The combined induction of liver progenitor cells and the suppression of stellate cells by small molecules reverts chronic hepatic dysfunction

Rationale: We developed a cocktail of soluble molecules mimicking the in vivo milieu supporting liver regeneration that could convert mature hepatocytes to expandable liver progenitor-like cells in vitro. This study aimed to induce endogenous liver progenitor cells by the administration of the soluble molecules to provide an alternative approach for the resolution of liver fibrosis. Methods: In vitro cultured hepatocyte-derived liver progenitor-like cells (HepLPCs) were transplanted into CCL4-treated mice to investigate the therapeutic effect against liver fibrosis. Next, we used HGF in combination with a cocktail of small molecules (Y-27632, A-83-01, and CHIR99021 (HACY)) to induce endogenous CD24(+) liver progenitor cells and to inhibit the activation of hepatic stellate cells (HSCs) during CCL4-induced hepatic injury. RNA sequencing was performed to further clarify the features of HACY-induced CD24(+) cells compared with CCL4-induced CD24(+) cells and in vitro derived HepLPCs. Finally, we evaluated the expansion of HACY-induced CD24(+) cells in human hepatocyte-spheroids from fibrotic liver tissues. Results: HepLPCs exhibited the capacity to alleviate liver fibrosis after transplantation into CCL4-treated mice. The in vivo administration of HACY not only induced the conversion of mature hepatocytes (MHs) to CD24(+) progenitor cells but prevented the activation of HSCs, thus leading to enhanced improvement of liver fibrosis in CCL4-treated mice. Compared to CD24(+) cells induced by CCL4 alone, HACY-induced CD24(+) cells retained an enhanced level of hepatic function and could promote the restoration of liver function that exhibited comparable gene expression profiles with HepLPCs. CD24(+) cells were also observed in human liver fibrotic tissues and were expanded in three-dimensional (3D) hepatic spheroids in the presence of HACY in vitro. Conclusions: Hepatocyte-derived liver progenitor-like cells are crucial for liver regeneration during chronic hepatic injuries. The administration of HACY, which allowed the induction of endogenous CD24(+) progenitor cells and the inactivation of HSCs, exerts beneficial effects in the treatment of liver fibrosis by re-establishing a balance favoring liver regeneration while preventing fibrotic responses.