Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors.

Understanding the mechanism of how liver ductal cells (cholangiocytes) differentiate into hepatocytes would permit liver-regenerative medicine. Emerging liver ductal organoids provide an ex vivo system to investigate cholangiocyte-to-hepatocyte differentiation. However, as current gene manipulation methods require organoid dissociation into single cells and have only low efficiency, it is difficult to dissect specific gene functions in these organoids. Here we developed the adeno-associated virus (AAV) vector AAV-DJ as a powerful tool to transduce mouse and human liver ductal organoids. Via AAV-DJ-mediated up- or down-regulation of target genes, we successfully manipulated cholangiocyte-to-hepatocyte differentiation. We induced differentiation by overexpressing the hepatocyte-specifying regulator hepatocyte nuclear factor 4α (HNF4α) and blocked differentiation by stimulating Notch signaling or interfering with Smad signaling. Further screening for transcriptional factors critical for cholangiocyte-to-hepatocyte differentiation identified HOP homeobox (HOPX), T-box 15 (TBX15), and transcription factor CP2-like 1 (TFCP2L1) as master regulators. We conclude that this highly efficient and convenient gene manipulation system we developed could facilitate investigation into genes involved in cell lineage transitions and enable application of engineered organoids in regenerative medicine.

Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.

Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.

Morphological and histological study of the ostrich (Struthio Camelus L.) liver and biliary system.

The peculiarity of the digestive system of the ostrich (Struthio Camelus L.), which is characterized by the continuous production of bile, led us to undertake macroscopical and histological studies of the liver and its biliary system, since very little bibliographic data exist on the subject. For this purpose we observed the organs of male and female ostriches 16-18 months of age, in situ, in order to describe their location, relationships and morphology. Samples of the liver were processed for observation by light microscopy; samples of the hepatoenteric duct were processed for observation by light and electron microscopy. Our findings regarding the liver revealed the presence of two lobes: a left lobe, subdivided into three lobes, and a right undivided lobe. There was no gall-bladder. The histological picture showed unlimited hepatic lobules, with hepatocytes arranged in cord-like fashion two cells thick. A large hepatoenteric duct arose from the porta hepatis, and opened into a papilla in the descending limb of the duodenum. The mucosa of the duct was lined by simple columnar epithelium consisting of cells having the same morphological cytoplasmatic features but distinguished by either a light or a dark nucleus.

Transit-amplifying ductular (oval) cells and their hepatocytic progeny are characterized by a novel and distinctive expression of delta-like protein/preadipocyte factor 1/fetal antigen 1.

Hepatic regeneration from toxic or surgical injury to the adult mammalian liver, endorses different cellular responses within the hepatic lineage. The molecular mechanisms determining commitment of a cell population at a specific lineage level to participate in liver repair as well as the fate of its progeny in the hostile environment created by the injury are not well defined. Based on the role of the Notch/Delta/Jagged system in cell fate specification and recent reports linking Notch signaling with normal bile duct formation in mouse and human liver, we examined the expression of Notch1, Notch2, Notch3, Delta1, Delta3, Jagged1, and Jagged2, and delta-like protein/preadipocyte factor 1/fetal antigen 1 (dlk) in four well-defined experimental rat models of liver injury and regeneration. Although Delta3 and Jagged2 were undetectable by reverse transcriptase-polymerase chain reaction and Northern blot, we observed the most significant up-regulation of all other transcripts in the 2-acetylaminofluorene-70% hepatectomy (AAF/PHx) model, in which liver mass is restored by proliferation and differentiation of transit-amplifying ductular (oval) cells. The most profound change was observed for dlk. Accordingly, immunohistochemical analyses in the AAF/PHx model showed a specific expression of dlk in atypical ductular structures composed of oval cells. Delta-like protein was not observed in proliferating hepatocytes or bile duct cells after partial hepatectomy or ligation of the common bile duct whereas clusters of dlk immunoreactive oval cells were found in both the retrorsine and the AAF/PHx models. Finally, we used dlk to isolate alpha-fetoprotein-positive cells from fetal and adult regenerating rat liver by a novel antibody panning technique.