Technical advances in the development of zonation liver in vitro systems that incorporate localized Wnt activating signals

A Wnt microenvironment sustained by the hepatic central vein is essential for the segregation of liver functions into zones. Current liver culture systems lack localized Wnt cues and as a consequence fail to maintain the hepatocyte functional heterogeneity that is observed in the intact organ. In this study, organoid models and 2D-culture systems were used to identify cellular sources and Wnt presentation methods that could support the future development of zonated liver in vitro systems. Using soluble ligands, we show that primary hepatocyte (PH)-derived organoids but not bile duct (BD)-derived organoids may be used to recapitulate the resting liver. We provide evidence that differentiation of PH-organoids in the presence of Wnt9b and Rspo3 induce pericentral maturation. Finally, we show that immobilization of Rspo3 onto beads in combination with soluble Wnt9b may be a valid strategy to recreate the central vein Wnt microenvironment in vitro.

SCL Expression in the Mouse Embryo Detected With a Targeted lacZ Reporter Gene Demonstrates Its Localization to Hematopoietic, Vascular, and Neural Tissues

The helix-loop-helix transcription factor SCL (TAL1) is indispensable for blood cell formation in the mouse embryo. We have explored the localization and developmental potential of cells fated to express SCL during murine development using SCL-lacZmutant mice in which the Escherichia coli lacZreporter gene was ‘knocked in’ to the SCL locus. In addition to the hematopoietic defect associated with SCL deficiency, the yolk sac blood vessels in SCLlacZ/lacZ embryos formed an abnormal primary vascular plexus, which failed to undergo subsequent remodeling and formation of large branching vessels. Intraembryonic vasculogenesis in precirculationSCLlacZ/lacZ embryos appeared normal but, in embryos older than embryonic day (E) 8.5 to E9, absolute anemia leading to severe hypoxia precluded an accurate assessment of further vascular development. In heterozygous SCLlacZ/w embryos, lacZ was expressed in the central nervous system, vascular endothelia, and primitive and definitive hematopoietic cells in the blood, aortic wall, and fetal liver. Culture of fetal liver cells sorted for high and low levels of β galactosidase activity fromSCLlacZ/w heterozygous embryos indicated that there was a correlation between the level of SCL expression and the frequency of hematopoietic progenitor cells.

SCL Expression in the Mouse Embryo Detected With a Targeted lacZ Reporter Gene Demonstrates Its Localization to Hematopoietic, Vascular, and Neural Tissues

The helix-loop-helix transcription factor SCL (TAL1) is indispensable for blood cell formation in the mouse embryo. We have explored the localization and developmental potential of cells fated to express SCL during murine development using SCL-lacZmutant mice in which the Escherichia coli lacZreporter gene was ‘knocked in’ to the SCL locus. In addition to the hematopoietic defect associated with SCL deficiency, the yolk sac blood vessels in SCLlacZ/lacZ embryos formed an abnormal primary vascular plexus, which failed to undergo subsequent remodeling and formation of large branching vessels. Intraembryonic vasculogenesis in precirculationSCLlacZ/lacZ embryos appeared normal but, in embryos older than embryonic day (E) 8.5 to E9, absolute anemia leading to severe hypoxia precluded an accurate assessment of further vascular development. In heterozygous SCLlacZ/w embryos, lacZ was expressed in the central nervous system, vascular endothelia, and primitive and definitive hematopoietic cells in the blood, aortic wall, and fetal liver. Culture of fetal liver cells sorted for high and low levels of β galactosidase activity fromSCLlacZ/w heterozygous embryos indicated that there was a correlation between the level of SCL expression and the frequency of hematopoietic progenitor cells.