Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Abstract

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

Figure 1. Deletion of DDB1 in hepatocytes results in hepatic oval cells activation.

(A) H&E staining of liver sections from 4-week old DDB1^F/F and DDB1^F/F;Alb-Cre^+/+ mice. PV, portal vein. (B) IHC staining for DDB1 on liver sections from 4-week old DDB1^F/F and DDB1^F/F;Alb-Cre^+/+ mice. (C) Co-IF staining for DDB1 and A6 on 4-week old DDB1^F/F and DDB1^F/F;Alb-Cre^+/+ liver sections.

Figure 2. Expression of oval cell markers in DDB1 mutant mouse liver.

(A) Co-IF staining for Cytokeratin-19 (CK19) and Albumin (Alb) (upper panels), E-cadherin and DDB1 (middle panels), α-fetoprotein (AFP) and CD133 (lower panels). (B–D) Co-IF staining for EpCAM and A6 on liver sections from 4-week old DDB1^F/F;Alb-Cre^+/+ (B), 6-week old DDB1^F/F;Alb-Cre^+/− (C), and adult DDB1^F/F;Mx1-Cre^+/− mice at 6 weeks after receiving poly(I:C) injection (D).

Figure 3. Deletion of p21 partially restores proliferation of DDB1-deficient hepatocytes and alleviates OC activation.

(A) Western blot for some substrates of DDB1-Cul4A ubiquitin ligase using lysates of hepatocytes isolated from DDB1^F/F and DDB1^F/F;Alb-Cre^+/+ mice. (B) Western blot for DDB1 and p21 using lysates of cytoplasmic fraction (C) and nuclear fraction (N) prepared from MEFs. (C) IHC staining for DDB1 and Ki67 on liver sections from DDB1^F/F;Alb-Cre^+/−, DDB1^F/F;p21^−/− and DDB1^F/F;Alb-Cre^+/−;p21^−/− mice. (D) Co-IF staining for A6 and DDB1 on DDB1^F/F;p21^−/− and DDB1^F/F;Alb-Cre^+/−;p21^−/− liver sections. Arrows in DDB1^F/F;Alb-Cre^+/−;p21^−/− mice indicate A6 positive oval cells.

Figure 4. EpCAM-positive cells from DDB1 mutant liver proliferate in vitro and repopulate the liver in vivo.

(A) Analysis by flow cytometry of EpCAM⁺ F4/80⁻ cells in non-parenchymal fractions prepared from 4-week old DDB1^F/F and DDB1^F/F;Alb-Cre^+/+ mice. The ratio of EpCAM⁺ F4/80⁻ cells from a representative experiment is shown in each panel. (B) In vitro culture of EpCAM⁺ cells sorted by FACS from DDB1 mutant liver and seeded on Matrigel. Cells form colonies after 1, 3, and 9 days of culture (upper panels) and exhibit EpCAM positivity (lower panels). (C) Co-IF staining for A6 and AFP on colonies from cultured EpCAM⁺ cells. (D) Experimental scheme for in vivo differentiation of EpCAM⁺ OCs. Adult DDB1^F/F;Mx1-Cre^+/−;Rosa26-lacZ mice were generated and injected with poly(I:C). EpCAM⁺ cells were isolated by FACS 6 weeks later, and injected intrasplenically to nude mice that had been fed with DDC diet. The recipient nude mice received poly(I:C) injection 2 weeks later and liver collected for analysis on the following day. (E) Isolation of EpCAM⁺ F4/80⁻ cells from donor or control mouse liver. The ratio of EpCAM⁺ F4/80⁻ cells is shown in each panel. (F) IHC staining for ß-gal on liver sections from recipient nude mice. Arrows indicate positively stained hepatocytes.

Figure 5. DDB1 mutant mice exhibit minor liver damage compared with DDC-treated mice.

(A): Co-IF staining for A6 and EpCAM on liver sections from DDC-treated mice. (B): Gross appearance of liver from mice fed with DDC diet for 4 weeks. (C): H&E staining of the liver in (B). (D): IHC staining for CD45 (upper panels) and F4/80 (lower panels) on liver sections from DDB1^F/F, DDB1^F/F;Alb-Cre^+/+ and DDC-treated mice. (E): Percentage of EpCAM⁺ cells from DDB1^F/F;Alb-Cre^+/+ and DDC-diet liver, determined by FACS analysis. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as the means±SEM; n = 3. (F): Serum alanine aminotransferase (ALT) levels. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as means±SEM; n = 3 **P<0.01.

Figure 6. Characterization of OCs from DDB1 mutant mice and DDC-treated mice.

(A–C): Quantitative real-time PCR analysis for selected genes expressed in hepatocytes isolated from wild type mice, and EpCAM⁺ cells isolated from DDC-treated and DDB1^F/F; Alb-Cre^+/+ mice. All data are normalized to 18s rRNA level. Data are representative of 4 independent experiments with 3–4 mice per group. *P<0.05 (A): Expression levels of hematopoietic markers Sca1, Thy1 and Cd44, and OC markers CD133, Connexin43 and Ncam1. (B): Expression levels of cholangiocyte marker Ck19 and Spp1, and hepatocyte marker Alb. (C): Expression levels of candidate OC markers, Reelin, EdnrB and Cd206. (D): IHC staining for reelin on liver sections from DDB1^F/F, DDB1^F/F;Alb-Cre^+/+ and DDC-treated mice. Arrows in DDC-treated liver indicate non-specific brown deposits.