Introduction Bioengineering of a fully functional tissue requires precise recapitulation of normal tissue development. Specifically for the liver, one may use bipotent human liver progenitor cells (hFLCs) capable of differentiation into hepatocytes and cholangiocytes. The goal of the current study was to develop a system that would efficiently recapitulate embryonic development of hepatic parenchymal tissue and bile ducts, using decellularized liver extracellular matrix (ECM) as scaffolds.Materials andMethods hFLCs were seeded on decellularized liver ECM discs (300 μm thickness, 8 mm diameter) and were cultured for up to 3 weeks in presence of hepatic differentiation medium. Immunofluorescence microscopy was used to determine the extent of progenitor cell differentiation into hepatocytes and cholangiocytes. Urea, albumin and drug metabolism were quantified as paramaters of liver function. Furthermore, a γ-secretase inhibitor was added to the culture media and bile duct and hepatocyte development was monitored. Results hFLCs seeded on acellular liver ECM discs differentiated into hepatocytes and cholangiocytes. The cells showed predominant albumin expression along with loss of α-feto protein (AFP) expression at 3 weeks (Fig. 1D,E). The cells also expressed other mature hepatocyte markers like HNF-4α, α-1-antitrypsin and cytochrome P450 1A2, 2A and 3A (Fig. 1B-E). The cells in the ductular structures expressed bile duct specific markers like CK19, SOX9, EpCAM, ASBT, β-catenin and the presence of apical primary cilia (stained with α-acetylated tubulin), thus demonstrating differentiation towards cholangiocyte lineage along with maintaining apico-basal polarity (Fig. 1B-E). Urea and albumin secretion was higher in the liver disc organoids compared to control hFLCs cultured in petri dishes. Several metabolites of the drugs diazepam and 7-ethoxycoumarin were also detected by LC-MS/MS, showing broad cytochrome P450 activity in these organoids. The addition of a γ-secretase inhibitor severely impacted the number and maturation of bile ducts formed, mirroring a biliary atresia model. Discussion and Conclusions Our results demonstrate the efficient generation of bioengineered human liver tissue with hFLC that recapitulates stepwise development of hepatocyte and bile duct formation (Fig. 1A). Altogether, this study demonstrates the potential of this technology to study and mimic human liver development. These models provide novel approaches for liver bioengineering, drug discovery and toxicology (including drug teratogenesis evaluation in vitro), and ultimately for the treatment of liver disease.