Understanding the mechanisms governing organogenesis provides unique insight into strategies to engineer ?in- a-dish? mini-organ (aka organoid) systems from human stem cells, with the potential for studying developmental disorders, drug screening, and therapeutic transplantation. We have thus capitalized on knowledge of embryogenesis and have a history of innovation for engineering complex endodermal tissues from human induced pluripotent stem cells (iPSCs), including gut, pancreatic and liver organoids with vascular and macrophage components. Our vascularized human liver organoids, furthermore, were able to restore metabolic function and maintain life of mice subjected to lethal acute liver failure. In this application, we will address the next level challenge by proposing highly innovative 3-dimensional (3D) technologies of multi-organ engineered from iPSC. In choosing a model system, we selected the hepato-biliary-pancreatic (HBP) system based on their endoderm derivation, anatomical relatedness via interconnected ducts, and well-defined tissue subdomains. The overall objective of the present proposal is to direct human iPSCs to generate balanced 3-D organogenesis of the HBP system as a contiguous multi-organ system. In our human iPSC-differentiated HBP organogenesis model, we discovered that, strikingly, the interaction between foregut-midgut enabled autonomous multi-organ, i.e. HBP, patterning at the boundary and this critically required retinoic acid signals. Based on these exciting preliminary data, we hypothesize that the boundary engagement of foregut-midgut interactions activates multi-organ bud programming by localized retinoic acid signaling. Our approach will first leverage the foregut-midgut boundary culture differentiated from human iPSCs to develop HBP organoid (HBPO) and will determine the mechanisms by which retinoic acid establishes tissue boundaries and segregation of multi-organ domains. Secondly, we will devise an efficient vascularization method for HBPO by our pioneering self- condensation culturing methodology, wherein endothelial cells are incorporated into organoids to facilitate in vivo engraftment. HBPO will be molecularly engineered to display dual organ subdomain reporter expression to precisely map hepatobiliary domains. This will guide the development of surgical methods to direct the anastomosis of extrahepatic biliary components into the recipient gut system. Finally, the metabolic maturation, functionality, and persistence of this HBPO transplantable system will be challenged by testing therapeutic efficacy to meet the metabolic needs of liver disease. Upon completion of the proposed studies, we will demonstrate that the experimental multi-organ integrated model serves as a tractable, manipulatable and easily accessible model for the study of human organogenesis and disease!in a way that cannot be accomplished by direct human studies. In addition to serving as system to model human diseases, the proposed multi-organ engineering has the potential to establish native and persistent physiological functions in diverse organ systems with the goal of developing a novel class of organoid based organ replacement therapy.
) This proposal aims to engineer three-dimensional multi-organs from human stem cells with vital connections to neighboring tissues. If successful, this technology will provide the basis for studying pathogenesis of diverse human diseases, and ultimately establish an innovative replacement strategy for patients with organ failure.