestine. The half-time for stomach emptying in rodents is approximately 10 minutes, whilst in comparison humans have an estimated stomach emptying half-time of 1-2 hours. This is considerably longer and therefore drug absorption is significantly quicker in animals compared to humans [
Over the years, concerns have been increasingly expressed regarding the progression attrition of pharmaceutical products in the long pipeline between “hit” identification and the market [
Stem-cell-derived cardiomyocytes: paving the way to safer and more effective cardiovascular medicines
The intriguing and exciting prospects for stem cells with properties of self-renewal, clonogeneticity, and multi-potentiality, have been hailed for many years to offer enormous potential in the study of disease and have generated great expectations in relation to possible applications [
As both hESCs and hiPSCs originate from a human source, they provide a more reliable representation of human cardiovascular tissue compared to animal tissue, offering physiological relevant expression, metabolism and responses. Stem cell technology also allows the propagation of cardiomyocytes to a high quantity, providing a readily available source of human cardiomyocytes opening new doors for the study of cardiovascular disease. The limited cell derivation and cell number in cardiac tissue has often caused difficulties in the study of cardiovascular disease in existing cardiac cell models. Moreover, with access to differentiated cardiomyocytes from patients with various cardiovascular diseases or healthy human cells engineered to consist of disease specific genetics, paves the way for greater predictive accuracy in pharmaceutical research, pre-clinical studies and toxicity assessment.
A well-known, however not yet resolved issue is the warranted safety of novel drug compounds within the drug industry. However, the development of a toxicity screen with human pluripotent stem cells offers a promising solution in providing a far greater indication of a drug’s potential toxicity than conventional animal models. The fear of replicating drugs such as thalidomide, tested with no effect on prenatal development on rodents but presenting lasting devastating defects on the development of several children [
The potential of human embryonic stem-cell-derived (hESC) cardiomyocytes
The revolutionary identification of mouse embryonic stem cells (mESC) by Professor Martin Evans in 1981 initiated the development of stem cell research and lead to the identification of human embryonic stem cells (hESCs) [
hESCs are derived from human blastocyst-stage embryos that are surplus to the requirements of in vitro fertilisation and have the capacity to self-renew indefinitely and provide a readily available source of all cell types of the human body as they have the ability to differentiate into all three embryonic germ layers, including the endoderm, mesoderm and ectoderm [
hESCs are most frequently used in cell replacement therapy, including bone marrow transplantation in the treatment of leukaemia. However recent advances have demonstrated the successful application of embryonic stem cells in cardiovascular drug discovery and testing, as well as disease modelling. In order to evaluate the efficiency of a new drug at preventing or reversing a CVD process, the hESCs are firstly differentiated into a cardiomyocyte cell line (hESC-CM). The targeted disease is then induced within the cells, usually through transfection using DNA plasmids to generate transgenic cells. This creates a model, which is similar to that of a transgenic animal; however through the use of hESC-CMs the ethical considerations that accompany the use of animals are avoided (Figure 1).
iPSCs – transforming the outlook of cardiovascular diseases
In 2007, the discovery of induced pluripotent stem cell (iPSC) technology was considered a milestone in regenerative medicine and offered a novel opportunity to model cardiovascular disease and evaluate new therapeutics. Since the concept of iPSC technology was first explored, approximately seventy hiPSC models of rare and complex human diseases have been published and current momentum is further increasing this [
iPSC technology has unprecedented potential to reconfigure genetically identical tissues whether healthy or in a disease state to screen possible therapeutic compounds to determine efficacy in particular diseases. The scope for modelling disease using iPSCs is greatly diverse. iPSCs differential capacity to become all cell types of the body serves as a potentially unlimited ex vivo source of human derivatives. The genetic diversity offers a magnitude of distinct disease phenotypes, some of which may not be possible in animal models. This will facilitate a powerful insight into mechanisms behind an array of diseases allowing for the effective study in the view of developing diagnostic or therapeutic applications [
Current applications of human pluripotent stem cells in drug screening
Studies into the effects of various compounds for preventing and reversing CVD in cardiomyocytes, derived from hESCs, is currently ongoing. The research involves the differentiation of stem cells into individual mini-hearts, composed of cardiomyocyte cells. This has provided grounding for cardiovascular drug discovery and development testing to be carried out [
Previous research has identified the involvement of the CDK9-related kinase pathway as a key factor in the induction of cardiomyocyte hypertrophic growth (Figure 2). The study suggests that the positive transcription elongation factor b (P-TEFb), which consists of several regulatory subunits, including CDK9, is involved in the disease process when a ‘normal’ healthy heart becomes hypertrophied. The catalytic CDK9 component of the P-TEFb subunit was found to play a key role in the hyper-phosphorylation of the enzyme RNA polymerase II (pol II), in response to hypertrophic stimuli, and trigger transcriptional elongation and ultimately lead to hypertrophy. It has therefore been suggested that cardiac hypertrophy can be blocked through the use of small CDK9 inhibiting molecules [
Research into the effectiveness of CDK9 inhibiting compounds as pharmaceutical drugs for targeting P-TEFb-dependent cardiac hypertrophy, has previously been performed using murine models [
The use of Roscovitine, a drug known to inhibit CDK-related activity in tumour cells, has demonstrated positive results in the prevention of cardiac hypertrophy [
Furthermore, new stem cell technologies such as in vitro-differentiated human pluripotent stem cell (IVD hPSC)-derived mini organs, have the potential to transform the drug discovery process [
The array of human disease modelled using hiPSCs is forever growing with a current differentiation repertoire of over 200 types of somatic cells [
Numerous proof-of-principle examples of late have been reported of IVD hPSC-cells from patients with monogenic disorders or engineered disease gene mutations recapitulating disease phenotypes in vitro [
Obstacles in the face of stem cell technology
One of the greatest obstacles to overcome when using hESCs in drug discovery and development is the control of differentiation. As embryonic stem cells are undifferentiated, it is often difficult to control the exact differentiation of the cells. This means that, due to chromosomal abnormalities, the cells could potentially become tumorous if they begin to grow and divide uncontrollably. It has been suggested that hESCs should be molecular cytogenetically characterised in an attempt to understand tumour initiation and progression. [
Limitations of hESCs lies with the high expense associated with propagating and maintaining the cells, as well as the extensive time required to successfully differentiate/mature the stem cells into cardiomyocytes. Many have also doubted the suitability of iPSCs technology in drug development simply due to time constraints as the production of the desired cell type can be a protracted process which conflicts with the fast moving drug development industry. Moreover iPSC technology also faces legal obstacles - complexity in designing appropriate consent forms for the use of immortalised cell lines [
Through time, hESCs may develop uncontrollable karyotypic changes that result in physiological changes. Ultimately, the cells may undergo genetic selection and adaption to the in vitro culture media environment. hiPSCs have an extremely low efficacy to harvest, and are therefore increasingly expensive to reprogramme, cultivate and maintain compared to hESCs and animal cell lines. Another line of thought is that the phenotypes expressed by hiPSCs-derived cells are developmentally immature compared to their in vivo-derived counterparts and may not exhibit full functionality [
As hiPSCs originate, and are reprogrammed, from somatic cells that are already differentiated, any existing genetic mutations within the patient’s genome will be passed through the hiPSCs. The presence of a secondary genetic condition could potentially affect the responsiveness of the cell model to drug treatment [
With a large volume of protocols inevitably a large level of variance will follow in relation to repeatability and robustness in generating scalable, homogenous cell populations. In contrast the IVD protocols for numerous cell types are lacking. The lack of standardisation is an issue and one that needs to be resolved if pluripotent stem cell technology is to become an integral part of drug development [
Fulfilling the potential of stem cell technology in the drug development industry necessitates the standardisation of protocols. Once achieved the possibilities and applications for stem cell technology are vast.
Using human pluripotent stem cells in cardiovascular medicine not only addresses the contentious issue of animal models but offers great scalability with the potential of high-throughput screening. The use of patient specific hiPSCs on a small scale could allow for the identification of side effects prior to the drug being administered to the patient. However, a more feasible approach for the use of hiPSCs in drug toxicity is on a large-scale due to the current expense of creating and maintaining pluripotent stem cell lines [
In 2008, the President’s Council of Advisors on Science and Technology (PCAST) articulated the concept of “personalised medicine”. Personalised or precision medicine explores the notion of designing a medicine based on a patient’s genetic make-up and specific disease characteristics in the hope of increasing therapeutic benefits while reducing the risk of adverse effects [
iPSC technology affords an opportunity to model patient-specific iPSCs derived from individuals with known susceptibilities or resistances to different drugs or diseases to assess and predict patient drug responses. This “patient in a dish” concept can expand further to uncover the niche of genetic and potential epigenetic factors that influence the variable drug responses witnessed within the population and within different subsets of patients [
The benefit to the drug industry financially, through the use of human pluripotent stem cells-derived cardiomyocytes offering a more sensitive assay for candidate drug toxicity and safety compared to previous conventional methods, is recognised. hiPSCs or hESCs screening has the potential to lessen the costly recall of already approved drugs and promote the development of a new generation of safer drugs through an alternative and less expensive strategy [
In addition to introducing low costs of development, human pluripotent stem cells offer the potential to increase the production of candidate compounds in a shorter turnaround time with the detection of life-threatening toxicity in a multitude of tissues. Of particular interest is the ability of iPSCs to recapitulate the process of development from embryo to adult tissue, offering a niche to test drug toxicity in developing tissues and opening a new possibility for safety testing for teratogenicity [
The field of cardiovascular research is advancing rapidly and research into the development of successful therapeutic drug treatments is ongoing. The development of human pluripotent stem cells for use in the field of medical and pharmaceutical research has revolutionized the drug discovery process. By using human derived stem cells, we are now able to gain a deeper understanding of the effects of drug compounds on the human system prior to human clinical trials. Although research into the uses of stem cells in drug discovery, and more specifically cardiovascular research, is still under investigation, the high reproducibility, readily available source of cells and potential to generate into multiple cell types suggests promising outcomes for the future of stem cell research.
- Ezzati M, Obermeyer Z, Tzoulaki I, Mayosi BM, Elliot P, Leon DA. Contributions of risk factors and medical care to cardiovascular mortality trends. Nat Rev Cardiol 2015, 12(9): 508-30.
- Engle SJ, Puppala D. Integrating Human Pluripotent Stem Cells into Drug Development. Cell Stem Cell 2013; 12(6): 669-675.
- Takebe T, Taniguchi H. Human iPSC-Derived Miniature Organs: A Tool for Drug Studies. Clin Pharmacol Ther 2014; 96(3): 310-313.
- Inoue H, Yamanaka S. The Use of Induced Pluripotent Stem Cells in Drug Development. Clin Pharmacol Ther 2011; 89(5): 655-661.
- Ko HC, Gelb BD. Concise Review: Drug Discovery in the Age of the Induced Pluripotent Stem Cell. Stem Cells Transl Med 2014; 3(4): 500-509.
- Sinnecker D, Laugwitz KL, Moretti A. Induced pluripotent stem cell-derived cardiomyocytes for drug development and toxicity testing. PharmacolTher 2014; 143(2): 246-252.
- Stoyanova V, Zhelev N, Ghenev E, Bosheva M. Assessment of left ventricular structure and function in rats subjected to pressure-overload hypertrophy in time. Kardiol Pol 2009; 67(1): 27-34.
- Stoyanova V, Zhelev N. Alterations in protein P53 expression during the development of pressure overload-induced left ventricular hypertrophy in rats. Biotechnol. & Biotechnol Eq 2008; 22(4): 977- 983.
- Stoyanova V.K, Zhelev N.Z., Yanev I.B, Ghenev E.D, Nachev C.K Time course and progression of pressure overload-induced cardiac hypertrophy in rats. Folia Med 2005; 47(2): 52-7.
- Nau H. Species differences in pharmacokinetics and drug teratogenesis. Environ Health Perspect 1986; 70: 113-129.
- Lin JH. Species similarities and differences in pharmacokinetics. Drug Metab Dispos 1995; 23(10): 1008-1021.
- Arabadjiev A, Petkova R, Chakarov S, Momchilova A, Pankov R. Do We Need More Human Embryonic Stem Cell Lines? Biotechnol. & Biotechnol Eq 2010; 24(3): 1921-1927.
- Petkova R, Arabadjiev A, Chakarov S, Pankov R. Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse? Biotechnol. & Biotechnol Eq 2014; 28(2): 184-191.
- Arabadjiev A, Petkova R, Chakarov S, Pankov R, Zhelev N. We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells. BioDiscovery 2014; 14: 2.
- Liu W, Deng Y, Liu Y, Gong W, Deng W. Stem Cell Models for Drug Discovery and Toxicology Studies. J Biochem Mol Toxicol 2013; 27(1): 17-27.
- Evans M. Discovering pluripotency: 30 years of mouse embryonic stem cells. Nat Rev Mol Cell Biol 2011; 12(10): 680-686.
- Arabadjiev B, Petkova R, Momchilova A, Chakarov S, Pankov R. Of mice and men – differential mechanisms of maintaining the undifferentiated state in mESC and hESC. BioDiscovery 2012; 3(1).
- Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282(5391): 1145-1147.
- Zhelev NZ, Tummala H, Trifonov D, D'Ascanio I, Oluwaseun OA, Fischer PM. Recent advances in the development of cyclin-dependent kinase inhibitors as new therapeutics in oncology and cardiology. Curr Opin Biotechnol 2013; 24(1): 25.
- Maron BJ. Hypertrophic Cardiomyopathy: A systemic Review. JAMA 2002; 287(10): 1308-1320.
- Schneider MD, Sano M, Abdellatif M, Oh H, Xie M, Bagella L et al. Activation and Function of Cyclin T-Cdk9 (Positive Transcription Elongation Factor-b) in Cardiac Muscle-Cell Hypertrophy. Nat Med 2002; 8(11): 1310-1317.
- Romano G. Deregulations in the Cyclin-Dependent Kinase-9-Related Pathway in Cancer: Implications for Drug Discovery and Development. ISRN Oncol 2013; 2013: 305371.
- Cherrier T, Douce V, Eilebrecht S, Riclet R, Marban C, Dequiedt F et al. CTIP2 is a Negative Regulator of P-TEFb. Proc Natl Acad Sci U S A 2013; 110(31): 12655-12660.
- Zhelev NZ, Trifonov D, Tummala H, Clements S. Effect of Roscovitine on Cardiac Hypertrophy in Human Stem Cell Derived Cardiomyocytes. Curr Opin Biotechnol 2013; 24(1): 114-115.
- Zhelev N., Tupone MG., Cecceroni L., Cavicchi L., D'Ascanio I., Khalil HS., Uth K., Mitev V. Discovery and development of Seliciclib. How systems biology approaches can lead to better drug performance. J Biotechnol 2014; 185: 10.
- Khalil, H.S., Mitev V., Vlaykova T., Cavicchi L., Zhelev N. Discovery and development of Seliciclib. How systems biology approaches can lead to better drug performance. J Biotechnol 2015; 202: 40-9.
- Zhelev N., Trifonov D., Wang S., Hassan M., El Serafi I., Mitev V. From Roscovitine to CYC202 to Seliciclib – from bench to bedside: discovery and development. BioDiscovery 2013; 10: 1.
- Liang P, Lan F, Lee AS, Gong T, Sanchez-Freire V, Wang Y et al. Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity. Circulation 2013; 127(16): 1677-91.
- Jung CB, Moretti A, Mederos Y, Schnitzler M, Lop L, Storch U et al. Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. EMBO Mol Med 2012; 4(3): 180-191.
- Matsa E, Dixon JE, Medway C, Georgiou O, Patel MJ, Morgan K. Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes. Eur Heart J 2013; 35(16): 1078-1087.
- Karamysheva TV, Prokhorovich MA, Lagarkova MA, Kiselev SL, Liehr T, Rubtsov NB. Chromosome rearrangements in sublines of human embryonic stem cell lines hESM01 and hESM03. BioDiscovery 2013; 7(1).
- Burridge PW, Keller G, Gold JD, Wu JC. Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming. Cell Stem Cell 2012; 10(1): 16-28.
- Ebben JD, Zorniak M, Clark PA, Kuo JS. Introduction to Induced Pluripotent Stem Cells: Advancing the Potential for Personalized Medicine. World Neurosurg 2012; 76(3-4): 270-275.
- Mordwinkin NM, Lee AS, Wu JC. Patient-Specific Stem Cells and Cardiovascular Drug Discovery. JAMA 2013; 310(19): 2039-2040.