Liver progenitor cell and KDR.

Liver disease affects hundreds of millions of patients worldwide. Liver transplantation is currently the only curable treatment for liver failure caused by cirrhosis, chronic hepatitis B or C, bile duct disease, genetic disease, autoimmune liver disease, primary liver cancer, alcoholic liver disease, or fatty liver disease. Currently, more than 6000 liver transplants are performed each year in the United States. However, there are over 16 000 Americans on the waiting list for a liver transplant. Hepatocyte transplantation is considered a potential treatment for liver diseases and a bridge for patients awaiting liver transplantation, but its application has been hampered by a limited supply of hepatocytes. Hepatocytes derived from human pluripotent stem cell (hPSC) differentiation cultures could provide an unlimited supply for cell replacement therapy. However, generation of mature functional cells from these stem cells remains a challenge. Better understanding of the early molecular mechanisms regulating normal liver development could help optimize the generation of functional hPSC-derived hepatic cells.

Liver develops from the anterior foregut endoderm. Hepatic specification occurs at E8.5 in the mouse embryo and around the third week of pregnancy in humans, leading to the development of fetal hepatoblast cells. These are bipotential progenitors for hepatocytes and cholangiocytes that constitute the main cell components of the adult liver. In both species, hepatoblasts express α-fetoprotein (AFP) and albumin (ALB) and migrate into the adjacent septum transversum mesenchyme to form the liver bud. The processes of hepatic specification and hepatoblast cell expansion are dependent on endothelial cells that surround the hepatic endoderm as early as E8.5 in the mouse embryo.,

We identified within the mouse embryonic foregut endoderm a novel hepatic progenitor cell that precedes the hepatoblast stage and that surprisingly expresses the receptor VEGFR2, also named FLK-1 or KDR. Previously, KDR was thought to be mainly restricted to mesodermal cell derivatives including progenitors for blood and endothelial cells., KDR expression is downregulated as endoderm specifies to a hepatic fate. A lineage tracing study in the mouse tracking the fate of KDR-expressing cells (Kdr-Cre x Rosa26-FloxpSTOPFloxp-eYFP) revealed that they derive in fetal E13.5 livers a large subset of hepatoblasts (30% to 50%), which subsequently gave rise to many hepatocytes (positive for HNF4α, ALB and DPP4) and cholangiocytes (positive for SOX9 and CK19) in adult livers. This study provided in vivo evidence for the existence of the KDR+ hepatic progenitor.

We found that the KDR+ hepatic progenitor was conserved between mouse and human, as a similar KDR-expressing progenitor was also identified in human embryonic stem cell (hESC) hepatic differentiation cultures and human fetal livers. KDR+ progenitors develop from hESC-derived endoderm, which was highly purified based on expression (and exclusion) of defined cell-surface markers. The hESC-derived endoderm generated homogenous clusters of cells expressing the endoderm marker FOXA2. As the cultures differentiated further, hepatic cells, identified by the presence of the hepatic proteins AFP and ALB and high transcript levels for hepatic genes including CYP3a4 and AAT, develop concomitantly with the KDR+ hepatic progenitors with an average ratio of 1:1. The KDR+ cells are true hepatic progenitors, as they differentiate into functional hepatic cells that can be efficiently infected with the hepatitis C virus, a unique feature of functional hepatocytes. Differentiation of KDR+ progenitors into hepatic cells was dependent on three-dimensional cell-cell contact and KDR activity, as inhibition of KDR signaling with an inhibitory antibody against KDR decreased significantly AFP and ALB levels.

Given that hepatic cells and KDR+ progenitors develop together in hESC cultures, we investigated whether KDR+ cells could also serve as supportive cells for the hepatic cells to specify and mature. Coculture strategies between both cell types revealed that KDR+ cells induced a dramatic increase of AFP and ALB transcript levels (~10-fold and ~50-fold, respectively) indicative of their supportive effect on hepatic specification and maturation of the developing hepatic cells. Blocking KDR function using either a small molecule inhibitor or an inhibitory antibody completely abrogated induction of ALB levels in cocultures, demonstrating the requirement of KDR signaling for maturation of hepatic cells in a non-cell autonomous fashion. The functionality of KDR in liver development is consistent with the lack of liver bud formation in Kdr deficient mice.

In summary, our study identified a conserved murine and human hepatic progenitor that expresses KDR, which acts as a functional receptor to promote hepatic fate in a cell autonomous fashion, and hepatic cell maturation in a non-cell autonomous fashion. The identification of this novel hepatic progenitor raises some interesting questions regarding the impact of these cells in liver regeneration following injury and more specifically their direct relation with the stem-progenitor cell pool present in adult liver.