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.

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.

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.

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.

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.

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.

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.

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.

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.