Direct reprogramming of adult hepatocytes to generate LGR5+ endodermal progenitor

Abstract

We successfully converted hepatocytes isolated from adult mice into expandable and stable leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive endodermal progenitor cells (EndoPCs). This was accomplished in vitro through transient exposure to four transcriptional factors (OCT3/4, SOX2, KLF4, and cMYC) and STAT3 activators. EndoPCs were generated by a process that involved an epithelial-mesenchymal transition (EMT) without the use of any components of the canonical WNT/{beta}-catenin or LGR5/R-spondin signaling pathways. We showed that the proliferation and capacity for self-renewal of EndoPCs in 2D long-term culture were controlled by three interrelated signaling pathways: gp130/JAK/STAT3, LGR5/R-spondin, and WNT/{beta}-catenin. After long-term maintenance in two- and three-dimensional culture systems, EndoPCs were able to differentiate into liver-restricted lineages such as hepatocyte-like cells and bile duct-like structures in vitro and in vivo. After intra-muscular injection, EndoPCs generated macroscopically visible and well-vascularized liver-like tissue, which contained Alb+ liver parenchyma-like structures and substantial, KRT7/KRT19+ bile duct-like cell organizations. ConclusionWe have developed an efficient method for producing LGR5+ adult endodermal stem cells. These cells will be useful for the in vitro study of the molecular mechanisms of liver development and have important potential for therapeutic strategies, including approaches based on bioengineered liver tissue. These cells also open up new avenues for experiments focused on disease modeling, toxicology studies, and regenerative medicine applications.