The rapidly evolving field of stem cell bioengineering and organoids technology face key challenges: a) stem cell derived tissues are stalled developmentally and show fetal-stage phenotypes b) they often lack key subsets of vascular endothelial and stromal cells derived from different germ layers c) There is a lack of toolset to quantitatively assess cell or tissue identity and d) to guide morphogenetic events towards their native adult phenotypes. In this proposal, we will address these issues by undertaking an integrative synthetic biology and systems biology approach. We recently generated novel human fetal liver organoids ex vivo using human iPSCs. Our approach entails genetic engineering of human iPSCs via overexpression of GATA6 transcription factor in pluripotent media with bFGF and TGF-β. Through this strategy, we showed development of human endodermal and mesodermal, intercellular communications, co-differentiation and self-organization of cultures into a multi- cell type fetal liver organoid. We will employ this tissue as a unique testbed to develop and address biotechnology challenges for in vitro maturation, assessment and engineering of organoids. Through aim 1 of this proposal we will develop and validate a set of genetic toolset to drive cell-fate reprogramming of the multicellular tissue. Through aim 2, we will establish a computational platform to quantitatively assess liver organoids and to identify transcriptional regulators of stage specific development IMPACT: We tackle improvement and assessment of human organoids ex vivo, two key challenges in stem cell bioengineering. The successful completion of this study will result in a hypothesis-driven framework for rational engineering and advancement of stem cell-derived tissues ex vivo. Our study will also generate synthetic liver tissues with close proximity to adult human liver. It reduces dependence on animal experiments and increases access to refined human tissues.