Bioengineered liver organoids were generated by seeding freshly isolated hFLC through the vena cava and portal vein of the intact liver scaffold. These seeded scaffolds remained in a perfusion bioreactor up to two weeks. In parallel, hFLCs were seeded on decellularized liver ECM discs (300 μm thick, 8 mm diameter) and were cultured for 3 weeks in hepatic differentiation medium. Confocal microscopy

was used to determine the extent of progenitor cell differentiation into hepatocytes and cholangiocyies in disk organoids and whole liver scaffold. Urea, albumin and drug metabolism AZD3965 were guantified as paramaters of liver function. hFLCs seeded on liver ECM discs differentiated into hepatocytes and cholangiocytes. The cells showed predominant albumin expression along with loss of AFP expression at 3 weeks. The cells also expressed other mature hepatocyte markers like HNF-4α, α-1AT and CYP450 1A2, 2A and 3A. The cells in the ductular structures expressed bile duct specific markers like CK19, SOX9, EpCAM, ASBT, β-catenin and the presence of primary cilia, thus demonstrating differentiation check details towards cholangiocyte lineage along with maintaining apicobasal polarity. Similarly, hFLC seeded in whole liver scaffolds showed progressive tissue formation and organization with clusters of hepatocytes expressing albumin, AFP, CYP450 3A and 2A, E-cad, Hep-1

and EpCAM, and several long ductular structures staining positive for biliary markers CK19, EpCAM, Interleukin-3 receptor ASBT spawning for 200-400μm in length. Urea and albumin secretion was higher in the whole bioengineered liver and liver disc organoids compared to control hFLCs cultured in petri dishes. Several metabolites of diazepam and 7-ethoxycoumarin were also detected by LC-MS/MS, showing broad

CYP450 activity in both culture systems. Our results demonstrate the efficient generation of bioengineered human liver tissue with hFLC that recapitulates stepwise development of hepatocyte and bile duct formation. 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, and ultimately for the treatment of liver disease. Disclosures: The following people have nothing to disclose: Pedro M. Baptista, Dipen Vyas, Emma Moran, Anthony Atala, Shay Soker There is considerable interest in the use of bone marrow(BM) cells in liver cirrhosis, however the role of purified haematopoietic stem cells(HSC) and use of repeated infusions have not been studied. We also set out to determine whether increased retention of HSC within the injured liver by modulating their response to Sphingosine 1-phosphate(S1P) would augment their anti-fibrotic effect. Liver injury was induced in BoyJ(CD45.