Successful induction of human chemically induced liver progenitors with small molecules from damaged liver.

Abstract

Human chemically induced liver progenitors (hCLiP) induced by small molecules produced by mature hepatocytes can potentially overcome issues related to hepatocyte transplantation, such as graft rejection or donor shortage. However, to our knowledge, no studies have explored the induction of hCLiP from mature hepatocytes (MHs) in damaged liver, indicated for liver transplantation. Liver tissues were collected from surgically resected livers, including damaged livers, of 86 patients at our department, and hepatocytes were isolated using the collagenase perfusion method. Hepatocytes isolated from 33 of these 86 donors were cultured in YAC medium containing Y-27632 (ROCK inhibitor), A-83-01 (TGF-β type I receptorinhibitor), and CHIR99021 (GSK-3inhibitor) to induce hCLiP, and their functions were assessed. Hepatocytes were isolated regardless of the liver fibrosis classifications (viability: F0,1: 87.2 ± 13.2%; F2,3: 87.8 ± 13.1%; and F4: 86.3 ± 4.2%). Most hepatocytes cultured in the YAC medium acquired the liver progenitor cell (LPC) gene. The expression of MH markers (ALB, HNF4α, G6PC, and CYP1A2) was lower in hCLiP than in MHs before reprogramming. Reverse transcription-polymerase chain reaction revealed that hCLiP markers (e.g., EpCAM, SOX9, CK19, and CD133) exhibited higher expression in LPCs than in MHs. Furthermore, hCLiPs had the ability to differentiate into hepatocytes, and were engrafted on the liver surface as mature hepatocytes. Hepatocytes could be isolated from damaged liver. Furthermore, hCLiP may be obtained from hepatocytes isolated from damaged liver and may differentiate into MHs in vitro. Autologous hCLiP can potentially be transplanted without tumorigenesis and remodel damaged liver.