Hepatocytes undergo punctuated expansion dynamics from a periportal stem cell niche in normal human liver

Background & Aims: While normal human liver is thought to be generally quiescent, clonal hepatocyte expansions have been observed, though neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease-associated dysregulation. Methods: Herein, we use in vivo lineage tracing and methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next-generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially distinct areas of laser-captured, microdissected, clones, in tandem with computational modelling in morphologically normal human liver. Results: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mitochondrial DNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mitochondrial DNA variants reveal large numbers of spatially restricted mutations in conjunction with limited numbers of clonal mutations. Conclusions: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in the normal human liver. Impact and implications: The liver is mainly composed of hepatocytes, but we know little regarding the source of these cells or how they multiply over time within the disease-free human liver. In this study, we determine a source of new hepatocytes by combining many different lab-based methods and computational predictions to show that hepatocytes share a common cell of origin with bile ducts. Both our experimental and computational data also demonstrate hepatocyte clones are likely to expand in slow waves across the liver in a specific trajectory, but often lie dormant for many years. These data show for the first time the expansion dynamics of hepatocytes in normal liver and their cell of origin enabling the accurate measurment of changes to their dynamics that may lead to liver disease. These findings are important for researchers determining cancer risk in human liver

Clonal transitions and phenotypic evolution in Barrett esophagus

BACKGROUND & AIMS: Barrett's esophagus (BE) is a risk factor for esophageal adenocarcinoma but our understanding of how it evolves is poorly understood. We investigated BE gland phenotype distribution, the clonal nature of phenotypic change, and how phenotypic diversity plays a role in progression. METHODS: Using immunohistochemistry and histology, we analyzed the distribution and the diversity of gland phenotype between and within biopsy specimens from patients with nondysplastic BE and those who had progressed to dysplasia or had developed postesophagectomy BE. Clonal relationships were determined by the presence of shared mutations between distinct gland types using laser capture microdissection sequencing of the mitochondrial genome. RESULTS: We identified 5 different gland phenotypes in a cohort of 51 nondysplastic patients where biopsy specimens were taken at the same anatomic site (1.0-2.0 cm superior to the gastroesophageal junction. Here, we observed the same number of glands with 1 and 2 phenotypes, but 3 phenotypes were rare. We showed a common ancestor between parietal cell-containing, mature gastric (oxyntocardiac) and goblet cell-containing, intestinal (specialized) gland phenotypes. Similarly, we have shown a clonal relationship between cardiac-type glands and specialized and mature intestinal glands. Using the Shannon diversity index as a marker of gland diversity, we observed significantly increased phenotypic diversity in patients with BE adjacent to dysplasia and predysplasia compared to nondysplastic BE and postesophagectomy BE, suggesting that diversity develops over time. CONCLUSIONS: We showed that the range of BE phenotypes represents an evolutionary process and that changes in gland diversity may play a role in progression. Furthermore, we showed a common ancestry between gastric and intestinal-type glands in BE

YAP immunofluorescent staining is non-specific in LPCs.

<p>Wild-type (WT) non-transformed BMEL A-EGFP cells were stably infected with lentiviruses bearing control (Con) or YAP-targeting (KD) shRNAs. Stably infected cells were grown on coverslips and fixed before being stained with YAP (a, e, i) and counterstained with Hoechst stain (b, f, j). Overlay of YAP and Hoechst images are shown in (c, g, k). Undetectable immunofluorescence was seen with the Alexa Fluor-488 antibody alone (d, h, l).</p

YAP immunofluorescent staining is non-specific in MEFs.

<p>Wild-type (WT) MEFs were stably infected with lentivirus harbouring control (Con) or YAP-targeting (KD) shRNAs. <b>A</b>) Lysates of stably infected cells were separated by SDS-PAGE, transferred to membrane and immunoblotted for YAP and β-actin as indicated. Size markers are shown in kilodaltons. <b>B</b>) Stably infected cells were grown on coverslips and fixed before being stained with YAP (a, e, i) and counterstained with Hoechst stain (b, f, j). Overlay of YAP and Hoechst images are shown in (c, g, k). Immunofluorescence was not detected using Alexa Fluor-488 secondary antibody alone (d, h, l). <b>C)</b> Con MEFs were pre-labelled with MT then washed, trypsinized and mixed 1:1 with unlabelled YAP KD MEFs before being replated onto glass coverslips. Cells were allowed to settle for 4 h before being fixed and stained for YAP (a) and counterstained with Hoechst stain. MT was visualised (d) and merged images of YAP/MT (b), and Hoechst/MT (c) are shown, with enlarged insets (boxed regions in b and c) shown in (e) and (f), respectively.</p

YAP protein is undetectable in shRNA-mediated YAP knockdown LPCs.

<p>Wild-type (WT) non-transformed BMEL A-EGFP LPCs (BMEL) were stably infected with lentiviruses bearing control (Con) or YAP-targeting (KD) shRNAs. After selection with puromycin stably infected cells were harvested. Cell lysates were separated by SDS-PAGE, transferred to membrane and immunoblotted for YAP and β-actin as indicated. Size markers are shown in kilodaltons.</p

YAP immunofluorescent staining correlates with YAP abundance in D645 cells.

<p>Stably infected D645 cells were treated with or without 100 nM 4HT for 24 h to induce hYAP1 expression as indicated. A). Cell lysates were separated by SDS-PAGE, transferred to membrane and immunoblotted for YAP and β-actin, as indicated. B) Cells were grown on coverslips and fixed before being stained with YAP (c, g) and counterstained with Hoechst stain (b, i). Merge of YAP and Hoechst images are shown in (d, h). Undetectable immunofluorescence was seen with the Alexa Fluor-488 antibody alone (a, e).</p

A modified choline-deficient, ethionine-supplemented diet reduces morbidity and retains a liver progenitor cell response in mice

The choline-deficient, ethionine-supplemented (CDE) dietary model induces chronic liver damage, and stimulates liver progenitor cell (LPC)-mediated repair. Long-term CDE administration leads to hepatocellular carcinoma in rodents and lineage-tracing studies show that LPCs differentiate into functional hepatocytes in this model. The CDE diet was first modified for mice by our laboratory by separately administering choline-deficient chow and ethionine in the drinking water (CD+E diet). Although this CD+E diet is widely used, concerns with variability in weight loss, morbidity, mortality and LPC response have been raised by researchers who have adopted this model. We propose that these inconsistencies are due to differential consumption of chow and ethionine in the drinking water, and that incorporating ethionine in the choline-deficient chow, and altering the strength, will achieve better outcomes. Therefore, C57Bl/6 mice, 5 and 6 weeks of age, were fed an all-inclusive CDE diet of various strengths (67% to 100%) for 3 weeks. The LPC response was quantitated and cell lines were derived. We found that animal survival, LPC response and liver damage are correlated with CDE diet strength. The 67% and 75% CDE diet administered to mice older than 5 weeks and greater than 18 g provides a consistent and acceptable level of animal welfare and induces a substantial LPC response, permitting their isolation and establishment of cell lines. This study shows that an all-inclusive CDE diet for mice reproducibly induces an LPC response conducive to in vivo studies and isolation, whilst minimizing morbidity and mortality