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 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.

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.

Aldolase triggers metabolic reprogramming in colorectal cancer in hypoxia and stiff desmoplastic microenvironments.

Colorectal cancer (CRC) progression is highly associated with desmoplasia. Aerobic glycolysis is another distinct feature that appears during the CRC phase of the adenoma-carcinoma sequence. However, the interconnections between the desmoplastic microenvironment and metabolic reprogramming remain largely unexplored. In our in vitro model, we investigated the compounding influences of hypoxia and substrate stiffness, two critical physical features of desmoplasia, on the CRC metabolic shift by using engineered polyacrylamide gels. Unexpectedly, we found that compared to cells on a soft gel (approximately 1.5 kPa, normal tissue), cells on a stiff gel (approximately 8.7 kPa, desmoplastic tissue) exhibited reduced glucose uptake and glycolysis under both normoxia and hypoxia. In addition, the increasing substrate stiffness activated focal adhesion kinase (FAK)/phosphoinositide 3-kinase signaling, but not the mitochondrial respiratory inhibitor HIF-1α. However, the presence of aldolase B (ALDOB) reversed the CRC metabolic response to mechanosignaling; enhanced glucose uptake (approximately 1.5-fold) and aerobic glycolysis (approximately 2- to 3--fold) with significantly decreased mitochondrial oxidative phosphorylation. ALDOB also changed the response of CRC traction force, which is related to tumor metastasis, under hypoxia/normoxia. In summary, our data suggest a counter influence of hypoxia and substrate stiffness on glucose uptake, and ALDOB upregulation can reverse this, which drives hypoxia and stiff substrate to enhance the CRC aerobic glycolysis synergistically. The results not only highlight the potential impacts on metabolic reprogramming led by physical alterations in the microenvironment, but also extend our understanding of the essential role of ALDOB in CRC progression from a biophysical perspective.

Dynamic bioenergetic alterations in colorectal adenomatous polyps and adenocarcinomas.

BACKGROUND: Energy metabolism in carcinogenesis is poorly understood. It is widely accepted the majority of colorectal cancers (CRCs) arise from adenomatous polyps (APs). We aimed to characterize the bioenergetic alterations in APs and CRCs. METHODS: Fifty-six APs, 93 CRCs and adjacent normal mucosae were tested. Oxygen consumption rate (OCR) was measured representing mitochondrial oxidative phosphorylation (OxPhos), and extracellular acidification rate (ECAR)was measured representing glycolysis. Mitochondrial DNA (mtDNA) variants and mutations were studied. Over-expressed metabolic genes in APs were identified by microarray and validated by qRT-PCR, Western blots and immunohistochemistry. Identified genes were knocked down in WiDr and colo205 CRC cell lines, and their expression was analyzed in APs/CRCs with enhanced glycolysis. FINDINGS: ECAR, not OCR, was significantly increased in APs. While no difference of ECAR was found between CRCs and normal mucosae, OCR was significantly reduced in CRCs. OCR/ECAR ratio was decreased in APs over 1 cm, APs with a villous component and CRCs, indicating their glycolytic tendencies. The number of mtDNA mutations was increased in APs and CRCs, but not correlated with metabolic profiles. Two metabolic genes ALDOB and SLC16A4 were up-regulated in APs. Both ALDOB-knockdown and SLC16A4-knockdown CRC cell lines showed increased basal motichondrial OxPhos and decreased basal glycolysis. Moreover, the increase of mitochondrial ATP-linked respiration and the decrease of glycolytic capacity were showed in SLC16A4-knockdown cells. Finally, APs/CRCs with enhanced glycolysis had increased SLC16A4 expression. INTERPRETATION: ATP production shifts from OxPhos to glycolysis in the process of AP enlargement and villous transformation. OxPhos defects are present in CRCs but not in APs. APs and CRCs tend to accumulate mtDNA mutations, but these are not correlated with bioenergetic profiles. Finally, the ALDOB and SLC16A4 may contribute to the glycolytic shift in APs/CRCs.

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.

pH regulators to target the tumor immune microenvironment in human hepatocellular carcinoma.

Interfering with tumor metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Featuring a rapid proliferation rate and exacerbated glycolysis, hepatocellular carcinoma (HCC) creates a highly hypoxic microenvironment with excessive production of lactic and carbonic acids. These metabolic conditions promote disease aggressiveness and cancer-related immunosuppression. The pH regulatory molecules work as a bridge between tumor cells and their surrounding milieu. Herein, we show that the pH regulatory molecules CAIX, CAXII and V-ATPase are overexpressed in the HCC microenvironment and that interfering with their pathways exerts antitumor activity. Importantly, the V-ATPase complex was expressed by M2-like tumor-associated macrophages. Blocking ex vivo V-ATPase activity established a less immune-suppressive tumor microenvironment and reversed the mesenchymal features of HCC. Thus, targeting the unique cross-talk between tumor cells and the tumor microenvironment played by pH regulatory molecules holds promise as a strategy to control HCC progression and to reduce the immunosuppressive pressure mediated by the hypoxic/acidic metabolism, particularly considering the potential combination of this strategy with emerging immune checkpoint-based immunotherapies.

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.

An Immunopathological Evaluation of the Porcine Cholecyst Matrix as a Muscle Repair Graft in a Male Rat Abdominal Wall Defect Model.

With the increasing use of animal-based biomaterials for regenerative medical applications, the need for their safety assessment is paramount. A porcine cholecyst-derived scaffold (CDS), intended as a muscle repair graft, prepared by a nondetergent/enzymatic method was engrafted in a rat abdominal wall defect model. Host tissue-scaffold interface samples were collected 2, 8, and 16 weeks postimplantation and evaluated by histopathology, immunohistochemistry, and electron microscopy. The nature of the tissue reaction was compared with those induced by a jejunum-derived scaffold (JDS) prepared by the same method and a commercial-grade small intestinal submucosa (CSIS) scaffold. A study of the immunopathological response in major lymphoid tissues and immunophenotyping for M1 and M2 macrophages was performed at the host tissue-scaffold interface. Further, "irritancy scores" for CDS and JDS were determined using CSIS as the reference material. Both CDS and JDS appeared to be potential biomaterials for muscle grafts, but the former stimulated a skeletal muscle tissue remodeling response predominated by M2 macrophages. The data support the notion that biomaterials with similar biocompatibility, based on local tissue response on implantation, may cause differential immunogenicity. Additionally, CDS compared to JDS and CSIS was found to be less immunotoxic.

Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury.

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

Cholangiocytes: No Longer Cinderellas to the Hepatic Regenerative Response.

Biliary ductal cells proliferate from the portal areas of chronically damaged livers, but their significance to regeneration has been controversial. A recent article in Nature by Raven et al. (2017) now shows that blocking hepatocyte replication is essential for the hepatic differentiation of ductular cells after liver damage.

A gold nanoparticle coated porcine cholecyst-derived bioscaffold for cardiac tissue engineering.

Extracellular matrices of xenogeneic origin have been extensively used for biomedical applications, despite the possibility of heterogeneity in structure. Surface modification of biologically derived biomaterials using nanoparticles is an emerging strategy for improving topographical homogeneity when employing these scaffolds for sophisticated tissue engineering applications. Recently, as a tissue engineering scaffold, cholecyst derived extracellular matrix (C-ECM) has been shown to have several advantages over extracellular matrices derived from other organs such as jejunum and urinary bladder. This study explored the possibility of adding gold nanoparticles, which have a large surface area to volume ratio on C-ECM for achieving homogeneity in surface architecture, a requirement for cardiac tissue engineering. In the current study, gold nanoparticles (AuNPs) were synthesized and functionalised for conjugating with a porcine cholecystic extracellular matrix scaffold. The conjugation of nanoparticles to C-ECM was achieved by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide chemistry and further characterized by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy and thermogravimetric analysis. The physical properties of the modified scaffold were similar to the original C-ECM. Biological properties were evaluated by using H9c2 cells, a cardiomyoblast cell line commonly used for cellular and molecular studies of cardiac cells. The modified scaffold was found to be a suitable substrate for the growth and proliferation of the cardiomyoblasts. Further, the non-cytotoxic nature of the modified scaffold was established by direct contact cytotoxicity testing and live/dead staining. Thus, the modified C-ECM appears to be a potential biomaterial for cardiac tissue engineering.

Regenerating the liver: not so simple after all?

Under normal homeostatic conditions, hepatocyte renewal is a slow process and complete turnover likely takes at least a year. Studies of hepatocyte regeneration after a two-thirds partial hepatectomy (2/3 PH) have strongly suggested that periportal hepatocytes are the driving force behind regenerative re-population, but recent murine studies have brought greater complexity to the issue. Although periportal hepatocytes are still considered pre-eminent in the response to 2/3 PH, new studies suggest that normal homeostatic renewal is driven by pericentral hepatocytes under the control of Wnts, while pericentral injury provokes the clonal expansion of a subpopulation of periportal hepatocytes expressing low levels of biliary duct genes such as Sox9 and osteopontin. Furthermore, some clarity has been given to the debate on the ability of biliary-derived hepatic progenitor cells to generate physiologically meaningful numbers of hepatocytes in injury models, demonstrating that under appropriate circumstances these cells can re-populate the whole liver.

The Influence of Bone Marrow-Secreted IL-10 in a Mouse Model of Cerulein-Induced Pancreatic Fibrosis.

This study aimed to understand the role of IL-10 secreted from bone marrow (BM) in a mouse model of pancreatic fibrosis. The severity of cerulein-induced inflammation, fibrosis, and the frequency of BM-derived myofibroblasts were evaluated in the pancreas of mice receiving either a wild-type (WT) BM or an IL-10 knockout (KO) BM transplantation. The area of collagen deposition increased significantly in the 3 weeks after cerulein cessation in mice with an IL-10 KO BM transplant (13.7 ± 0.6% and 18.4 ± 1.1%, p < 0.05), but no further increase was seen in WT BM recipients over this time. The percentage of BM-derived myofibroblasts also increased in the pancreas of the IL-10 KO BM recipients after cessation of cerulein (6.7 ± 1.1% and 11.9 ± 1.3%, p < 0.05), while this figure fell in WT BM recipients after cerulein withdrawal. Furthermore, macrophages were more numerous in the IL-10 KO BM recipients than the WT BM recipients after cerulein cessation (23.2 ± 2.3 versus 15.3 ± 1.7 per HPF, p < 0.05). In conclusion, the degree of fibrosis, inflammatory cell infiltration, and the number of BM-derived myofibroblasts were significantly different between IL-10 KO BM and WT BM transplanted mice, highlighting a likely role of IL-10 in pancreatitis.

Immunomodulatory Factors Control the Fate of Melanoma Tumor Initiating Cells.

Melanoma is a highly heterogeneous tumor for which recent evidence supports a model of dynamic stemness. Melanoma cells might temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context. However, factors driving these functional changes are still unknown. We focused on the role of cyto/chemokines in shaping TICs isolated directly from tumor specimens of two melanoma patients, namely Me14346S and Me15888S. We analyzed the secretion profile of TICs and of their corresponding melanoma differentiated cells and we tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We found that TICs, grown in vitro as melanospheres, had a complex secretory profile as compared to their differentiated counterparts. Some factors, such as CCL-2 and IL-8, also produced by adherent melanoma cells and melanocytes did not influence TIC properties. Conversely, IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation while IL-10, produced by Me15888S, strongly promoted TIC self-renewal through paracrine/autocrine actions. Complete neutralization of IL-10 activity by gene silencing and antibody-mediated blocking of the IL-10Rα was required to sensitize Me15888S to IL-6-induced differentiation. For the first time these results show that functional heterogeneity of melanoma could be directly influenced by inflammatory and suppressive soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Thus, understanding the tumor microenvironment (TME) role in modulating melanoma TIC phenotype is fundamental to identifying novel therapeutic targets to achieve long-lasting regression of metastatic melanoma. Stem Cells 2016;34:2449-2460.

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.

Cell lineage tracing in human epithelial tissues using mitochondrial DNA mutations as clonal markers.

The study of cell lineages through heritable genetic lineage tracing is well established in experimental animals, particularly mice. While such techniques are not feasible in humans, we have taken advantage of the fact that the mitochondrial genome is highly prone to nonpathogenic mutations and such mutations can be used as clonal markers to identify stem cell derived clonal populations in human tissue sections. A mitochondrial DNA (mtDNA) mutation can spread by a stochastic process through the several copies of the circular genome in a single mitochondrion, and then through the many mitochondria in a single cell, a process called 'genetic drift.' This process takes many years and so is likely to occur only in stem cells, but once established, the fate of stem cell progeny can be followed. A cell having at least 80% of its mtDNA genomes bearing the mutation results in a demonstrable deficiency in mtDNA-encoded cytochrome c oxidase (CCO), optimally detected in frozen tissue sections by dual-color histochemistry, whereby CCO activity stains brown and CCO deficiency is highlighted by subsequent succinate dehydrogenase activity, staining the CCO-deficient areas blue. Cells with CCO deficiency can be laser captured and subsequent mtDNA sequencing can ascertain the nature of the mutation. If all cells in a CCO-deficient area have an identical mutation, then a clonal population has been identified; the chances of the same mutation initially arising in separate cells are highly improbable. The technique lends itself to the study of both normal epithelia and can answer several questions in tumor biology. WIREs Dev Biol 2016, 5:103-117. doi: 10.1002/wdev.203 For further resources related to this article, please visit the WIREs website.