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.

The cellular origins of cancer with particular reference to the gastrointestinal tract.

Stem cells or their closely related committed progenitor cells are the likely founder cells of most neoplasms. In the continually renewing and hierarchically organized epithelia of the oesophagus, stomach and intestine, homeostatic stem cells are located at the beginning of the cell flux, in the basal layer of the oesophagus, the isthmic region of gastric oxyntic glands and at the bottom of gastric pyloric-antral glands and colonic crypts. The introduction of mutant oncogenes such as KrasG12D or loss of Tp53 or Apc to specific cell types expressing the likes of Lgr5 and Mist1 can be readily accomplished in genetically engineered mouse models to initiate tumorigenesis. Other origins of cancer are discussed including 'reserve' stem cells that may be activated by damage or through disruption of morphogen gradients along the crypt axis. In the liver and pancreas, with little cell turnover and no obvious stem cell markers, the importance of regenerative hyperplasia associated with chronic inflammation to tumour initiation is vividly apparent, though inflammatory conditions in the renewing populations are also permissive for tumour induction. In the liver, hepatocytes, biliary epithelial cells and hepatic progenitor cells are embryologically related, and all can give rise to hepatocellular carcinoma and cholangiocarcinoma. In the exocrine pancreas, both acinar and ductal cells can give rise to pancreatic ductal adenocarcinoma (PDAC), although the preceding preneoplastic states are quite different: acinar-ductal metaplasia gives rise to pancreatic intraepithelial neoplasia culminating in PDAC, while ducts give rise to PDAC via. mucinous cell metaplasia that may have a polyclonal origin.

The effect of prophylactic hemoclip placement and risk factors of delayed post-polypectomy bleeding in polyps sized 6 to 20 millimeters: a propensity score matching analysis.

BACKGROUND: Delayed post-polypectomy bleeding (PPB) is a major complication of polypectomy. The effect of prophylactic hemoclipping on delayed PPB is uncertain. The aim of this study was to evaluate the effectiveness of prophylactic hemoclipping and identify the risk factors of delayed PPB. METHODS: Patients with polyps sized 6 to 20 mm underwent snare polypectomy from 2015 to 2017 were retrospectively reviewed. The patients with prophylactic hemoclipping for delayed PPB prevention were included in the clipping group, and those without prophylactic hemoclipping were included in the non-clipping group. The incidence of delayed PPB and time to bleeding were compared between the groups. Multivariate analysis was used to identify the risk factors of delayed PPB. Propensity score matching was used to minimize potential bias. RESULTS: After propensity score matching, 612 patients with 806 polyps were in the clipping group, and 576 patients with 806 polyps were in the non-clipping group. There were no significant differences in the incidence of delayed PPB and days to bleeding between two groups (0.8% vs 1.3%, p = 0.4; 3.4 ± 1.94 days vs 4.13 ± 3.39 days, p = 0.94). In the multivariate analysis, the polyp size [Odds ratio (OR):1.16, 95% confidence interval (CI):1.01-1.16, p = 0.03), multiple polypectomies (OR: 4.64, 95% CI:1.24-17.44, p = 0.02) and a history of anticoagulant use (OR:37.52, 95% CI:6.49-216.8, p < 0.001) were associated with delayed PPB. CONCLUSIONS: In polyps sized 6 to 20 mm, prophylactic hemoclip placement did not decrease the risk of delayed PPB. Patients without risk factors including multiple polypectomies and anticoagulant use are no need to performing prophylactic hemoclipping.

Bile ductular reactions in the liver: similarities are only skin deep.

Extensive bile ductular reactions (DRs) accompany many cholestatic liver diseases such as primary biliary cholangitis and primary sclerosing cholangitis (PSC) as well as parenchymal liver cell diseases such as alcoholic liver disease, non-alcoholic steatohepatitis and HCV and HBV infections. DRs originate from bile ducts or hepatocytes after damage and can be identified by expression of markers associated with cholangiocytes, often being associated with disease progression and fibrosis. In a recent issue of The Journal of Pathology, Govaere et al employed high-throughput RNA sequencing to compare the transcriptomic profiles of DR cells from liver diseases of different aetiology; HCV infection affecting hepatocytes and PSC initially affecting biliary epithelial cells. Both DR transcriptomes were markedly different from that of their neighbouring hepatocytes and 330 genes were significantly differently expressed between the DRs of the HCV and PSC liver diseases. Exploring such gene expression profiles could enable therapeutic targeting of DRs, on the one hand to inhibit liver fibrosis and inflammation and conversely to promote hepatocyte and cholangiocyte regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The many ways to mend your liver: A critical appraisal.

In the latter half of the 20th century, our understanding of mammalian liver regeneration was shaped by the manner of compensatory hyperplasia occurring after a partial rat liver resection. This response involves almost all hepatocytes and thus is unlikely to be the outcome of the multiple cycling of a small stem cell population. It was most intense in the outer third of lobule, the location closest to the afferent arterial blood supply. With the advent of heritable genetic labelling techniques, usually applied to mice, hitherto unrecognized hepatocytes with clonogenic potential have been discovered, contributing to homoeostatic renewal and/or regenerative responses after tissue loss. This review combines observations from cell lineage tracing studies with other data to summarize the Four proposed anatomical locations for hepatocyte stem cells: the periportal zone, the pericentral zone, a randomized distribution and finally within the intrahepatic biliary tree. As in other endodermal-derived tissues, it appears that there are both homoeostatic stem cells and regenerative stem cells, while some normally homoeostatic stem cells can become more active to boost regeneration.

Diverse routes to liver regeneration.

The liver's ability to regenerate is indisputable; for example, after a two-thirds partial hepatectomy in rats all residual hepatocytes can divide, questioning the need for a specific stem cell population. On the other hand, there is a potential stem cell compartment in the canals of Hering, giving rise to ductular reactions composed of hepatic progenitor cells (HPCs) when the liver's ability to regenerate is hindered by replicative senescence, but the functional relevance of this response has been questioned. Several papers have now clarified regenerative mechanisms operative in the mouse liver, suggesting that the liver is possibly unrivalled in its versatility to replace lost tissue. Under homeostatic conditions a perivenous population of clonogenic hepatocytes operates, whereas during chronic damage a minor population of periportal clonogenic hepatocytes come to the fore, while the ability of HPCs to completely replace the liver parenchyma has now been shown.

Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous, not bone marrow-derived cells.

To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony-stimulating factor (P-GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl2 ). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P-GCSF+, EGF+, P-GCSF+EGF+, HgCl2 , HgCl2 +P-GCSF+, HgCl2 +EGF+ and HgCl2 +P-GCSF+EGF+. Following HgCl2 , injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF-treated groups were resistant to this acute kidney injury. A four-in-one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S-phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl2 damage, with no effects of exogenous EGF or P-GCSF. Only 0.5% proximal tubular cells were seen in S-phase in the undamaged group kidneys; this increased to 7-8% after HgCl2 damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S-phase after HgCl2 damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl2 damage, and the major cause of this protective effect was division of indigenous cells, whereas BM-derived cells were less responsive. P-GCSF did not influence damage or regeneration.

Identification of lineage-uncommitted, long-lived, label-retaining cells in healthy human esophagus and stomach, and in metaplastic esophagus.

BACKGROUND & AIMS: The existence of slowly cycling, adult stem cells has been challenged by the identification of actively cycling cells. We investigated the existence of uncommitted, slowly cycling cells by tracking 5-iodo-2'-deoxyuridine (IdU) label-retaining cells (LRCs) in normal esophagus, Barrett's esophagus (BE), esophageal dysplasia, adenocarcinoma, and healthy stomach tissues from patients. METHODS: Four patients (3 undergoing esophagectomy, 1 undergoing esophageal endoscopic mucosal resection for dysplasia and an esophagectomy for esophageal adenocarcinoma) received intravenous infusion of IdU (200 mg/m(2) body surface area; maximum dose, 400 mg) over a 30-minute period; the IdU had a circulation half-life of 8 hours. Tissues were collected at 7, 11, 29, and 67 days after infusion, from regions of healthy esophagus, BE, dysplasia, adenocarcinoma, and healthy stomach; they were analyzed by in situ hybridization, flow cytometry, and immunohistochemical analyses. RESULTS: No LRCs were found in dysplasias or adenocarcinomas, but there were significant numbers of LRCs in the base of glands from BE tissue, in the papillae of the basal layer of the esophageal squamous epithelium, and in the neck/isthmus region of healthy stomach. These cells cycled slowly because IdU was retained for at least 67 days and co-labeling with Ki-67 was infrequent. In glands from BE tissues, most cells did not express defensin-5, Muc-2, or chromogranin A, indicating that they were not lineage committed. Some cells labeled for endocrine markers and IdU at 67 days; these cells represented a small population (<0.1%) of epithelial cells at this time point. The epithelial turnover time of the healthy esophageal mucosa was approximately 11 days (twice that of the intestine). CONCLUSIONS: LRCs of human esophagus and stomach have many features of stem cells (long lived, slow cycling, uncommitted, and multipotent), and can be found in a recognized stem cell niche. Further analyses of these cells, in healthy and metaplastic epithelia, is required.

Nature and mediators of parietal epithelial cell activation in glomerulonephritides of human and rat.

Bowman's capsule parietal epithelial cell activation occurs in several human proliferative glomerulonephritides. The cellular composition of the resulting hyperplastic lesions is controversial, although a population of CD133(+)CD24(+) progenitor cells has been proposed to be a major constituent. Mediator(s) involved in proliferation and migration of progenitor cells into the Bowman's space have been poorly explored. In a series of 36 renal biopsies of patients with proliferative and nonproliferative glomerulopathies, dysregulated CD133(+)CD24(+) progenitor cells of the Bowman's capsule invade the glomerular tuft exclusively in proliferative disorders. Up-regulation of the CXCR4 chemokine receptor on progenitor cells was accompanied by high expression of its ligand, SDF-1, in podocytes. Parietal epithelial cell proliferation might be sustained by increased expression of the angiotensin II (Ang II) type-1 (AT1) receptor. Similar changes of CXCR4, SDF-1, and AT1 receptor expression were found in Munich Wistar Frömter rats with proliferative glomerulonephritis. Moreover, an angiotensin-converting enzyme inhibitor normalized CXCR4 and AT1 receptor expression on progenitors concomitant with regression of crescentic lesions in a patient with crescentic glomerulonephritis. These results suggest that glomerular hyperplastic lesions derive from the proliferation and migration of renal progenitors in response to injured podocytes. The Ang II/AT1 receptor pathway may participate, together with SDF-1/CXCR4 axis, to the dysregulated response of renal precursors. Thus, targeting the Ang II/AT1 receptor/CXCR4 pathways may be beneficial in severe forms of glomerular proliferative disorders.

CD133 as a biomarker for putative cancer stem cells in solid tumours: limitations, problems and challenges.

The cancer stem cell (CSC) hypothesis, despite the limitations of the currently available models and assays, has ushered in a new era of excitement in cancer research. The development of novel strategies for anti-tumour therapy relies on the use of biomarkers to identify, enrich, and/or isolate the cell population(s) of interest. In this context, various cell characteristics and antigen expression profiles are discussed as surrogate markers. The cell surface expression of the human prominin-1 (CD133) antigen, in particular of the AC133 epitope, is among those that have been most frequently studied in solid cancers, although no mechanism has yet been proposed to link CD133 expression with the CSC phenotype. Some inconsistencies between published data can be ascribed to different analytical tools as well as methodological limitations and pitfalls, highlighted in the present review. Therefore, a comprehensive overview on the current state of knowledge in this growing and exciting field with an emphasis on the most recent studies is presented. We highlight the link between the tumour microenvironment, tumour cell plasticity, and CD133 expression, and evaluate the utility of CD133 expression as a prognostic marker.

Stem cells in cancer: instigators and propagators?

There is growing realization that many - if not all - cancer-cell populations contain a subpopulation of self-renewing stem cells known as cancer stem cells (CSCs). Unlike normal adult stem cells that remain constant in number, CSCs can increase in number as tumours grow, and give rise to progeny that can be both locally invasive and colonise distant sites - the two hallmarks of malignancy. Immunodeficient mouse models in which human tumours can be xenografted provide persuasive evidence that CSCs are present in human leukaemias and many types of solid tumour. In addition, many studies have found similar subpopulations in mouse tumours that show enhanced tumorigenic properties when they are transplanted into histocompatible mice. In this Commentary, we refer to CSCs as tumour-propagating cells (TPCs), a term that reflects the assays that are currently employed to identify them. We first discuss evidence that cancer can originate from normal stem cells or closely related descendants. We then outline the attributes of TPCs and review studies in which they have been identified in various cancers. Finally, we discuss the implications of these findings for successful cancer therapies.

Bone marrow-derived cells contribute to cerulein-induced pancreatic fibrosis in the mouse.

Bone marrow (BM) cells may transdifferentiate into circulating fibrocytes and myofibroblasts in organ fibrosis. In this study, we investigated the contribution and functional roles of BM-derived cells in murine cerulein-induced pancreatic fibrosis. C57/BL6 female mice wild-type (WT) or Col 1α1(r/r) male BM transplant, received supraphysiological doses of cerulein to induce pancreatic fibrosis. The CD45(+)Col 1(+) fibrocytes isolated from peripheral blood (PB) and pancreatic tissue were examined by in situ hybridization for Y chromosome detection. The number of BM-derived myofibroblasts, the degree of Sirius red staining and the levels of Col 1α1 mRNA were quantified. The Y chromosome was detected in the nuclei of PB CD45(+)Col 1(+) fibrocytes, confirming that circulating fibrocytes can be derived from BM. Co-expression of α-smooth muscle actin illustrated that fibrocytes can differentiate into myofibroblasts. The number of BM-derived myofibroblasts, degree of collagen deposition and pro-collagen I mRNA expression were higher in the mice that received Col 1α1(r/r) BM, (cells that produce mutated, collagenase-resistant collagen) compared to WT BM, indicating that the genotype of BM cells can alter the degree of pancreatic fibrosis. Our data indicate that CD45(+)Col 1(+) fibrocytes in the PB can be BM-derived, functionally contributing to cerulein-induced pancreatic fibrosis in mice by differentiating into myofibroblasts.

Hepatocyte turnover and regeneration: virtually a virtuoso performance.

The liver and exocrine pancreas share a common structure, with functioning units (hepatic plates and pancreatic acini) connected to the ductal tree. Here we show that Sox9 is expressed throughout the biliary and pancreatic ductal epithelia, which are connected to the intestinal stem-cell zone. Cre-based lineage tracing showed that adult intestinal cells, hepatocytes and pancreatic acinar cells are supplied physiologically from Sox9-expressing progenitors. Combination of lineage analysis and hepatic injury experiments showed involvement of Sox9-positive precursors in liver regeneration. Embryonic pancreatic Sox9-expressing cells differentiate into all types of mature cells, but their capacity for endocrine differentiation diminishes shortly after birth, when endocrine cells detach from the epithelial lining of the ducts and form the islets of Langerhans. We observed a developmental switch in the hepatic progenitor cell type from Sox9-negative to Sox9-positive progenitors as the biliary tree develops. These results suggest interdependence between the structure and homeostasis of endodermal organs, with Sox9 expression being linked to progenitor status.

Cancer stem cells: problems for therapy?

Many, if not all, tumours contain a sub-population of self-renewing and expanding stem cells known as cancer stem cells (CSCs). The symmetric division of CSCs is one mechanism enabling expansion in their numbers as tumours grow, while epithelial-mesenchymal transition (EMT) is an increasingly recognized mechanism to generate further CSCs endowed with a more invasive and metastatic phenotype. Putative CSCs are prospectively isolated using methods based on either a surface marker or an intracellular enzyme activity and then assessed by a 'sphere-forming' assay in non-adherent culture and/or by their ability to initiate new tumour growth when xenotransplanted into immunocompromised mice-hence, these cells are often referred to as tumour-propagating cells (TPCs). Cell sub-populations enriched for tumour-initiating ability have also been found in murine tumours, countering the argument that xenografting human cells merely select human cells with an ability to grow in mice. Cancer progression can be viewed as an evolutionary process that generates new/multiple clones with a fresh identity; this may be a major obstacle to successful cancer stem cell eradication if treatment targets only a single type of stem cell. In this review, we first briefly discuss evidence that cancer can originate from normal stem cells or closely related descendants. We then outline the attributes of CSCs and review studies in which they have been identified in various cancers. Finally, we discuss the implications of these findings for successful cancer therapies, concentrating on the self-renewal pathways (Wnt, Notch, and Hedgehog), aldehyde dehydrogenase activity, EMT, miRNAs, and other epigenetic modifiers as potential targets for therapeutic manipulation.

Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response.

BACKGROUND AND METHODS: In advanced liver damage, hepatic regeneration can occur through proliferation of a resident hepatic progenitor cell (HPC) population. HPCs are located within a designated niche in close association with myofibroblasts and bone marrow (BM) derived macrophages. Extra-cellular matrix (ECM) laminin invariably surrounds HPCs, but the functional requirement of this matrix-cell association is untested in vivo. Using the collagen Iα1((r/r)) mouse (r/r), which produces mutated collagen I resistant to matrix metalloproteinase degradation and has an exaggerated fibrotic response to liver injury, we test the relationship between collagen degradation, laminin deposition, and the HPC response. RESULTS: Chronic fibrotic carbon tetrachloride (CCl4) injury can induce a florid HPC response associated with dense laminin deposition. In the recovery phase after chronic CCl4 injury, r/r mice have a markedly attenuated HPC response compared to wild-types, together with persistence of collagen I and failure to deposit ECM laminin. Similar results were found in r/r mice given the choline-deficient ethionine supplemented diet, another model of the HPC response. In cross-over sex-mismatched BM transplantation (BMT) experiments between r/r mice and wild-types, the blunted HPC response of r/r mice was not rescued by wild-type BMT and likewise not conferred on to wild-type recipients by r/r BMT, demonstrating that the attenuated HPC response in r/r mice is a property intrinsic to the liver. CONCLUSION: Failure of ECM remodelling after chronic fibrotic liver injury hinders the ability of the liver to activate HPCs. Laminin-progenitor cell interactions within the HPC niche are a critical for HPC mediated regeneration.

The histogenesis of regenerative nodules in human liver cirrhosis.

UNLABELLED: Here, we investigate the clonality and cells of origin of regenerative nodules in human liver cirrhosis using mitochondrial DNA (mtDNA) mutations as markers of clonal expansion. Mutated cells are identified phenotypically by deficiency in the entirely mtDNA encoded cytochrome c oxidase (CCO) enzyme by histochemical and immunohistochemical methods. Nodules were classified as either CCO-deficient or CCO-positive, and among 526 nodules from 10 cases, 18% were homogeneously CCO-deficient, whereas only 3% had a mixed phenotype. From frozen sections, hepatocytes were laser-capture microdissected from several sites within individual CCO-deficient nodules. Mutations were identified by polymerase chain reaction sequencing of the entire mtDNA genome. In all cases except for one, the nodules were monoclonal in nature, possessing up to four common mutations in all hepatocytes in a given nodule. Moreover, the identification of identical mutations in hepatic progenitor cells abutting CCO-deficient nodules proves that nodules can have their origins from such cells. CONCLUSION: These data support a novel pathway for the monoclonal derivation of human cirrhotic regenerative nodules from hepatic progenitor cells.

Chronic inflammation and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) invariably develops within a setting of chronic inflammation caused by either hepatotropic viruses, toxins, metabolic liver disease or autoimmunity. Mechanisms that link these two processes are not completely understood, but transcription factors of the NF-κB family and signal transducer and activator of transcription 3 (STAT3), cytokines such as IL-6 and IL-1α and ligands of the epidermal growth factor receptor (EGFR) family are clearly pivotal players. HCC may have its origins in either hepatocytes or hepatic progenitor cells (HPCs), and HCCs, like other solid tumours appear to be sustained by a minority population of cancer stem cells.