The objective of this proposal is to develop cardiac and neural 3D microtissues from human pluripotent stem cell (PSC) sources that can be used to model human development and disease as well as potentially be used directly for regenerative medicine therapies. During the past year we have made significant progress in creating 3D cardiac microtissues from heart muscle and supporting cells derived from PSCs and/or human tissue. We have begun to characterize the effects of combining the different cell types in a controlled manner in order to understand how the different cells influence each other and also affect tissue function. We anticipate that these investigations will lead to better models of engineered cardiac tissue that more accurately reflect properties of real heart tissue. In order to develop new 3D models of neural tissue, particularly spinal cord, we have successfully differentiated multiple neuronal cell types, including a new excitatory spinal interneuron (V2a) that has not previously been reported from human PSCs. We are now preparing to combine different types of spinal neurons using our 3D technologies in an attempt to create a model of human spinal cord. Lastly, we are using engineered PSC lines to examine how cells self-organize to create complex structures during tissue development. These latter studies we anticipate could lead to novel ways to pattern the multicellular organization of 3D tissues derived from PSCs.