HeLiVa platform: integrated heart-liver-vascular systems for drug testing in human health and disease

Our project team is developing an integrated microphysiological platform with functionally connected vascular, liver and cardiac microtissues derived from a single line of human pluripotent stem cells. The platform enables functional representation of human physiology in conjunction with real-time biological readouts (via imaging and homologous reporters for all three cell phenotypes) and compatibility with high-throughput/high-content analysis. In this paper, we summarize progress made over the first year of the grant.

drug development failures are due to unforeseen absorption, distribution, metabolism, excretion, and toxicity profi les that might have been predicted if models of human liver tissue were available earlier in drug development [6]. Likewise, existing heart models of cardiotoxicity do not always correlate with clinical risks. For example, preclinical hERG assays are quite helpful in identifying compounds with action potential prolongation, but are not suffi cient for predicting clinical QT-proarrhythmia [4]. Human microtissues platforms are now under develop ment to improve our capability for disease modeling [7,8] and toxicology studies [9,10].
Overall, the lack of predictive drug-screening systems is a critical barrier to bringing drugs to patients. In spite of major advances, existing culture systems still lack many of the structural and signaling features of native tissues, the temporal and spatial sequences of molecular and physical regulatory factors, and the dynamic forces and systemic factors provided by blood circulation. Even in the best culture settings where distinct cell types of a specifi c organ system are co-cultured to promote tissuespecifi c transport and signaling, the eff ects of organorgan interactions, metabolic/oxygen gradients, and cellular traffi cking through tissues are still not included [9,11]. Also, engineered tissues have only scratched the surface in attempting to model human disease. Animal models often fail to capture human-specifi c features, and off er only limited control of and insight into specifi c mechanisms. As a result, disconnect between in vitro studies, translational animal models, and human clinical studies decreases the eff ectiveness of the resulting therapeutic strategies. Functional human tissue units, engineered to combine biological fi delity with the use of high-throughput platforms and real-time measurement of physiological responses, would be transformative to drug screening and predictive modeling of disease.
Modeling integrated human physiology in vitro is a formidable goal that has not been reached with any of the existing cell/tissue systems. Tissue engineering is now becoming increasingly successful in more authentically representing the actual environmental milieu of develop ment, regeneration and disease progression, and in providing real-time insights into cellular and morphogenic events. While it is unreasonable to expect threedimensional tissue models to exactly match native tissues, the models can still recapitulate certain physiological functions and be used to investigate the effi cacy, safety and mode of action of therapeutic agents. Instead of attempting to achieve the entire complexity of an organ, we seek to identify the simplest functional tissue unit allowing predictive in vitro studies of normal physiology and disease. Such a minimally functional tissue unit would replicate the tissue-specifi c architecture (as a basis for function in most tissues) and a subset of most relevant functions.
Derivation of multiple microtissues (such as heart, liver, vasculature) starting from a single population of human induced pluripotent stem (iPS) cells allows us to reach into a large genotype pool and use both the healthy cells and cells with genetic mutations for drug screening and modeling of disease. Modular microtissue platforms can then capture the salient features of multiple human tissues, by integrating tissue-specifi c cues within the micro tissue unit (to induce physiologic cell function) with the vascular network (to assemble miniature yet functional tissue modules). Th ese microphysiological plat forms can be further interfaced with functional imaging, to enable real-time monitoring of physiological responses at molecular, cellular and tissue levels. Such interactive microtissue systems off er enormous complexity and diversity of responses to drugs and disease that can be modeled (for example, cardiotoxicity of drugs metabolized by liver in a diverse genotype pool of patients, infl ammation, combinations of disease genotypes and disease environments).
We propose that human tissues with utility for microphysiological studies need to combine biological complexity (multicellular composition, normal and disease phenotypes, tissue-specifi c architectures, vascu larization, normal human biology) and compatibility with high-throughput screening platforms (small size, easy handling, online readouts). With this in mind, we are developing integrated tissue platforms that faithfully represent the human vascular network, metabolizing liver lobule and working cardiac muscle

Project goals and objectives
In the current (UH2) phase of work, we are establishing iPS cell-based vascular, liver and cardiac microtissues providing tissue-specifi c architectures with an integrated vascular network, microfl uidic endothelialized connections between the tissue modules, functional representation of human biology of health, injury and disease (real-time biological readouts), and compatibility with high-throughput multi-tissue platforms for studies of drug toxicity and over long periods of time (≥4 weeks). Th e work is being done using iPS cells (to provide a large diversity of normal and disease genotypes) with nondestructive monitoring of the tissue architecture and function (for real-time insights into the progression of biological responses). In the subsequent (UH3) phase of work, we aim to deploy an integrated cardiac-hepaticvascular platform and demonstrate its utility for predictive studies of human physiology.
Towards these goals, we are pursuing a set of coordinated aims with constant feedback and monitoring of the milestones. Aim 1 is to develop cell-type specifi c labeling and sensing systems for online assessment of tissue architecture and cell function. Aim 2 is to develop a perfusable branching vascular network serving as a model of the vascular bed and for assembling vascularized liver and cardiac tissues. Aim 3 is to develop a liver module by assembling hepatic microtissues in hydrogel around the vascular tree. Aim 4 is to develop a cardiac module by assembling matured cardiac microtissues in hydrogel around the vascular tree. Aim 5 is to conduct studies of disease susceptibility, for the individual tissue modules and in the multi-tissue platform. Aim 6 is to investigate human physiology and disease in multi-tissue platforms. Th is way, we aim to develop a new technology for studies of drugs in human tissue models. If successful, the proposed approach would radically enhance the translation of drug discovery into human applications.

Induced pluripotent stem reporter lines of endothelial cells, cardiomyocytes and hepatocytes
Our laboratories have continued interest and strong expertise in biophysical regulation of the fate and function of stem cells and their diff erentiated progeny using molecular, cellular, matrix-derived and physical factors. We will continue to use these approaches for directed diff erentiation of stem cells into endothelial, cardiac and hepatic lineages, and maturation of the resulting diff erentiated cells.
To create clinically relevant human tissue models, we aim to generate all cell types needed for tissue construction from the same batch of iPS cells. In order to allow use of molecular and functional imaging and study physiological processes at multiple hierarchical levels and in real time, we are now incorporating biosensors (reporters) into the iPS cells to monitor specifi c cell phenotypes in culture (for example, distinguish between endothelial cells and cardiomyocytes), and to monitor functional readouts for tissue cells. Th us far, we have established culture conditions for routine and robust generation of endothelial cells that exhibit co-expression of cell surface markers (that is, CD31, VE-cadherin) and functional properties (endothelial nitric oxide synthase production, tube formation) from multiple iPS cell lines with at least 10 to 40% effi ciency [19]. We have recently adopted the culture conditions used by the Bhatia and Vunjak-Novakovic laboratories to maintain the specifi c iPS cell line that we are using to create the reporter lines (iPS C2a). We are now optimizing the conditions for endothelial cell-specifi c diff erentiation of this specifi c cell line. We are now generating reporter cell lines using Talens [28] for homologous recombination in the iPS C2a cells (iPS endothelial cells, VEcad/GFP; iPS cardio myo cytes, beta-MHC/FRFP; and iPS hepatocytes, CYP3A4/RFP).

Perfusable vascular networks
By this time, we have developed a general approach to rapidly construct perfusable vascular networks to Chamber design for a cardiac microtissue forming around a sugar lattice (that dissolves to leave vascular network channels) and two posts (designed to subject cardiac tissue to mechanical strain, and also to serve for optical measurement of force generation by the cells), between two electrodes for electrical stimulation (the whole row of chambers shares a single pair of electrodes). (C) Chamber design for a liver microtissue, also forming around a sugar lattice (that dissolves to leave vascular network channels). For all chambers, ports are provided for fl uid inlets and outlets and sample retrieval. http://stemcellres.com/content/4/S1/S8 support three-dimensional culture of tissues. We printed three-dimensionally the rigid fi lament networks of a carbohydrate glass, and used them as a cytocompatible sacrifi cial template in engineered tissues containing living cells to generate networks lined with endothelial cells and perfused with blood under high-pressure pulsatile fl ow [20][21][22][23]. We have developed several methods to generate channel architectures within matrix scaff olds using either sacrifi cial fi laments printed in sugars [20] or gelatin [22], or using needles that are withdrawn from the material [23]. In all of these cases, seeding human endothelial cells into these channels results in the formation of well-developed endothelium that exhibits characteristics of functional endothelium. We have observed intraluminal fl ows at high rates relative to extraluminal fl ow, and identifi ed tight junctions that were permeable to transluminal leakage only when cells are exposed to vasoactive permeating factors.
To facilitate assembly of the HeLiVa platform, we have also developed a mechanism that allows easy plug-in of input and output microfl uidic tubing to ultimately drive the perfusion within the vascular bed and connect multiple organ systems [22]. In parallel, we have begun to explore methods to integrate the vascular perfusion approaches with the cardiac microtissue platform.

Liver microtissues
We initially focused on generating hepatic microtissues from cryopreserved human hepatocytes, and then extended the established methods to a variety of cell types including iPS cell-derived hepatocytes. An integral part of this eff ort was the development of platforms for scalable control of tissue architectures [24,27]. To help maintain hepatocyte phenotype, we fi rst co-cultured the primary hepatocytes with supportive fi broblasts by seeding the two cell types into an array of pyramidal microwells. Defi ned clusters of hepatocytes and fi broblasts (approximately fi ve hepatocytes and fi ve fi broblasts) in each microwell formed multicellular aggregates over 24 hours. Th ese primary hepatocyte aggregates were then removed, encapsulated in microfl uidically generated droplets of polyethylene glycol diacrylate prepolymer, and photopolymerized to form hepatocyte-laden hydrogel microtissues. Resulting liver microtissues are viable as evaluated by live-dead staining, and exhibit stable phenotypic function, secreting albumin for over 2 weeks. Towards an iPS cell-derived liver module, we have generated morphologically identifi able hepatocytes from human iPS cells with diff erentiation effi ciencies >90%, as quantifi ed by albumin and α 1 -antitrypsin immuno fl uorescence, and identifi ed small molecules for human hepatocyte expansion and iPS cell diff erentiation [16]. iPS cell-derived hepatocytes were additionally validated through characterization of urea production, albumin and α 1 -antitrypsin secretion, and phase I drug metabolism activity. Notably, liver microtissues were successfully used to model hepatitis C infection [25].

Heart microtissues
We established protocols for diff erentiation of iPS cell lines into contractile cardiomyocytes, for three lines of iPS cells. Over the last 6 months we focused on one specifi c line, iPS C2a, which is now shared between all four laboratories. After systematically investigating several variations of staged molecular induction in three diff erent settings (embryoid bodies, monolayers and pseudo-monolayers), we came to our protocol of choice that yields ~65% of diff erentiated cardiomyocytes (as evaluated by Troponin staining and contractile function). By micromanipulation (picking of beating areas), the yield of the protocol can be increased to >80%, as com pared with our target go-no-go criterion of >30%. Simple microfl uidic platforms [12,27] were instrumental in the development of protocols for directed diff er en tiation of stem cells. Th e resulting iPS cardiomyocytes, although functional, remain immature. To promote cardiomyocyte maturation, we applied electromechanical conditioning, following molecular induction (7 days, 5 V/cm, 2 ms square waves, frequency = 0, 0.5 Hz, 1 Hz, 2 Hz). Th e protocols were fully established for human embryonic stem cell cardiomyocytes and are now translated to iPS C2a cardiomyocytes. Electrical stimulation increased the strain of contractions in cardiac microtissues. In parallel, the contractions became synchronized and the strain generated by cardiac microtissues increased as the stimulation frequency increased.

Platform integration
We have developed the second working prototype of our platform, in which we have tested the assembly and perfusion of cardiac microtissues formed from iPS C2a cells, and recently started testing with iPS hepatocytes. Th e culture chambers have a space for the formation of cardiac microtissues from a suspension of iPS cardiomyocyte micro-aggregates in hydrogel. Th e hydrogel forms around the sugar lattice, between two electrodes, and around two posts (that at the same time stretch the cardiac microtissues, and enable optical measurement of the force generated by the cells from the defl ection of the posts). To design the posts (material composition, geometry) we conducted analyses of the strain distribution in contracting cardiac organoids. We are now assembling subsections of the integrated microphysiologic platform, for further testing and for obtaining feedback to the individual chamber design.

Future directions
We have established methods to derive, starting from the same population of iPS cells (using the line C2a as the main experimental model), functional endothelial cells, cardiomyocytes and hepatocytes of high fi delity. We have also developed methods for maturation of diff erentiated cells, using small molecules (for hepatocytes) and physical signals (for cardiomyocytes). Th e fi rst prototypes of microfl uidic platforms for the formation and cultivation of vascular, cardiac and hepatic microtissues are now being used to demonstrate utility of human iPS cellderived microtissues for physiological and pharmacological studies. Our immediate goals are to refi ne our microtissues by incorporating the vascular bed, to establish modular multi-tissue platforms, and to use these platforms in studies of interactive responses of cardiac, vascular and hepatic microtissues to pharmacological agents, physiological and pathological stimuli.

Competing interests
The authors declare that they have no competing interests.