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

Abstract

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.

CD24+LCN2+ liver progenitor cells in ductular reaction contributed to macrophage inflammatory responses in chronic liver injury

“…S5b). Although HepLPCs transplantation reduced liver injury as reported before [ 21 ], CD24 + LCN2 + LPC transplantation enhanced ductular reaction and liver fibrosis in H&E, Sirius Red, CK19, and α-SMA staining, and increased the serum levels of ALT and AST compared to control mice on day 40 (Fig. 5 b–d, Additional file 1 : Fig.…”

“…Furthermore, we observed that DDC-hepPDs displayed a closer clustering with HepLPCs rather than Cd24a + Lcn2 + LPCs or BecLPCs, suggesting the correlation between the in vitro-cultured LPCs and the state of their in vivo counterparts. In accordance with this correlation, we also compared the expression profile of isolated Cd24a + LPCs after CCl 4 treatment in vivo (CCl 4 -LPC) [ 21 ] with that of in-vitro cultured Cd24a + Lcn2 + LPCs (Fig. 3 e).…”

“…1 a, Additional file 1 : Fig. S1a, Tables S4 and S5) [ 21 ], suggesting the activation and expansion of ductular reactive cells[ 1 ]. Consistently, sc-RNA analysis of NPCs after CCl 4 -induced liver injury identified a subset of facultative CD24 + LPCs cells expressing SOX9/CK19/HNF1B/FOXA2/ALB/ASS1 (Fig.…”

“…The transcriptional profile of cultured CD24 + LCN2 + LPCs was comparable to that of endogenous CD24 + LPCs in ductular reaction foci [ 21 ]. Furthermore, the presence of CD24 + LCN2 + LPCs was confirmed by sc-RNA seq data of NPCs and immunofluorescent staining of chronic liver injury models.…”

“…When foregut endoderm cells were ready to express the liver-specific gene, Alb [ 5 ], Alb CreERT /R26 GFP mice bred from B6.129S-Albtm1.1(CreERT2) Smoc ( Alb CreERT ) and C57BL/6-Gt (ROSA26) Sortm1(CAG−DTR−EGFP) /Bcgen ( ROSA26 DTR−EGFP ) were used for genetic labeling of albumin (ALB)-expressing cells and characterized by genotyping. These tdTom-positive (tdTom + ) hepatocytes and ALB and EpCAM-negative (GFP − EpCAM − ) cells were isolated using a two-step collagenase perfusion protocol, as described previously [ 21 ].…”