Cardiomyocytes derived from human pluripotent stem cells for drug screening
Introduction
The average time to develop a new drug is between 10 and 15 years and the associated costs can reach the magnitude of one billion USD (Kola & Landis, 2004). The attrition rate of drugs is staggeringly high: more than 90% of the drugs tested in clinical trials fail to be approved. Compounds fail late in clinical testing or even after approval because of a lack of sufficient efficacy or unanticipated toxicity; many more compounds fail early in the discovery process (Rubin, 2008). Disease models that will more faithfully represent the actual human diseases are critical for the two key aspects of developing new drugs: discovering new effective entities and screening for their safety.
Given the high costs incur in development of new candidates and the high rate of drug candidate attrition that occurs late in development, pharmaceutical companies have become increasingly cautious of embarking on new drug development. Thus, the last decade has witnessed a significant decrease in new drug discovery, with only half as many new candidates reported compared to the previous decade (Hughes, 2009). The reason for this is likely multifactorial, but the costs of drug development has increased exponentially. Of particular concern, many of the late attritions of drugs in development have been attributed to unforeseen cardiotoxic side effects of the drugs being tested in humans that were not realized in preclinical animal models (Kola & Landis, 2004).
The heart has proven to be particularly sensitive to off-target, life-threatening toxic effects of non-cardiac drugs. Over the past several years, reports of unexpected drug induced ventricular arrhythmias associated with sudden cardiac death have led to the withdrawal of a number of drugs from the market, while for many others, additional safety labels have been required to warn of potential risks (Braam et al., 2010). The withdrawal of the non-steroidal anti-inflammatory drug Vioxx from the market by Merck in 2004 due to cardiovascular safety concerns illustrates the tremendous cost in lost revenues and patient litigations caused by late-stage candidate failure and underscores the need for better and more predictive models (Bresalier et al., 2005).
Predictive models available during preclinical drug development include in vivo and ex vivo animal models, non-human primary cell cultures and/or immortalized cell lines, all of which fail to fully recapitulate relevant human biology (as will be discussed later). These suboptimal test systems enable the progression of compounds with sub-maximal efficacy and/or increased toxicity along the development pipeline. Recent advances in human stem cell biology have paved the way for incorporation of human cell models into drug discovery and toxicity studies.
The first derivation of human embryonic stem cells (hESCs) from pre-implantation embryos was more than a decade ago (Thomson et al., 1998). These pluripotent cells can be isolated and maintained by well-established protocols, with scalable options, proliferating indefinitely while retaining the ability to differentiate into virtually any cell of the human body; including unquestioned cardiomyogenic potential (Kehat et al., 2001, Xu et al., 2002, Mummery et al., 2003). Another breakthrough took place when Takahashi and Yamanaka (2006) discovered that by the overexpression of four retrovirally-transduced transcription factors (Oct4, Sox2, c-Myc, and Klf4) in mouse dermal fibroblasts grown in conditions favoring ESC expansion, they were able to generate induced pluripotent stem cells (iPSCs). The promise of applying this technology to human cells was rapidly realized with the successful generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts using either the same combination of transcription factors (Takahashi et al., 2007, Park et al., 2008) or a slightly different cocktail of lentivirally-transduced genes (Oct4, Sox2, Nanog and Lin28) (Yu et al., 2007). The iPSCs field continues to progress very rapidly and pluripotency has now been induced by using recombinant virus, recombinant proteins and/or small molecules in many cell types, including those carrying simple or complex genetic diseases (Colman and Dreesen, 2009, Ichida et al., 2009, Zhou et al., 2009, Moretti et al., 2010, Novak et al., 2011). The potential of human pluripotent stem cells (hPSCs), including both hESCs and hiPSCs, as a promising human cells source for the field of drug screening and discovery will be discussed in the current review.
Section snippets
Adverse cardiac effects
The cardiovascular system is a critical focus of drug screening when considering drug safety, since adverse effects on the heart might be not only chronic as for other organs (e.g., liver), but can be reflected immediately and become life threatening. Cardiotoxicity is a general term that describes myocyte damage leading to decreased cardiac function, and arises broadly from electrophysiological and biochemical mechanisms. Nonetheless, acute drug effects on cardiac electrophysiology do not
Cardiac differentiation
Cardiac differentiation of hESCs is mainly achieved by three approaches: (1) spontaneous differentiation through embryoid bodies (EBs) grown in suspension, during which contracting areas containing cardiomyocytes start to appear within a few days (Kehat et al., 2001); (2) co-culture with endoderm-like cells or cells secreting endoderm-like signals (Mummery et al., 2003); and (3) direct addition of growth factors, hormones, or small molecules thought to be involved in heart development to ESCs
Drug testing using human pluripotent stem cells-derived cardiomyocytes
One of the most critical determinants of normal cardiac function is the intact response to hormones and transmitters of the autonomic nervous system. The muscarinic and adrenergic receptors mediate the autonomic effects on the heart (i.e., sinus node firing rate, action potential configuration, contractility), by specifically affecting a variety of ion currents, such as If. For that reason, many reports have documented the response of hESC-CMs to both adrenergic and cholinergic agents, as part
Conclusions
Recent advances in human stem cell biology have paved the way for incorporation of human cell models into drug discovery and toxicity studies, as can be perceived from the above mentioned studies included in this review. Yet, the task is not completed. The efficiency and reproducibility of the differentiation protocols have to be improved and enable cost-effective production suitable for the industry. The laboratory-scale cell-based assays (e.g., patch-clamp, MEA, Ca2+ measurements) have to be
Acknowledgment
This work was supported by the Israel Science Foundation (ISF), the Ministry of Health — Chief Scientist, the Rappaport Family Institute for Research in the Medical Sciences and The Sohnis and Forman Families Stem Cells Center.
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