Project Summary: Regeneration and development operate on the sub-millimeter scale, using evolved design principles to drive self-assembly of replacement and new organs. Our goal with this project is to follow the principle of 'the organizer', where a small group of cells are microsurgically grafted into a naive host tissues to pattern and induce organ primordia from the naive tissue. To achieve this we first identify methods to produce stable laminar sheets of naive tissue (i.e. stable host tissues), and to develop novel tools to assemble these laminar sheets, (i.e. in place of manual microsurgical grafts) to enable formation of planar polarized 3D structures. The principles that propel modern tissue engineering are based on classic embryological studies of morphogenesis in organoids. These classic studies relied on amphibian models where specified cells could be isolated from embryos, formed into aggregates, and differentiated into distinctive tissues. Following programs of sorting and engulfment, and differentiation, cells self-assemble planar, polarized laminar sheets with distinct polarity, e.g. anterior-to-posterior, to form tissues consisting of multiple cell types at high cell density. It is now widely recognized that similar, conserved programs of self-assembly shape human tissues during embryogenesis, regeneration, and cancer progression. However, current efforts to engineer complex 3D structures suffer from an inability to reproducibly and reliably generate organized multi-laminar tissues of multiple cell types at high cell density. To date, no tissue engineering approach is capable of recreating multi-laminar polarized 3D structures analogous to those that form at even the earliest stages in the embryo. Using our experience with and expertise in embryonically assembled tissues, we leverage principles of the organizer, and engineer stable multi-laminar tissues with planar polarity. Based on our published methods for shaping 3D embryonic tissues into laminar sheets, we will to expose the cellular that stabilize those sheets and to develop assembly methods to produce planar polarized tissues. Rapid translation to human biomedical research and tissue are made possible by leveraging the speed and accessibility of the amphibian embryonic model to test and translate key findings to human embryonic stem cell models of the organizer. We envision that the long term outcome of this project will transform efforts to engineer and manufacture tissues that can be sourced from human cell types and iPSCs for a wide range of clinical and research applications.