Hepatic progenitor cells in human liver tumor development

In recent years, the results of several studies suggest that human liver tumors can be derived from hepatic progenitor cells rather than from mature cell types. The available data indeed strongly suggest that most combined hepatocellular-cholangiocarcinomas arise from hepatic progenitor cells that retained their potential to differentiate into the hepatocytic and biliary lineages. Hepatic progenitor cells could also be the basis for some hepatocellular carcinomas and hepatocellular adenomas, although it is very difficult to determine the origin of an individual hepatocellular carcinoma. There is currently not enough data to make statements regarding a hepatic progenitor cell origin of cholangiocarcinoma. The presence of hepatic progenitor cell markers and the presence and extent of the cholangiocellular component are factors that are related to the prognosis of hepatocellular carcinomas and combined hepatocellular- cholangiocarcinomas, respectively.

Characterization of hepatic progenitors from human fetal liver during second trimester

AIM： To enrich hepatic progenitors using epithelial cell adhesion molecule （EpCAM） as a marker from human fetal liver and investigate the expression of human leukocyte antigen （HLA） and their markers associated with hepatic progenitor cells. METHODS： EpCAM ＋ve cells were isolated using magnetic cell sorting （MACS） from human fetuses （n = 10） at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein （AFP）, CD29 （integrin ~1）, CD49f （integrin c^6） and CD90 （Thy 1） was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ （A, B, C） and class Ⅱ （DR） expression was studied by flow cytometry only. RESULTS： FACS analysis indicated that EpCAM ＋ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class Ⅱ （DR） and CD45. RT PCR showed that EpCAM ＋ve cells expressed liver epithelial markers （CK18）, biliary specific marker （CK19） and hepatic markers （albumin, AFP）. On immunocytochemical staining, EpCAM ＋ve cells were shown positive signals for CK18 and albumin. CONCLUSION： Our study suggests that these EpCAM ＋ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.

Syngeneic implantation of mouse hepatic progenitor cell-derived three-dimensional liver tissue with dense collagen fibrils

BACKGROUND Liver transplantation is a therapy for irreversible liver failure;however,at present,donor organs are in short supply.Cell transplantation therapy for liver failure is still at the developmental stage and is critically limited by a shortage of human primary hepatocytes.AIM To investigate the possibility that hepatic progenitor cells(HPCs)prepared from the portal branch-ligated hepatic lobe may be used in regenerative medicine,we attempted to enable the implantation of extracellular matrices containing organoids consisting of HPC-derived hepatocytes and non-parenchymal cells.METHODS In vitro liver organoid tissue has been generated by accumulating collagen fibrils,fibroblasts,and HPCs on a mesh of polylactic acid fabric using a bioreactor;this was subsequently implanted into syngeneic wild-type mice.RESULTS The in vitro liver organoid tissues generated transplantable tissues in the condensed collagen fibril matrix and were obtained from the mouse through partial hepatectomy.CONCLUSION Liver organoid tissue was produced from expanded HPCs using an originally designed bioreactor system.This tissue was comparable to liver lobules,and with fibroblasts embedded in the network collagen fibrils of this artificial tissue,it is useful for reconstructing the hepatic interstitial structure.

Long-term and non-invasive in vivo tracking of DiD dye-labeled human hepatic progenitors in chronic liver disease models

BACKGROUND Chronic liver diseases(CLD)are the major public health burden due to the continuous increasing rate of global morbidity and mortality.The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option.However,identifying the fate of transplanted cells in vivo represents a crucial obstacle.AIM To evaluate the potential applicability of DiD dye as a cell labeling agent for longterm,and non-invasive in vivo tracking of transplanted cells in the liver.METHODS Magnetically sorted,epithelial cell adhesion molecule positive(1×106 cells/mL)fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency(SCID)mice.Near-infrared(NIR)imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d.The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing.RESULTS NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7,15,30,45,60,and 80 post-transplantation.Furthermore,positive staining of cytokeratin,c-Met,and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells.Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase,glutamate-pyruvate transaminase,and bilirubin.The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80.CONCLUSION DiD-labeling is promising for long-term and non-invasive in vivo cell tracking,and understanding the regenerative mechanisms incurred by the transplanted cells.

Xenogeneic hepatic progenitor cell transplantation ameliorate CCl_4 /partial hepatectomy-induced rat acute liver failure

Objective： Hepatic progenitor cell transplantation has shed light on the treatment of liver failure. The present study was designed to evaluate whether xenogeneic liver epithelial progenitor cells （LEPCs） transplantation could promote liver recovery in a rat model of acute liver failure. The engraftment and hepatocytic differentiation of transplanted hepatic progenitor cells in the rat spleen was also investigated. Methods： LEPCs were propagated in vitro for long and transduced with lentiviral vector carrying mCherry gene. Intraperitoneal injection of CC14 followed by 2/3 partial hepatectomy three days later were used to establish rat models of acute liver failure. Rats were intrasplenically injected with mCherry modified LEPCs （n=20, 1× 107 cells/0.5 mL） or the same volume of medium （n=20）. Serum liver enzymes （ALT, AST） and liver histology were evaluated for 21 days after transplantation. The engraftment of transplanted LEPCs in the spleens was tested by polymerase chain reaction （PCR） amplification targeting mCherry gene. The differentiation into hepatocytic lineage of transplanted LEPCs was investigated usingimmunohistochemistry staining against Alb. Results： LEPCs were effectively transduced with lentiviral vector showing a transduction efficiency of 90%. Compared with control, cell-injected group displayed significantly lower levels of ALT and AST （P〈0.05） and better histological features including less swelling change and hepatocyte death. PCR amplification of mCherry sequences confirmed the engraftment of LEPCs in the spleens. Alb-positive cells first appeared 5 days after cell transplantation and the number of Alb-positive cells increased substantially （P〈0.05）, which revealed the hepatocytic differentiation process Conclusion： Xenogeneic hepatic progenitor cells can engraft and differentiate into hepatocytes in the splenic parenchyma. Intrasplenic delivery of hepatic progenitor cells ameliorates CCh/partial hepatectomy-induced liver injury in rats

Autophagy in hepatic progenitor cells modulates exosomal miRNAs to inhibit liver fibrosis in schistosomiasis

Schistosoma infection is one of the major causes of liver fibrosis.Emerging roles of hepatic progenitor cells(HPCs)in the pathogenesis of liver fibrosis have been identified.Nevertheless,the precise mechanism underlying the role of HPCs in liver fibrosis in schistosomiasis remains unclear.This study examined how autophagy in HPCs affects schistosomiasis-induced liver fibrosis by modulating exosomal miRNAs.The activation of HPCs was verified by immunohistochemistry(IHC)and immunofluorescence(IF)staining in fibrotic liver from patients and mice with Schistosoma japonicum infection.By coculturing HPCs with hepatic stellate cells(HSCs)and assessing the autophagy level in HPCs by proteomic analysis and in vitro phenotypic assays,we found that impaired autophagy degradation in these activated HPCs was mediated by lysosomal dysfunction.Blocking autophagy by the autophagy inhibitor chloroquine(CQ)significantly diminished liver fibrosis and granuloma formation in S.japonicum-infected mice.HPC-secreted extracellular vehicles(EVs)were further isolated and studied by miRNA sequencing.miR-1306-3p,miR-493-3p,and miR-34a-5p were identified,and their distribution into EVs was inhibited due to impaired autophagy in HPCs,which contributed to suppressing HSC activation.In conclusion,we showed that the altered autophagy process upon HPC activation may prevent liver fibrosis by modulating exosomal miRNA release and inhibiting HSC activation in schistosomiasis.Targeting the autophagy degradation process may be a therapeutic strategy for liver fibrosis during Schistosoma infection.

Hepatic progenitor cell activation in liver repair

The liver possesses an extraordinary ability to regenerate after injury.Hepatocyte-driven liver regeneration is the default pathway in response to mild-to-moderate acute liver damage.When replication of mature hepatocytes is blocked,facultative hepatic progenitor cells(HPCs),also referred to as oval cells(OCs)in rodents,are activated.HPC/OCs have the ability to proliferate clonogenically and differentiate into several lineages including hepatocytes and bile ductal epithelia.This is a conserved liver injury response that has been studied in many species ranging from mammals(rat,mouse,and human)to fish.In addition,improper HPC/OC activation is closely associated with fibrotic responses,characterized by myofibroblast activation and extracellular matrix production,in many chronic liver diseases.Matrix remodeling and metalloprotease activities play an important role in the regulation of HPC/OC proliferation and fibrosis progression.Thus,understanding molecular mechanisms underlying HPC/OC activation has therapeutic implications for rational design of anti-fibrotic therapies.

Efficient generation of functional hepatocytelike cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Effect of liver regeneration on malignant hepatic tumors

Liver regeneration after major surgery may activate occult micrometastases and facilitate tumor growth,leading to liver tumor recurrence.Molecular changes during liver regeneration can provide a microenvironment that stimulates intrahepatic tumor propagation through alterations in cellular signaling pathways,where activation and proliferation of mature hepatocytes,hepatic progenitor cells,non-parenchymal liver cells might favor both liver regeneration and tumor growth.This review highlights recent advances of tumor growth and development in the regenerating liver,possible mechanisms and clinical implications.

Clinical features,histology,and histogenesis of combined hepatocellular-cholangiocarcinoma

Combined hepatocellular-cholangiocarcinoma(CHC) is a rare tumor with poor prognosis,with incidence ranging from 1.0%-4.7% of all primary hepatic tumors.This entity will be soon renamed as hepato-cholangiocarcinoma.The known risk factors for hepatocellular carcinoma(HCC) have been implicated for CHC including viral hepatitis and cirrhosis.It is difficult to diagnose this tumor pre-operatively.The predominant histologic component within the tumor largely determines the predominant radiographic features making it a difficult distinction.Heterogeneous and overlapping imaging features of HCC and cholangiocarcinoma should raise the suspicion for CHC and multiple core biopsies(from different areas of tumor) are recommended before administering treatment.Serum tumor markers CA19-9 and alpha-fetoprotein can aid in the diagnosis,but it remains a challenging diagnosis prior to resection.There is sufficient data to support bipotent hepatic progenitor cells as the cell of origin for CHC.The current World Health Organization classification categorizes two main types of CHC based on histo-morphological features:Classical type and CHC with stem cell features.Liver transplant is one of the available treatment modalities with other management options including transarterial chemoembolization,radiofrequency ablation,and percutaneous ethanol injection.We present a review paper on CHC highlighting the risk factors,origin,histological classification and therapeutic modalities.

SOX9 in biliary atresia: New insight for fibrosis progression

Background:Liver fibrosis is a hallmark determinant of morbidity in biliary atresia(BA)even in successfully operated cases.Responsible factors for this rapid progression of fibrosis are not completely defined.Aberrant expression of the transcription factor SOX9 and hepatic progenitor cells(HPCs)proliferation have roles in fibrogenesis in cholestatic disorders.However,they were not investigated sufficiently in BA.We aimed to delineate the relation of SOX9 and HPCs to fibrosis and its progression in BA.Methods:Forty-eight patients with BA who underwent an initial diagnostic liver biopsy(LB)and consequent intraoperative LB were recruited and compared to 28 cases with non-BA cholestasis that had an LB in their diagnostic workup.Liver fibrosis,tissue SOX9 and HPC expressions were studied in both BA and non-BA-cholestasis cases.Liver fibrosis,SOX9,and HPCs’dynamic changes in BA cases were assessed.Relation of fibrosis and its progression to SOX9 and HPCs in BA was assessed.Results:SOX9 and HPCs in ductular reaction(DR)form were expressed in 100%of BA and their grades increased significantly in the second biopsy.The rapidly progressive fibrosis in BA,represented by fibrosis grade of the intraoperative LB,correlated significantly to SOX9-DR and HPC-DR at the diagnostic(r=0.420,P=0.003 and r=0.405,P=0.004,respectively)and the intraoperative(r=0.460,P=0.001 and r=0.467,P=0.001,respectively)biopsy.On the other hand,fibrosis,SOX9-DR,and HPC-DR were significantly lower in non-BA cases at a comparable age(P<0.001,P=0.006,and P=0.014,respectively).Conclusions:Fibrosis in BA is rapidly progressive within a short time and is significantly correlated to SOX9 and HPCs.Assessment of targeting SOX9 and HPCs on fibrosis progression is warranted.

Effect of Yiguanjian decoction on cell differentiation and proliferation in CCl_4-treated mice

AIM： To investigate the cellular mechanisms of action of Yiguanjian （YGJ） decoction in treatment of chronic hepatic injury. METHODS： One group of mice was irradiated, and received enhanced green fluorescent protein （EGFP）- positive bone marrow transplants followed by 13 wk of CCh injection and 6 wk of oral YGJ administration. A second group of Institute for Cancer Research mice was treated with 13 wk of CCI4 injection and 6 wk of oral YGJadministration. Liver function, histological changes in the liver, and Hyp content were analyzed. The expres- sion of m-smooth muscle actin （α-SMA）, F4/80, albumin （AIb）, EGFP, mitogen-activated protein kinase-2 （PKM2）, Ki-67, fetoprotein （AFP）, monocyte chemotaxis pro- tein-1 and CC chemokine receptor 2 were assayed. RESULTS： As hepatic damage progressed, EGFP-po- sitive marrow cells migrated into the liver and were mainly distributed along the fibrous septa. They showed a conspicuous coexpression of EGFP with ^-SMA and F4/80 but no coexpression with AIb. Moreover, the expression of PKM2, AFP and Ki-67 was enhanced dy- namically and steadily over the course of liver injury. YGJ abrogated the increases in the number of bone marrow-derived fibrogenic cells in the liver, inhibited expression of both progenitor and mature hepatocyte markers, and reduced fibrogenesis. CONCLUSION： YGJ decoction improves liver fibrosis by inhibiting the migration of bone marrow cells into the liver as well as inhibiting their differentiation and suppressing the proliferation of both progenitors and hepatocytes in the injured liver.

大鼠肝卵圆细胞增殖模型建立及其纯化鉴定

为建立大鼠卵圆细胞增殖模型,分离纯化卵圆细胞并对其特性进行研究,SD大鼠喂饲2-乙酰氨基芴(2-acetyailamifluorene,2-AAF),剂量分别为5、10、15、20mg/kg,连续给药6d,于第7天行三分之二部分肝切除,术后继续给药1周,每隔3d取肝组织行常规组织学观察,作细胞增殖试验及免疫组化染色。通过免疫磁珠标记C-kit阳性细胞以纯化卵圆细胞(Magneticactivatedcellsorting,MACS),用免疫细胞化学及RT-PCR对纯化细胞进行鉴定。结果:10、15、20mg/kg三种剂量2-AAF均可成功建立卵圆细胞增殖模型,卵圆细胞增殖于第8天较明显,至第11天达高峰,第14天有所减少。卵圆细胞胞核Brdu染色阳性,其除表达卵圆细胞特异性抗原OV6外,尚表达肝细胞标记白蛋白及胆管细胞标记CK19。胚胎期肝细胞抗原AFP、连接蛋白43在卵圆细胞中亦有高表达,同时还表达造血干细胞标记C-kit。以MACS纯化的C-kit阳性卵圆细胞,90%以上为OV6阳性,并有AFP、白蛋白、CK19mRNA转录。结论:2-AAF剂量为10～20mg/kg时可获得满意的大鼠卵圆细胞增殖模型,增生的卵圆细胞为具有双向分化潜能的肝干细胞/前体细胞,利用C-kit标记通过MACS可纯化这种肝干细胞/前体细胞。

Molecular pathways of liver regeneration:A comprehensive review

The liver is a unique parenchymal organ with a regenerative capacity allowing it to restore up to 70%of its volume.Although knowledge of this phenomenon dates back to Greek mythology(the story of Prometheus),many aspects of liver regeneration are still not understood.A variety of different factors,including inflammatory cytokines,growth factors,and bile acids,promote liver regeneration and control the final size of the organ during typical regeneration,which is performed by mature hepatocytes,and during alternative regeneration,which is performed by recently identified resident stem cells called“hepatic progenitor cells”.Hepatic progenitor cells drive liver regeneration when hepatocytes are unable to restore the liver mass,such as in cases of chronic injury or excessive acute injury.In liver maintenance,the body mass ratio is essential for homeostasis because the liver has numerous functions;therefore,a greater understanding of this process will lead to better control of liver injuries,improved transplantation of small grafts and the discovery of new methods for the treatment of liver diseases.The current review sheds light on the key molecular pathways and cells involved in typical and progenitor-dependent liver mass regeneration after various acute or chronic injuries.Subsequent studies and a better understanding of liver regeneration will lead to the development of new therapeutic methods for liver diseases.

Ductular reaction in non-alcoholic fatty liver disease:When Macbeth is perverted

Non-alcoholic fatty liver disease(NAFLD)or metabolic(dysfunction)-associated fatty liver disease is the leading cause of chronic liver diseases defined as a disease spectrum comprising hepatic steatosis,non-alcoholic steatohepatitis(NASH),liver fibrosis,cirrhosis,and hepatic carcinoma.NASH,characterized by hepatocyte injury,steatosis,inflammation,and fibrosis,is associated with NAFLD prognosis.Ductular reaction(DR)is a common compensatory reaction associated with liver injury,which involves the hepatic progenitor cells(HPCs),hepatic stellate cells,myofibroblasts,inflammatory cells(such as macrophages),and their secreted substances.Recently,several studies have shown that the extent of DR parallels the stage of NASH and fibrosis.This review summarizes previous research on the correlation between DR and NASH,the potential interplay mechanism driving HPC differentiation,and NASH progression.

In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepato- cytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxan- induced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/ progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/ progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.

Prognostic significance of combining high mobility group Box-1 and OV-6 expression in hepatocellular carcinoma

The inflammatory environment and existence of cancer stem cells are critical for progression and intrahepatic recurrence of hepatocellular carcinoma （HCC） after curative resections. Here, we investigated the prognostic significance of combining high mobility group box 1 （HMGB1） expression and hepatic progenitor marker OV6 in hepatocellular carcinoma. Expression of HMGB 1 and OV6 was evaluated using immunohistochemistry profiling in tissue microarrays containing samples from 208 HCC patients. Invasive clinical or pathological factors were found in patients with high expression of HMGB 1 or OV6. Higher HMGB1 was associated with poorer clinical outcomes, and independently related to elevated 5-year recurrence incidence （85.5% vs. 62.4%, P〈0.001）. We also found that more OV6 positive staining was correlated with poor prognosis of HCC patients （P〈0.001）. Notably, expression of HMGB 1 was positively correlated with OV6 in density （R2=0.032, P〈0.001） and reversely related to HCC outcomes. Abnormal expression of HMGBI in combination with positive staining of OV6 displayed poorer prognostic performance than single biomarker alone （area under curve （AUC） survival=0.696）. Therefore, HMGB 1 and OV6 positive staining are promising prognostic parameters for HCC, and we propose that HMGB1 and OV6 may cooperate with each other and predict poor prognosis of HCC.

p53 functional loss,stemness and hepatocellular carcinoma

The tumor suppressor p53 is a key player in the control of genomic integrity and homeostasis in connection with p63 and p73,the two other members of the p53 family.Loss of functional p53 leads to the proliferation and survival of mature cells and progenitor or stem cells that accumulate genetic alterations,thus favoring tumorigenesis.p53 loss of function,observed in a wide variety of human tumor types,is frequently caused by missense mutations more frequently found in the DNA binding domain,but can also be due to the expression of a plethora of viral and cellular negative regulators.Human hepatocellular carcinoma(HCC)represents a specific situation,first because the TP53 gene mutations pattern exhibits a“hot spot”rarely found in other tumor types that is linked to Aflatoxin B1 exposure and,second,because many HCCs do not exhibit any TP53 mutation.Here,we provide an overview of the current knowledge about the inhibition of p53 functions by the N-terminal(ΔN)truncated forms of the family,and their role in the emergence and maintenance of pre-malignant cells with stem cell characteristics and in HCC development.We focus in particular on the Nanog-IGF1R-ΔNp73 axis that is associated with stem-like features in HCC cells and that may provide an attractive new therapeutic target and help to develop new biomarkers for HCC risk stratification,as well as preventive strategies.