Next Generation Organoids for Biomedical Research and Drug Discovery

“Mice are not little people” – a refrain becoming louder as the strengths and weaknesses of animal models of human disease become more apparent.  At the same time, three emerging approaches are headed toward integration:  powerful systems biology analysis of cell-cell and intracellular signaling networks in patient-derived samples; 3D tissue engineered models of human organ systems, often made from stem cells; and micro-fluidic and meso-fluidic devices that enable living systems to be sustained, perturbed and analyzed for weeks in culture.  This talk will highlight the integration of these rapidly moving fields to understand difficult clinical problems, with an emphasis on translating academic discoveries into practical use.  At one end of the spectrum, there is a tremendous need for synthetic microenvironments to propagate and differentiate human organoids. Examples of such microenvironments for expansion and use of gut and endometrial organoids will be highlighted. Further,   technical challenges in modeling complex diseases with “organs on chips” approaches include the need for relatively large tissue masses and organ-organ cross talk to capture systemic effects, as well as new ways of thinking about scaling to capture multiple different functionalities from drug clearance to cytokine signaling crosstalk. An example of how gut-liver interactions can be parsed at these levels will be featured.

Multicellular ecosystems such as biofilms, tissues, and whole organisms operate as highly integrated systems that link physical structure and biological function. In mammalian tissues, structure determines the effectiveness by which muscles generate force, lungs oxygenate blood, or glandular organs produce bile, milk, or saliva. Even at the level of single cells, tissue structure constrains how cells interact with surrounding extracellular matrix, neighboring cells, and physical forces, and these “microenvironmental” cues in turn regulate cell function, such as proliferation, differentiation, migration, and suicide. Understanding the underlying control systems that give rise to these ecosystems is central to our ability to rationally perturb or synthetically design and build them. Using engineered microenvironments, we have begun to expose the complex interplay that occurs between structure, force, signaling, and function in cells and multicellular systems. We show that these control loops are central to cell and multicellular function and assembly; use these insights to build in vitro organotypic models that mimic native tissue functions; and examine opportunities and challenges for how to connect these insights to impact regenerative medicine therapies.

Salivary gland dysfunction is a consequence of off-target radiation due to head and neck cancer treatments, Sjogren’s syndrome, and many commonly-prescribed drugs. Progress in mechanistic understanding of gland dysfunction and development of therapeutic strategies are hampered by the lack of in vitro models, as salivary gland cells (SGCs) rapidly (in <1 day) lose critical secretory function in vitro. The goal of this work is to engineer functional human salivary gland tissue chips for high-throughput drug screening.

To develop functional salivary gland tissues, we are synergizing two technologies: hydrogels and microbubble array (MBs). Hydrogel-encapsulated SGCs exhibit long-term survival and form polarized structures expressing Mist1, a secretory marker. Although these data are promising, Mist1 expression is reduced compared to native glands. MBs are micron-scale spherical cavities molded in polydimethylsiloxane. MBs have the advantage of length scales and curvatures similar to gland acinar units to promote cell-cell contact and concentration autocrine-paracrine factors to enhance tissue function.

Gland acinar-intercalated-ductal (AIDUC) tissue maintains Mist1 gene expression and functional calcium flux compared to native glands. Mist1 and sodium-potassium-chloride channel (NKCC)1 are increased 20-40x in gel-MB and 5-10x compared to AIDUCs in tissue culture plates over 14 days. Calcium signaling is maintained for >14 days in both murine and human SGCs cultured in gel-MB. Furthermore, both mouse and human SGCs adopt morphologies similar to native gland, as indicated by immunofluorescence.

Salivary gland tissue chips are promising platforms for drug screening to promote or protect salivary gland function.

Modeling integrated human physiology in vitro is a formidable goal that is becoming increasingly plausible by the emergence of human “organs on a chip” platforms. The advances in stem cell biology and tissue engineering [e.g., 1-3] are now enabling the formation of miniature tissues and organs that are grown in vitro, matured, and functionally integrated to emulate human physiology and disease [4]. These platforms take advantage of patient-specific iPS cells and gene editing methodologies, to capture the mechanisms of disease and drug action. In contrast to tissues grown for transplantation, the “organs on a chip” provide a fast-track opportunity for tissue engineering, with direct impacts on biological research and human medicine.

Here we describe the design and operation of a modular human platform in which the individual tissue modules (heart, liver, bone, skin, solid tumor) are connected by perfusable vasculature. The functional integration is achieved by (i) maintaining a local regulatory niche for each tissue, (ii) connecting tissue units by a blood substitute containing circulating cells, and (iii) establishing endothelial barrier between the vascular and tissue compartments. In particular, the separation of each tissue from vascular perfusion by endothelial barrier, as in native tissues in the body, allows independent optimization of each culture environment for maturation and functionality, without limitations related to the use of a “common medium” for multiple tissue types. We describe the methods for maturation of the component tissues, and the maintenance of stable tissue phenotypes over 4 weeks of culture, with real-time measurements of cell and tissue function. The platform is highly configurable, and enables connections of tissue compartments into functional networks, while maintaining the tissue maturity and physiologic function, and providing communication via microfluidic vasculature. As all tissues are derived from the same source of human iPS cells, the platform allows studies of genetic diversity and disease phenotypes. To illustrate the platform utility, we report studies of disease modelling and drug testing using the interacting tissue systems.

The need for human, organotypic culture models coupled with the requirements of contemporary drug discovery and toxin screening (i.e. reproducibility, high throughput, transferability of data, clear mechanisms of action) frame an opportunity for a paradigm shift. The next generation of high throughput assay formats will require a broadly applicable set of tools for human tissue assembly and analysis. Toward that end, we have recently focused on: i) generating iPS-derived cells that properly represent the diverse phenotypic characteristics of developing or mature human somatic cells; ii) assembling organotypic cell culture systems that are robust and reproducible; iii) translating organotypic cell culture models to microscale systems for high throughput screening; and iv) combining genomic analyses with bioinformatics to gain insights into organotypic model assembly and the pathways influenced by drugs and toxins. This talk will emphasize assembly of organotypic vascular and brain tissues. These tissues mimic aspects of human organ structure, and can be used for reproducible identification of drug candidates and toxicants by both academic and industry scientists. We particularly emphasize reproducibility and data transferability, which are important for the widespread use of organotypic human models in toxicity testing, including in emerging industry applications. The talk will also describe assembled human tissues as models of rare neurodevelopmental disorders of the brain.

Human Pluripotent Stem Cell-derived cells/organoids provide a robust platform for in vitro disease modeling and drug screening. With the goal of modeling human disease of the large intestine, we developed an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs. We applied this strategy to hESC/iPSCs harboring colorectal cancer related mutations, including patients with familial adenomatous polyposis (FAP-iPSCs). We generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. We found that geneticin, a ribosome-binding antibiotic with translational ‘read-through’ activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.