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Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
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5

In Vitro Alternatives to Animal Models

iPSCs TO MODEL CHEMOTHERAPY-INDUCED CARDIOTOXICITY

Paul Burridge, assistant professor of pharmacology at Northwestern University, said that induced pluripotent stem cells (iPSCs) can help answer unique questions that are otherwise inaccessible. For example, iPSCs can be used to predict which patients will experience negative effects from a drug,

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
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identify the genetic cause and pathway of the drug response, and develop drugs to modify that drug response.

In the clinical care of the future, Burridge believes, vast amounts of data from each patient will allow large-scale comparisons and the identification of genetic mutations associated with specific phenotypes. Once a mutation is identified, iPSCs can be used to validate the association, allowing clinicians to predict which patients would suffer negative effects from certain drugs, and researchers to develop therapies based on an individual’s genetic information.

iPSCs are now simple and cheap to produce, said Burridge, and are a powerful tool for genomics. A very small volume of blood (e.g., a small blood draw or a finger prick) is expanded and transformed into pre-iPSCs. Burridge’s lab has developed chemically defined, cost-effective methodologies to generate around 100 iPSC lines annually. The time from blood draw to ready-to-use iPSCs is about 3.5 months, at a cost of $500. iPSCs are, in essence, human genomes captured in culture, capable of differentiating into any of the cell types in the body, said Burridge.

Burridge’s lab focuses specifically on differentiating iPSCs into cardiomyocytes to recapitulate cardiac diseases with known genetic causes. Beyond using these cells to study diseases with a known genetic makeup, his lab explores whether cardiomyocytes can be used to recapitulate patient-specific drug responses, by focusing on doxorubicin, a common chemotherapy drug (Burridge et al., 2016). Doxorubicin is used in nearly 1 million patients annually, despite well-established dose-dependent cardiotoxicity that occurs in 8 to 10% of patients. The dose of doxorubicin that breast cancer patients can receive is limited by its cardiotoxicity, so eliminating or predicting toxicity would allow patients to receive a higher dose and improve the efficacy of their chemotherapy treatment, said Burridge. Researchers tested iPSCs and cardiomyocytes from patients with cardiotoxicity symptoms and their controls and discovered that the iPSCs/cardiomyocytes accurately recapitulate a patient’s predilection to doxorubicin-induced cardiotoxicity. However, even though the cells recapitulated a patient’s phenotype, Burridge said, there were multiple causes of toxicity, including reactive oxygen species (ROS) production, DNA damage, calcium overload, sarcomeric disarray, and mitochondrial dysfunction.

To find out the genetic basis for doxorubicin’s cardiotoxic effects, Burridge’s lab collaborated with the Canadian Pharmacogenomics Network, which had performed a genome-wide association study (GWAS) on more than 400 patients, and discovered a retinoic acid receptor variant (RARG) that correlated with doxorubicin-induced cardiotoxicity (Aminkeng et al., 2015). Validating this variant using the iPSC model was not as simple as it seemed, said Burridge. The original GWAS population had to be interrogated to identify patients with only the RARG variant, among all other

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×

variants associated with cardiotoxicity. The selected patients had to be re-recruited, a process that involved “nurse practitioners driving hundreds of miles to collect blood in high school bathrooms.” Finally, said Burridge, the data showed that patients with the RARG variant exhibited higher sensitivity to doxorubicin. The data further confirmed that this variant was associated with the phenotypes seen earlier, including DNA damage and ROS production. Overexpression and knockout mouse models of the variant produced the same phenotype. Aiming for the gold standard answer, the research team performed a SNP correction, which reversed the doxorubicin hypersensitivity. Expanding on this information, Burridge and his colleagues are investigating whether specific RARG agonists could stimulate the RARG receptor to act as a cardio protectant for patients with doxorubicin toxicity

IN VITRO CARDIAC DISEASE MODELS

Megan McCain, assistant professor and the Chonette Early Career Chair in the Department of Biomedical Engineering and the Department of Stem Cell Biology and Regenerative Medicine at the University of Southern California, presented her work using in vitro models to study cardiac disease. She noted that the heart’s key function to contract and pump blood is achieved by the myocardium. The structure–function relationship of the cardiomyocytes is critical to the proper functioning of the organ. For example, in populations of elongated, rectangular cardiac myocytes with all of their microfibrils aligned in the same direction, when the microfibrils’ sarcomeres contract they do so in the same direction and thus achieve optimal efficiency. Understanding these structure–function relationships, which are present throughout the heart, said McCain, is essential to understanding the function of the healthy organ and how function can decline in certain diseases.

Conventional animal models for cardiac disease have several limitations. While these models allow researchers to study native physiology in intact organisms, it is difficult to study or control the microenvironment of the heart. Conventional in vitro models, such as cell cultures, allow researchers to study human cardiac cells at a low cost and with high throughput, but they lack the native tissue structure and thus have limited functional outputs.

Given the limitations of conventional models, McCain proceeded to describe her research on building disease models that are modular, scalable, and adaptable to other applications (such as drug screening), robust, quantitative, relevant to humans, and patient specific. She first described the Muscular Thin Films, developed in Kevin (Kit) Parker’s laboratory at Harvard University. Building this platform begins with laser engraving small

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×

windows onto a glass coverslip, which is then coated with a temperature-sensitive polymer that remains in solid state in the incubator but melts at room temperature. The coverslip is then coated with polydimethylsilioxane (PDMS), a silicone elastomer, and micro-printed with different patterns designed using computer-assisted design software. Cardiac myocytes are seeded, and they adopt the pattern that has been printed. This model allows researchers to control tissue architecture and creates films that actually contract once the temperature-sensitive layer melts away. Using an electrode to control the myocytes’ pacing rate, one can monitor and calculate the contractility. McCain noted that this type of in vitro modeling is mostly done with neonatal rat cardiomyocytes.

An in vitro model for inherited Barth syndrome cardiomyopathy, developed in collaboration with William Pu and Gang Wang at Children’s Hospital in Boston, depends on human cells. Barth syndrome is the result of a mutation in TAZ, a transacylase localized to the mitochonrdial membrane. There are few therapeutic approaches or animal models available, and patients commonly need a heart transplant by adolescence. Using iPSCs and Muscular Thin Films, Pu found that cardiomyocytes from patients with Barth syndrome had dramatically less contractility compared to normal controls. Researchers validated that the disease is directly caused by the mutation in the TAZ gene, after recovering the normal phenotype by re-expressing TAZ, said McCain.

Using these in vitro models allows for hypothesis-driven research, better understanding of mechanisms of disease, and, ultimately, the identification of potential therapeutic targets in a human-relevant platform, concluded McCain.

HUMAN MICROPHYSIOLOGICAL SYSTEMS

John Wikswo, the Gordon A. Cain University Professor, the A.B. Learned Professor of Living State Physics, and professor of biomedical engineering, molecular physiology and biophysics at Vanderbilt University, discussed microphysiological systems (MPSs) as models for studying the enormous complexity of biological systems on a scale that is small and controllable.

All current methods for testing drug efficacy and toxicity, said Wikswo, have disadvantages. Traditional in vitro experiments that are conducted on two-dimensional cell cultures test cells that “from a cynical point of view could be argued to have cancer, are inbred, are diabetic, are potatoes on a stiff plastic couch without exercise, enjoy neither gender nor sex, live almost entirely in the dark, gorge themselves on sugar once a day, may be slowly suffocating in an increasingly acidic environment, live in their own excrement, never bury their dead, may take a complete or only partial

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×

bath every day or two, and talk only to cells of like mind. Clearly we have learned a lot of biology from these cells, since one can get reproducible, statistically significant results, but are they relevant to human biology and disease?” While these experiments may end up with reproducible and statistically significant results, their relevance to human biology and disease is questionable, said Wikswo. Animal models also have genetic and physiological differences that affect their relevance to human biology as well. Although humans are ultimately the best model for human disease, MPSs such as organs-on-chips, tissue chips, 3D transwells, and self-assembling organoids can recapitulate the human-specific dynamic interactions between drugs and organs without harming human beings.

Organ-on-a-Chip

A number of organ-on-a-chip technologies have been developed for organs, including the lung, the brain, the liver, and the heart, as well as lymph node-on-a-chip and mammary-gland-on-a-chip. These chips are all designed slightly differently, and many researchers, Wikswo said, are trying to create a 2D, 2D+, or 3D architecture with perfusion to more accurately recapitulate the in vivo environment. Organs-on-chips are expensive to produce and are not yet fully validated. However, they potentially have some significant advantages over traditional methods of toxicity testing:

  • Better than 2D biology
  • Ideal for barrier functions
  • Can reproduce physiological flows
  • Provide thick extracellular matrix for scaffolding and drug/factor binding
  • Support organ–organ interactions
  • Sufficient tissue for multi-omics analyses of tens to thousands of variables
  • Require minimal media volumes
  • May ultimately reduce drug costs
  • May ultimately enable the construction of a single-patient homunculus (i.e., single-patient miniature human)
  • May ultimately enable the construction of animals-on-chips

Organoids

Organoids are three-dimensional, self-organizing microphysiological systems, models with tissue-level functions and disease phenotypes—essentially, miniature in vitro organs. Organoids, said Wikswo, have a number of advantages: they are better models than 2D biology, they can

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×

demonstrate development, and they are transplantable. However, individual organoids are hard to replicate and difficult to integrate with other organ systems.

Wikswo said it is his career goal to introduce two new “species” to the biomedical research ecosystem: Homo minutus (MicroHuman) and Homo chippiens (NanoHuman). These miniature “humans” (homunculi) could be used in any number of research areas, including drug development, environmental toxicology, physiology, and understanding disease mechanisms.

There are several areas of research in which MPSs could be immediately applied, said Wikswo. Within the category of disease biology and pharmacology, MPSs could be used to discover novel mechanisms of human diseases, to identify novel compounds and clinical candidates, and to unravel the mechanism of action of drug candidates. In clinical pharmacology, MPSs can help identify problematic human haplotypes and drug–drug interactions, and to improve prediction of human exposure for compounds and clinical formulations. In toxicology research, MPSs have value for early termination of toxic drugs and avoiding inappropriate drug terminations (Watson et al., 2017). In addition to these immediate applications, there are research applications that are on the cutting edge of MPS, including re-creating the full metastatic cascade and testing immuno-oncology drugs.

MPS models can be particularly helpful in the late preclinical stages of research and development. If a drug had entered phase I clinical trials with unexpected results, MPSs could be used for high content analysis to decipher the problem, said Wikswo. The U.S. Food and Drug Administration has stated its vision of “clinical trials in a dish,” where MPSs are used across multiple areas of drug development, including areas where animal models are currently used: “If properly validated, there is the potential for clinical trials in a dish to replace animal testing, some types of clinical trials, or to be used to select patient populations or even individual patients most likely to benefit or least likely to be harmed by therapies” (Strauss and Blinova, 2017). Another vision for MPSs, said Wikswo, is a human-on-a-chip, made from cells from patients, that would be able to predict the patient’s response to a drug and whether there would be negative effects—before the human patient is put at any risk (Watson, 2017).

QUANTITATIVE SYSTEMS PHARMACOLOGY AND MICROPHYSIOLOGICAL SYSTEMS

D. Lansing Taylor, distinguished professor and Allegheny Foundation Professor of Computational and Systems Biology and director of the University of Pittsburgh Drug Discovery Institute, said that there are two main challenges to developing precision therapeutics today. First, animal models do not always exhibit concordance with human mechanisms of disease

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×

and toxicology. Second, the variability in the genetic makeup of patients demands a research approach that focuses on this diversity in humans. Given these challenges, technologies such as iPSCs and humans-on-a-chip can serve as complementary research platforms for human-specific biology using an in vitro approach. Echoing the previous speaker, Taylor said that the human biological system is incredibly complex, with interactions and pathways between multiple organs, cells, and tissues, and many potential molecular targets for drugs. Quantitative systems pharmacology (QSP) is a system for “determining the mechanism(s) of disease progression and mechanism(s) of action of drugs on multi-scale systems through iterative and integrated computational and experimental methods to optimize the development of therapeutic strategies” (Stern et al., 2016, p. 522).

The Drug Discovery Institute at the University of Pittsburg, Taylor said, has developed a step-by-step platform to implement QSP in the drug discovery process. The process is complex but has 12 basic steps, each of which may inform the others and loop back in an iterative process:

  1. Analyze patients and clinical samples, informed by validated target knowledge and discovery
  2. Infer pathways of disease progression and identify potential drug targets
  3. Utilize machine learning to predict drugs that may interact with those targets
  4. Create and test phenotypic models of disease and safety, using technologies such as RNA interference (RNAi), CRISPR/Cas-9, and MPS
  5. Perform high-content screening and analysis of treatments
  6. Identify drugs that are active for the target
  7. Use medicinal chemistry to optimize targets
  8. Develop mammalian models of disease and safety
  9. Identify high-probability targets using chemical proteomics and RNAi
  10. Build initial computational models based on the available data
  11. Use the predictions from the computational models to inform optimal therapeutic strategy, pharmacodynamic biomarkers, and prognostic and predictive biomarkers
  12. Use the predictions from the computational models to inform clinical trial design and simulation

Human Microphysiological Systems

QSP can supplement and inform other forms of research, such as microphysiological models, iPSCs, and animal models. Taylor, building

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×

on Wikswo’s presentation about MPSs, described the effort to create a liver MPS, a tool to investigate both liver diseases as well as drug toxicity. The newest-generation liver MPS is a vascularized liver acinus MPS (vLAMPS), which consists of all primary human cells obtained from the same patient (i.e., hepatocytes, endothelial cells, stellate and Kupffer cells); it also includes a number of iPSCs and cells with a fluorescent biosensor, under continuous flow. The model is made primarily of glass and plastic, with minimal amounts of polymer, because polymers absorb hydrophobic molecules like drugs very well. This system is also scalable so multiple MPSs can be run simultaneously.

This MPS model has helped researchers induce immune-mediated hepatotoxicity and methotrexate-induced fibrosis. For the methotrexate experiment, researchers engineered stellate cells to express a green fluorescent protein. Subsequent imaging revealed fibrosis due to methotrexate exposure, identified by an increase in α-smooth muscle actin and collagen alpha 2(I) chain. The vLAMPS model, said Taylor, allows for cancer cell extravasation, immune cell infiltration, and delivery of factors from other organs. From his perspective, in order to fully understand liver disease and develop appropriate treatments, it is important to implement the full QSP platform, starting with patients, patient samples, and data, as well as develop multi-scaled experimental and computational models of liver diseases for drug discovery and toxicity testing.

Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 61
Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 62
Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 63
Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 64
Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 65
Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 66
Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 67
Suggested Citation:"5 In Vitro Alternatives to Animal Models." National Academies of Sciences, Engineering, and Medicine. 2018. Advancing Disease Modeling in Animal-Based Research in Support of Precision Medicine: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25002.
×
Page 68
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Precision medicine is focused on the individual and will require the rapid and accurate identification and prioritization of causative factors of disease. To move forward and accelerate the delivery of the anticipated benefits of precision medicine, developing predictable, reproducible, and reliable animal models will be essential. In order to explore the topic of animal-based research and its relevance to precision medicine, the National Academies of Sciences, Engineering, and Medicine convened a 2-day workshop on October 5 and 6, 2017. The workshop was designed to focus on the development, implementation, and interpretation of model organisms to advance and accelerate the field of precision medicine. Participants examined the extent to which next-generation animal models, designed using patient data and phenotyping platforms targeted to reveal and inform disease mechanisms, will be essential to the successful implementation of precision medicine. This publication summarizes the presentations and discussions from the workshop.

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