ISOLATION OF HEPATIC OVAL CELLS FROM DIFFERENT MODEL RATS INCLUDING DIABETIC RATS

Objective To acquire oval cells （progenitor stem cells ） from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley （ SD ） rats were divided into 5 groups randomly： control, 2-acetylaminofluorene （ 2-AAF ）, 2-AAF ＋ partial hepatectomy （ PH ）, 2-AAF ＋ carbon tetrachloride （ CCl4 ）, and diabetic groups. As two-step collagenase perfusion protocol of Seglen, oval cells were isolated by Percoll density gradient centrifugation. Thy1. 1 positive cells were sorted by flow cytometry, and then cultured in Dulbecco＇s minimum Eagle＇s medium （DMEM）. Immunofluorescence staining was applied to labelling Thyl. 1. Results Different rates of Thy1.1 positive oval cells were found in different rat model groups ： 0. 5 % in 2-AAF, 0. 3% in 2-AAF ＋ PH, 0. 2% in 2-AAF ＋ CCl4, 0. 1% in diabetic, and 0. 0% in control. Isolated cells adhered to plate with fusiform or polygon as epithelial cells. Conclusion Progenitor stem cells exist in injured liver tissue including those from diabetic rats.

Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

AIM： To elucidate the interaction between non- parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy （PH）. METHODS： We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2- acetylaminofluorene （2-AAF）/PH rat model. RESULTS： By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic Iobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells （HSCs）, laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver Iobule structures began to recover.CONCLUSION： Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

Intrahepatic transplantation of hepatic oval cells for fulminant hepatic failure in rats

AIM： To evaluate the effect of intrahepatic transplantation of hepatic oval cells （HOC） on fulminant hepatic failure （FHF） in rats.METHODS： HOC obtained from rats were labeled with green fluocescent protein （GFP） or 5, 6- carboxyfluorescein diacetate succinmidyl ester （CFDASE）. Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA- SE labeled HOC （5 × 10^6 cells each rat） were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin （ALB）, alanine aminotransferase （ALT）, aspartate aminotransferase （AST） and total bilirubin （TBil） levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation.RESULTS： The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 （9/15 vs 6/15） and 72 h （9/15 vs 4/15） after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum AIb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC.CONCLUSION： CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients.

Immunohistochemical study of hepatic oval cells in human chronic viral hepatitis

AIM: To detect immunohistochemically the presence of oval cells in chronic viral hepatitis with antibody against c-kit. METHODS: We detected oval cells in paraffin embedded liver sections of 3 normal controls and 26 liver samples from patients with chronic viral hepatitis, using immunohistochemistry with antibodies against c-kit, piclass glutathione S-transferase (pi-GST) and cytokeratins 19 (CK19). RESULTS: Oval cells were not observed in normal livers. In chronic viral hepatitis, hepatic oval cells were located predominantly in the periportal region and fibrosis septa,characterized by an ovoid nucleus, small size,and scant cytoplasm. Antibody against stem cell factor receptor, c-kit, had higher sensitivity and specificity than pi-GST and CK19. About 50%-70% of c-kit positive oval cells were stained positively for either pi-GST or CK19. CONCLUSION: Oval cells are frequently detected in human livers with chronic viral hepatitis, suggesting that oval cell proliferation is associated with the liver regeneration in this condition.

Cell culture isolation can miss the laboratory diagnosis of HSV ocular infection

AIMWe compared polymerase chain reaction (PCR) to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus (HSV) disease.

Enzymatic Cell Isolation and Explant Cultures of Rat Calvarial Osteoblast Cells

Osteoblast cells were isolated from the calvarial bones of newborn Wistar rats and cultured in vitro via both collagenase digestion method and explant technique, and a comparative study was carried out on the two culture methods. The biologic charwteristics of the osteoblast cells were studied via cell number counting, morphology observation, alkaline phosphatase staining of the cells and alizarine- red staining of the calcified nodules. The results show that osteoblast cells can be cultured in vitro via collagenase digestion method and explant technique, and the obtained cells ure of good biologic characteristics. In comparison with the explant technique, the operative procedure of the enzymatic digestion method is more complicated. The digestion time must be carefully controlled. However, with this method, one can obtain a lager number of cells in a short time. The operative procedure of the explant technique is simpler, but it usually takes longer time to obtain cells of desirable number.

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.

Mouse A6-positive Hepatic Oval Cells Derived from Embryonic Stem Cells

Summary： Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor（HGF） and epidermal growth factor（EGF） were used to induce differentiation of mouse embryonic stem（ES） cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells＇ differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1＋/CD34＋ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1＋/CD34＋ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1＋/CD34＋ cells on the day 8 were A6 positive. Highly purified A6＋/Sca-1＋/CD34＋ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group（up to 4.46%） was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells＇ membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

Advances in In-vitro Culture Technology of Human Fetal Hepatic Stellate Cells

Liver is an important organ for human metabolism and biological conversion. Medical research on hepatic disease clinic, drug metabolism and drug effi- cacy evaluation all needs an in-vitro model of liver as a research platform. Hepatic stellate cells are core cells for occurrence and development of liver fibrosis. Stud- ies at home and abroad deemed that human fetal hepatic stellate cells are an ideal material for the construction of an in-vitro research model for liver fibrosis. With clinical and basic research of liver going deeper, the requirements to quantity and quality of in-vitro models of fetal hepatic stellate cells become higher and higher. The advances in isolation, culture and cryopreservation technique of human fetal hepatic stellate cells were reviewed in this paper.

Effects of retinoic acid on proliferation,phenotype and expression of cyclin-dependent kinase inhibitors in TGF-β1-stimulated rat hepatic stellate cells

AIM To study the molecular mechanisms ofretinoic acid(RA)on proliferation andexpression of cyclin-dependent kinase inhibitors(CKI),i.e.p16,p21 and p27 in cultured rathepatic stellate cells(HSC)stimulated withtransforming growth factor beta 1(TGF-β1).METHODS HSC were isolated from healthy ratlivers and cultured.After stimulated with1 mg/L TGF-β1,subcultured HSC were treatedwith or without 1 nmol/L RA.MTT assay,immunocytochemistry(ICC)for p16,p21,p27and α-smooth muscle actin(α-SMA)protein,insitu hybridization(ISH)for retinoic acidreceptor beta 2(RAR-β2)and p16,p21 and p27mRNA and quantitative image analysis(partially)were performed.RESULTS RA inhibited HSC proliferation(41.50%,P【0.05),decreased the protein levelof α-SMA(55.09%,P【0.05),and induced HSCto express RAR-β2 mRNA.In addition,RAincreased the protein level of p16(218.75%,P【0.05)and induced p21 protein expression;meanwhile,p27 was undetectable by ICC in bothcontrol and RA-treated HSC.However,RA hadno influence on the mRNA levels of p16,p21 orp27 as determined by ISH.CONCLISION Up-regulation of p16 and p21 on post-transcriptional level may contribule, in part to RA inhibition of TGF-β1-initiated rat HSC activation in vitro.

Experimental study of bioartificial liver with cultured human liver cells

AIM To establish an extracorporeal bioartificial liver support system (EBLSS) using cultured human liver cells and to study its support effect for fulminant hepatic failure (FHF).METHODS The liver support experiment of EBLSS consisting of aggregates cultured human liver cells, hollow fiber bioreactor, and circulation unit was carried out in dizhepatic dogs.RESULTS The viability of isolated hepatocytes and nonparenchymal liver cells reached 96%. These cells were successfully cultured as multicellular spheroids with synthetic technique. The typical morphological appearance was retained up to the end of the artificial liver experiment. Compared with the control dogs treated with EBLSS without liver cells, the survival time of artificial liver support dogs was significantly prolonged. The changes of blood pressure, heart rate and ECG were slow. Both serum ammonia and lactate levels were significantly lowered at the 3rd h and 5th h. In addition, a good viability of human liver cells was noted after 5 h experiment.CONCLUSION EBLSS playing a metabolic role of cultured human hepatocytes, is capable of compensating the function of the liver, and could provide effective artificial liver support and therapy for patients with FHF.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

AIM To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3. CHO cells were transfected by pMSG-ns3 using calcium phosphate precipitation method and cultivated for 12 h-24 h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS After treated with 3×10-8mol/ L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.

Ex vivo-expanded bone marrow stem cells home to the liver and ameliorate functional recovery in a mouse model of acute hepatic injury

BACKGROUND:Stem cell transplantation provides a theoretical approach for liver regeneration medicine;it may promote liver regeneration and self-repair.However,the transplantation of bone marrow-mesenchymal stem cells expanded ex vivo as a therapy for liver disease has rarely been investigated.This study aimed to explore whether bone marrow stem cells expanded ex vivo home to the liver and foster hepatic recovery after CCl 4 injury.METHODS:Bone marrow cells from BALB/c mice were expanded ex vivo by multiple-passage cultivation,characterized by cytoflow immunofluorescence,and pre-labeled with PKH26 before intravenous infusion into animals treated with CCl 4.The integration of bone marrow cells into the liver was examined microscopically,and plasma hepatic enzymes were determined biochemically.RESULTS:Cultured bone marrow cells exhibited antigenic profiles comparable to those of primary medullary stem cells.Double immunofluorescence showed colocalization of these cells with proliferative activity and albumin expression in the liver of CCl 4 -treated mice.Densitometry showed increased in situ cell proliferation (50±14 vs 20±3 cells/high-power field,P<0.05) and albumin expression (149±25 vs 20±5 cells/high-power field,P<0.05) in the liver,as well as reduced serum aminotransferase levels (P<0.05) and better survival rates (P<0.05) in animals receiving cultured bone marrow cells relative to controls.CONCLUSIONS:Ex vivo-expanded bone marrow cells are capable of relocating to and proliferating in the chemically- injured liver.Transplantation of these pluripotent stem cells appears to improve serum indices of liver function and survival rate in mice after CCl4-induced hepatic damage.

Culture and Identification of Human Amniotic Mesenchymal Stem Cells

Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.

Ultrastructure of oval cells in children with chronic hepatitis B,with special emphasis on the stage of liver fibrosis:The first pediatric study

AIM:To investigate the ultrastructure of oval cells in children with chronic hepatitis B,with special emphasis on their location in areas of collagen fibroplasia. METHODS:Morphological investigations were conducted on biopsy material obtained from 40 children,aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis. RESULTS:Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells,the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells,i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells. CONCLUSION:We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.

Stimulation of oval cell and hepatocyte proliferation by exogenous bombesin and neurotensin in partially hepatectomized rats

AIM:To investigate the effect of the neuropeptides bombesin(BBS)and neurotensin(NT)on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor.METHODS:Seventy male Wistar rats were randomly divided into five groups:Ⅰ=controls,Ⅱ=sham operated,Ⅲ=partial hepatectomy 70%(PHx),Ⅳ=PHx+ BBS(30μg/kg per day),Ⅴ=PHx+NT(300μg/kg per day).Forty eight hours after liver resection,portal en-dotoxin levels and hepatic glutathione redox state were determined.α-fetoprotein(AFP)mRNA(in situ hybridisation),cytokeratin-19 and Ki67 antigen expression (immunohistochemistry)and apoptosis(TUNEL)were evaluated on liver tissue samples.Cells with morphological features of oval cells that were cytokeratin-19 (+)and AFP mRNA(+)were scored in morphometric analysis and their proliferation was recorded.In addition,the proliferation and apoptotic rates of hepatocytes were determined.RESULTS:In the control and sham operated groups,oval cells were significantly less compared to groups Ⅲ,ⅣandⅤ(P<0.001).The neuropeptides BBS and NT significantly increased the proliferation of oval cells compared to groupⅢ(P<0.001).In addition,BBS and NT induced a significant increase of hepatocyte proliferation(P<0.001),whereas it decreased their apoptotic activity(P<0.001)compared to groupⅢ.BBS and NT significantly decreased portal endotoxemia (P<0.001)and increased the hepatic GSH:GSSG ratio (P<0.05 and P<0.001,respectively)compared to groupⅢ.CONCLUSION:BBS and NT stimulated oval cell proliferation in a model of liver regeneration,without use of concomitant suppression of hepatocyte proliferation as oval cell activation stimuli,and improved the hepatocyte regenerative response.This peptides-induced combined stimulation of oval cell and hepatocyte proliferation might serve as a possible treatment modality for several liver diseases.

Simplified methods to isolate,culture and purify olfactory ensheathing cells

Conventional methods for harvesting, culturing and purifying olfactory ensheathing cells are complicated, time-consuming, and poorly reproducible. Olfactory bulbs were detached from adult Sprague Dawley rats and olfactory ensheathing cells were isolated using shearing, dispersion processes. After the primary cultures reached confluence, the cells were purified using a three-step process. The olfactory ensheathing cells attached and grew rapidly. The purity of the olfactory ensheathing cells increased following the three purification steps, eventually exceeding 95%. These cells could be maintained for an extended period time in culture. This simple, inexpensive, reproducible method of harvesting, culturing and purifying olfactory ensheathing cells shortens the culture cycle and provides sufficient olfactory ensheathing cells of controllable purity.

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.