Mechanism And Application Of Mature Hepatocytes-Derived CD24~+ Liver Progenitor Cells Induced By Small Molecules

Background&AimsThe liver stands out as one exceptional organ containing high regenerative potential after injury.Nevertheless,the persistent chronic damage such as virus infection,alcohol abuse and steatohepatitis leads to an abnormal steady state known as fibrosis which consists of liver architectural remolding and hepatic lobules destruction.Although inflammation typically precedes fibrosis,results from experimental models of this process have demonstrated that the common mechanisms responsible for liver fibrogenesis was not driven by inflammation at all times,but necessarily driven by the highly integrated processes of a complex interaction between inflammation and vicious cycle of cell death,contributing to chronic myofibroblast activation and excessive accumulation of ECM components.Focus should,then,be placed on the dynamics that govern regeneration and fibrosis to enhance epithelial replacement in humans.A more successful therapeutic approach would involve replacing damaged cells or restoring homeostasis to the areas that underlie the fibrotic response.Based on these,our study aims to explore the important role of activated liver precursor-like cells for liver regeneration during liver injury repair,and hopes to supplement liver progenitor cells through exogenous cell transplantation or small molecule injection,in order to promote hepatic function and revert liver fibrosis and cirrhosis.MethodsOur previous studies successfully screened a series of small molecule compounds as TEM medium to achieve the conversion from mouse and human primary hepatocytes to continuous proliferated cells in vitro,which we named as HepLPCs.Through RNA sequencing and bioinformatics analysis,HepLPCs was found to contain characteristics of liver progenitor cells with high expression of the precursor marker CD24.In vitro cultured-CD24~+HepLPCs was tested by methods of immunofluorescence,flow cytometry,PCR and Organoid formation to measured its capacity to large-scale proliferation,self-renewal and bipotency to differentiate into hepatocytic and cholangiocytic lineages in vitro;Then CD24~+HepLPCs were transplanted into CCL4-treated mice to test stem cell-based therapy effect.We then used HGF in combination with a cocktail of small molecules Y-27632,A-83-01,and CHIR99021(HACY)to induce the replenishment of endogenous CD24~+HepLPCs during CCL4-induced hepatic injury.HACY-induced CD24+HepLPCs expansion was also tested in human hepatocyte-spheroid from fibrotic liver tissuesResultsCD24+HepLPCs displayed characteristics of self-renewal capacity and intermediate hepatobiliary phenotypes,resulting in a significant improvement in liver fibrosis after transplantation.Consistently,injection of HACY into mice with CCL4 treatment induced conversion of mature hepatocytes(MHs)to CD24+progenitor-like cells in vivo and attenuated liver fibrosis.Compared to LPCs derived from NPCs,HACY-induced CD24+HepLPCs retained an increased hepatic function and could promote restoration of liver function.CD24+cells could also be observed in human liver fibrotic tissues and further expanded in 3D hepatic spheroid in the presence of HACY.ConclusionCD24~+HepLPCs are crucial for liver regeneration during chronic hepatic injuries.Injection of HACY,which allowed induction of endogenous CD24~+HepLPCs,has clinical utility in the treatment of patients with a broad range of hepatic fibrosis by providing an alternative approach for stem cel-based therapy.