Bipotential Adult Liver Progenitors Are Derived from Chronically Injured Mature Hepatocytes

SummaryAdult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass

Bile acid (BA) flux and hepatocyte proliferation after 2/3 partial hepatectomy (PH) in mouse.

<p>PH was performed in mice, and blood, liver and intestines were examined over a 7 day timecourse (3 mice per timepoint). Systemic serum BA rise immediately after PH indicating that the remnant 1/3 liver cannot handle the BA returning via enterohepatic circulation (EHC) from the intestine, thus moving from the EHC to the blood compartment. Liver Cyp7a is repressed soon after PH, followed later by a decrease in FGF15 RNA from the intestine.</p

RNA sequencing of liver after PH in rats with BF and BA infusion.

<p>Total RNA from liver of four rats per group/timepoint had complete RNA sequencing. Pathway analysis revealed an increase in gene expression related to DNA replication (A) in all groups at 24 hours post-PH, but significantly less at the 24 hour timepoint in the BF group compared to control and BF + BA infusion groups. Gene expression in the AP-1 pathway was increased at 4 hr post-PH in the control and BF + BA infusion groups (C), but the BF group had markedly and significantly decreased 4 hr expression compared to the other two groups. Gene expression in the Hippos pathway increased in all groups at 24 hours (D), but significantly less in the BF group. As expected the time zero bile acid gene expression pathway was increased compared to control and BF + BA infusion groups (E). Quantitative PCR for PCNA (B) and Cyp7a (F) confirmed similar patterns in these genes compared to the RNA sequencing data. A single asterisk (*) indicates a significant (p<0.05) difference between the control/BA replacement and the biliary fistula.</p

Bile acid flux and signaling after PH in biliary fistula and BA replacement model.

<p>Rats underwent sham biliary fistula (Control) or biliary fistula (BF) 24 hours prior to PH. Comparisons of serum bile acids (A), liver Cyp7a (B) and intestine Fgf15 (C) are shown between the experimental conditions (n = 4 rats per timepoint). A single asterisk (*) indicates a significant (p<0.05) difference between the control/BA replacement and the biliary fistula, while a double asterisk (**) indicates a significant difference between the control and the BA replacement.</p

Bile Acid Flux Is Necessary for Normal Liver Regeneration

<div><p>Background & Aims</p><p>Many signals governing liver regeneration (LR) following 2/3 partial hepatectomy (PH) are recognized, but the primary signal(s) remains unknown. The aim of the study was to confirm that the remnant liver after PH lacks capacity to secrete the BA pool returning via the enterohepatic ciruculation (EHC), which may in turn stimulate LR.</p><p>Methods</p><p>After standard PH, BA flux was documented and BA signaling (Fgf15) and synthesis (Cyp7a) determined by qPCR. Rat biliary fistula (BF) and Asbt knockout mouse models interrupted the EHC prior to PH, and standard assays for LR employed along with complete RNA sequencing. CCl₄ intoxication after BF tested the hypothesis in an alternate injury model.</p><p>Results</p><p>BA rise in systemic blood immediately following PH, confirming that the remnant liver cannot handle the BA returning via portal circulation. When the BA pool is drained prior to PH in the rat BF model, LR is markedly attenuated, a phenomenon reversed with duodenal BA replacement. Hepatocyte proliferation is similarly attenuated after PH in the Asbt knockout mouse as well as after CCl4₄ intoxication in rats with BF. Complete RNA sequencing in the rat PH model shows that early c-jun and AP-1 gene expression pathways are down regulated in the absence of BA, coincident with attenuated LR.</p><p>Conclusions</p><p>Absent BA return to the liver after PH or CCl₄ injury markedly attenuates LR, though hepatocyte proliferation still occurs, inferring that BA flux and signaling are not the sole signals governing LR. Transcriptional networks involving c-jun and AP-1 are involved in the BA-specific effects on hepatocyte proliferation.</p></div

Hepatocyte proliferation is significantly reduced in rats with BF after CCl₄ intoxication.

<p>After sham BF (controls), BF, or BF + BA infusion, rats were given 2 mg/kg CCl₄, and assessed afterwards. Comparisons of serum bile acids (A), liver Cyp7a (B) and intestine Fgf15 (C) are shown between the experimental conditions. Serum ALT levels after CCl₄ were similar in all groups when used to quantitate liver injury (D), but hepatocyte proliferation was attenuated in rats with BF compared to controls when assessed by Ki-67 staining (E,F). Proliferation was restored when a BA infusion was given to rats with a BF. Four rats per timepoint were used in these experiments. A single asterisk (*) indicates a significant (p<0.05) difference between the control/BA replacement and the biliary fistula experiments, while a double asterisk (**) indicates a significant difference between the control and the BA replacement.</p

Outline of biliary fistula, BA replacement rat model for PH and CCl₄ injury.

<p>Male Sprague-Dawley rats received biliary fistula (or sham) along with duodenal catheter placement and were allowed to recover for 24 hours prior to (A) PH or (B) CCl₄. The intestinal BA pool would therefore be drained prior to PH or CCl₄. In the “BF+BA” group, Na taurocholate was infused via the duodenal catheter 12 hours prior to PH or CCl₄, replacing the intestinal BA pool (but not other constituents of bile) before injury.</p

Comparison of Oligonucleotide Ligation Assay and Consensus Sequencing for Detection of Drug-Resistant Mutants of Human Immunodeficiency Virus Type 1 in Peripheral Blood Mononuclear Cells and Plasma

Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment (ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus in plasma and peripheral blood mononuclear cells (PBMC) was compared to consensus sequence dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low levels of select drug-resistant mutants