Human induced pluripotent stem cell-based microphysiological tissue models of myocardium and liver for drug development

Drug discovery and development to date has relied on animal models, which are useful but are often expensive, slow, and fail to mimic human physiology. The discovery of human induced pluripotent stem (iPS) cells has led to the emergence of a new paradigm of drug screening using human and disease-specific organ-like cultures in a dish. Although classical static culture systems are useful for initial screening and toxicity testing, they lack the organization of differentiated iPS cells into microphysiological, organ-like structures deemed necessary for high-content analysis of candidate drugs. One promising approach to produce these organ-like structures is the use of advanced microfluidic systems, which can simulate tissue structure and function at a micron level, and can provide high-throughput testing of different compounds for therapeutic and diagnostic applications. Here, we provide a brief outline on the different approaches, which have been used to engineer in vitro tissue constructs of iPS cell-based myocardium and liver functions on chip. Combining these techniques with iPS cell biology has the potential of reducing the dependence on animal studies for drug toxicity and efficacy screening.


Introduction
Current methods to evaluate drug safety and effi cacy are costly, ineffi cient, and rely on nonhuman animal models. Th e human body is an extremely complex system and even advanced animal models do not fully mimic the human physiology or recapitulate human disease states. Th e data obtained with animal models therefore cannot necessarily be extrapolated to human subjects. For example, in 2004 the US Food and Drug Administration reported that 92 out of every 100 drugs that successfully passed animal trials subsequently fail human trials [1]. Moreover, out of every 10,000 compounds that go through research and development, approximately 5 to 10 drugs make it to the clinical trials and ultimately only one receives US Food and Drug Administration approval [2]. Th e average time for this process is about 10 to 15 years and costs around $800 million to $1 billion. We and others have hypothesized that all of the aforementioned issues can be addressed by human diseasespecifi c in vitro microphysiological systems that reconstitute organ-level functions at the tissue level.
Advances in stem cell biology and microfl uidic fabrication technology have established the foundation to develop organ-inspired high-throughput in vitro assays. A critical breakthrough in biology was the discovery of human induced pluripotent stem (iPS) cells [3,4], which can be used for disease modeling and drug toxicity screening. Additionally, human iPS cells can be continuously expanded in culture in an undiff erentiated state and then diff erentiated into diff erent lineages; for example, cardiomyocytes (CMs), hepatocytes, adipocytes, or neurons [5]. By creating physiologically relevant microenvironments in microfl uidic devices and utilizing human iPS cells, it is now possible to establish diff erent three-dimensional tissue models, which can be used as drug screening systems ( Figure 1). Of these, heart and liver models are of utmost signifi cance (see Table 1). Cardiotoxicity accounts for about one-third of pharmaceuticals withdrawn because of safety issues [6], and the liver is the major site for drug metabolism and profoundly impacts the fi nal eff ect of the drug. In vitro dynamic physiological systems combining heart and liver tissue will thus make the drug discovery process more economic, predictable, and effi cient.

Abstract
Drug discovery and development to date has relied on animal models, which are useful but are often expensive, slow, and fail to mimic human physiology. The discovery of human induced pluripotent stem (iPS) cells has led to the emergence of a new paradigm of drug screening using human and disease-specifi c organ-like cultures in a dish. Although classical static culture systems are useful for initial screening and toxicity testing, they lack the organization of diff erentiated iPS cells into microphysiological, organ-like structures deemed necessary for high-content analysis of candidate drugs. One promising approach to produce these organ-like structures is the use of advanced microfl uidic systems, which can simulate tissue structure and function at a micron level, and can provide high-throughput testing of diff erent compounds for therapeutic and diagnostic applications. Here, we provide a brief outline on the diff erent approaches, which have been used to engineer in vitro tissue constructs of iPS cell-based myocardium and liver functions on chip. Combining these techniques with iPS cell biology has the potential of reducing the dependence on animal studies for drug toxicity and effi cacy screening.

Systems for high-content drug screening
Classical cell culture systems based on multiwell culture dishes can in principle be used for drug testing, but are not amenable to physiological perfusion required by organ-like cultures. Although the integration of electrophysiological measurement capacity and advanced compu tational frameworks have enabled automatized and parallelized drug screening [6], multiwell culture systems are limited in terms of biomimicry. Using microfl uidic technologies it is possible to create high-content drug screening systems, which simulate tissue structure and function at a micron level and bridge the gap between the in vivo and in vitro worlds. Bioinspired microfl uidic environ ments allow spatiotemporal control of various chemical and physical culture conditions that are unavailable with other methods, which in turn allow for manufacturing of more physiologically relevant drug screening platforms. Microfl uidic approaches also off er a number of advances, including a high degree of parallelization, miniaturization of large systems for convenient operation and reduction of reagent use, and unprecedented control of system architecture and dimensions [7,8]. Furthermore, because fl uid fl ow in microfl uidic channels is laminar, it can be mathematically modeled to make theoretical predictions of complex biological problems. Th ese mathematical models coupled with experimental analysis provide a robust system for understanding the physiological complexity of micro-organ function in vitro, and facilitate promising approaches for addressing many biological problems, including organon-chip arrays for drug screening and disease modeling.
In recent years, microfl uidic systems and microdevices have been employed for diff erent purposes involving CMs and hepatocytes. A recent study employed a microfl uidic system featuring hydrogel-coated channels to culture primary rat CMs in two dimensions [9]. Other applications included the modeling of specifi c conditions, such as ischemia/hypoxia [10]. Lee and coworkers have designed and tested an artifi cial liver sinusoid that mimics the three-dimensional organization of hepatocytes and employs an endothelial-like barrier to control the diff usion of nutrients and drug delivery [11]. By improving cell-cell contact, the structure allowed better maintenance of high-density three-dimensional hepatocytes. Micropatterning of human hepatocytes with fi broblasts or other stromal cells has been reported to improve hepatocyte function and allow hepatocyte prolifera tion [12]. We propose that for high content drug screening it is critical to create three-dimensional tissue constructs from normal and disease-specifi c human iPS cells to obtain faithful in vitro models that are predictive of in vivo conditions.

Tissue models of the myocardium and liver
To generate engineered three-dimensional tissue constructs in vitro, several diff erent methods have been proposed in the literature. In the case of heart tissue, the most proposed three-dimensional engineered heart tissue consists of natural-derived materials such as collagen and Matrigel™ with CMs, and in some cases fi broblasts and vascular endothelial cells embedded in natural materials such as collagen and Matrigel™ [13][14][15]. While these tissues physically contract in vitro, there is no proper organization of the cardiac cells. To address this lack of organization a few groups have placed neonatal rat CMs onto electrospun-aligned fi bers and showed that align ment improved anisotropic electrophysiological proper ties as compared with monolayers or unorganized CMs [16,17]. Although electrospinning permits fabrication of aligned fi bers, the low porosity limits cell infi ltration and does not create threedimensional tissues, hence the resulting aligned tissues are thin monolayers. Recently, one group has employed mouse embryonic stem cell-derived CMs and neonatal rat ventricular CMs to make a two-dimensional anisotropic cardiac muscle tissue by microfabrication of fi bronectin layers [18,19]. Combining this patterning method with cantilever-type microchips, these researchers were also able to quantify the exerted forces [20]. Yet, while they successfully showed that CMs aligned in the two-dimensional structure and generated contractile force, the model is not scalable to generate a threedimensional thick volume tissue. Similarly, nanoscale topography in two dimensions has been shown to aff ect anisotropic action potential propagation and tissue contractility of CM monolayers [21].
In the case of previously published eff orts to create an in vitro liver model, similar issues and limitations apply. Th e liver is a vital organ for metabolism and detoxi fication. To develop an in vitro liver model for drug screening, recapitulation of hepatocyte polarity and function and viability have been of main interest, and various approaches toward achieving these goals included cell-matrix interactions, cell culture format, extracellular mechanical properties and mass transport. Th e sandwich culture, where monolayered hepatocytes are placed between two layers of matrix such as collagen or Matrigel™, shows a cellular polarity in response to the extracellular environment and improved hepatic function and viability. For high-throughput screening applications, eff orts are underway to achieve three-dimensional culture of hepato cytes. For example, hepatocyte spheroids were constructed in suspension in a high-throughput manner [22], which maintained a more diff erentiated state as compared with monolayer culture. Th ree-dimensional hydro gels also have been extensively used to mimic the native liver niche (for example, porosity, mechanical modulus) and thus improve functionality and viability of the liver tissue construct in vitro [23]. In addition, extracellular matrix materials have been proven useful for encapsulating hepatocytes for the construction of threedimensional liver tissues in vitro. Collagen and glyco samino glyans are the main extracellular matrix components of the human livers, and provide mechanical integrity to the liver tissues and present several bioactive signals to cells [24]. Th e absence of suffi cient interactions with the extracellular matrix often causes cells to rapidly de-diff erentiate and die [25]. Furthermore, when engineer ing artifi cial cellular environments, it is impor tant to provide hepatocytes with quantities and qualities of oxygen and nutrients similar to the highly vascularized native liver [26][27][28].
In summary, most of the current in vitro cardiac and liver models are either two-dimensional aligned structures, three-dimensional nonaligned models, or not scalable to high-throughput drug screening. Consequently, an artifi cial environment able to support a functional three-dimensional myocardium and liver within a microdevice is one of the critical steps for eff ective and effi cient drug screening.

Future outlook and challenges
Th e urgent need for in vitro microphysiological systems has led to a steep growth of this intriguing new fi eld, and many promising approaches and inventive systems have been presented in recent years. However, there are several challenges. First, selecting the right cell source for experiments is critical. Most studies to date are based on neonatal rodent-derived CMs, which do not mimic the human CMs due to metabolic diff erences between humans and animals. Clearly, systems using cells that represent human cardiac tissue are required. Recent developments in diff erentiating CMs from human iPS cells open the possibility of obtaining such human CMs in the laboratory [29]. For iPS-derived hepatocytes, it is important to establish robust diff erentiation methods, which allow stable in vitro function, genomic stability, and scalability. Second, integrating multiple micro-organs on a single chip with physiological conditions and scaling those micro-organs as per the laws of biology will be challenging. In the human body, organs are interconnected via the circulatory system, and a drug designed for the heart may have off -target toxicity in the liver. Integrated in vitro micro physiological systems of human cardiac and liver tissue will not only serve as parallelized cardiotoxicity and liver toxicity tests, but also capture off -target toxicity. However, integra tion is not straightforward because each organ will have to be scaled in accordance to its size and function, and we do not know the minimal organoid size that satisfactorily recapitulates the drug response of the native organ. Although there are multiple approaches to address these issues -for example, allometric scaling, scaling based on histological sections, or functional scaling -none has been validated using multiple organoids on a chip [30].
Finally, when successfully integrated, a universal culture medium that mimics blood will be required to maintain tissue viability and function. Th is medium should be able to transport various dissolved gases -for example, carbon dioxide and oxygen -and should provide all of the necessary nutrients, growth factors, and proteins in a physiologically relevant manner to the integrated tissue system.
In summary, microengineered three-dimensional in vitro platforms for high-content drug screening for therapeutic and toxic eff ects have a realistic potential to reduce reliance on animal models and complement, if not to ultimately replace animal studies. Th e success, however, will depend on how eff ectively the engineering and the biology can be integrated to create a clinically relevant in vitro organ system. Abbreviations CM, cardiomyocyte; iPS, induced pluripotent stem.