Problem: This ongoing project seeks to establish methods and approaches to develop in-vitro 3D modeling systems for the growth and differentiation of hESC using rudimentary tissue engineering methods. Background: The primary focus of this work is to develop an approach to differentiate hES cell populations “in-situ” on 3D matrix materials of natural origin, in order to direct tissue-specific developmental outcomes. It is well established that standard embryoid bodies (EB’s) develop areas or regions of distinctive tissue-like differentiation which are capable of complex functions such as cardiac cells beating in unison. Additionally, in many EB’s differentiated cells also form multiple distinctive tissue-like patches. It is apparent that the 3D context and microenvironmental conditions established within EB’s are responsible for this spontaneous differentiation and tissue development. Hypothesis: This project seeks to test the concept that engineered 3D cultures can be used to generate the appropriate microenvironment for directed large-scale differentiation of hESC into specific tissue types. Research to date: In this report, we present results of our initial work that show significant hESC-derived ATs development using a decellularized extra-cellular matrix of marine origin. Colonies of the ATCC hESC line BG01V were introduced to feeder layer (HFF-1) covered and feeder-free scaffolds. These were then maintained in the presence of FGF for extended culture periods, followed by harvest and examination by light and electron microscopy to evaluate the resulting hESC ATs and their relationships with the feeder layer cells. Observations: BG01V stock colonies were maintained under standard conditions and medium sized undifferentiated colonies were transferred to scaffolds. ATs cultures were then maintained for more than 2 months in standard hESC media containing FGF. These cultures developed large-scale features consistent with embryoid body-like architectures, however in all cases there appeared to be a higher degree of polarity than commonly seen in standard EBs. Most of these structures appeared to be cystic and were relatively well-organized at the cellular level. When loaded directly onto scaffolds without feeder cells, colonies of hESC maintained a less organized morphology for longer periods, but eventually also differentiated into cystic structures. In all cases, there was a distinctive polarity seen, with the cystic region expanding away from the scaffolds and an apparent colony-like area of undifferentiated cells at or near the attachment point of the colony to the scaffolds. SEM studies also illustrated a complex interaction of hES-derived cell sheets and HFF-1 feeder layers at the scaffold interface. Conclusions: To date, our observations of 3D hESC ATs structures demonstrate that this culture approach does indeed present a unique set of microenvironmental cues to the cells, which appear to encourage EB-like development with a few significant distinctions. Future studies are planned to qualify the patterns of polarity and differentiation seen, the effective control of differentiation deep within the structures and the potential to induce or control specific patterns of differentiation in these 3D hESC ATs cultures.