Animals ; Cell Differentiation ; Fetus ; cytology ; Hematopoietic Stem Cells ; cytology ; Hepatocytes ; cytology ; Humans ; Liver ; cytology ; Stem Cells ; cytology ; physiology

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
Epithelial Cells/*physiology ; Epithelium/metabolism ; Hepatic Stellate Cells/*physiology ; Humans ; Liver/*cytology/injuries/*physiology ; Liver Cirrhosis/etiology/prevention & control ; Liver Regeneration/*physiology ; Mesenchymal Stromal Cells/physiology ; Myofibroblasts/physiology

OBJECTIVETo isolate and culture mesenchymal stem cells (MSCs) from human fetal livers and describe their biological characteristics.

METHODSMSCs were acquired using an optimized method. Cell cycles and the immunophenotype of the cells were analyzed by flow cytometry. The osteogenic and adipogenic differentiations were induced and identified by specific stainings, and hepatic differentiation by morphology and RT-PCR.

RESULTSThe target cells derived from human fetal livers adhered to the plate with fibroblast-like morphology, whose surface markers were CD90, CD44, CD147 positive, and CD34, CD45, HLA-DR negtive. In the differentiation study, these cells could be induced to differentiate into osteogenic, adipogenic and hepatocyte-like cells.

CONCLUSIONMultipotent MSCs can be isolated and cultured from human fetal livers.

Cell Differentiation ; physiology ; Cell Separation ; Cells, Cultured ; Fetus ; Humans ; Liver ; cytology ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo study the method of preparing the hollow fiber bioreactor for culturing pig hepatocytes.

METHODSHepatocytes were isolated from experimental suckling minipigs by two-step perfusion with collagenase, and seeded onto hollow fiber bioreactor, then cultured with an artificial capillary cell culture system. The albumin-excretion, lidocaine-transforming rate, lactate dehydrogenase (LDH) release and the cell viability in bioreactors were examined.

RESULTSThe porcine albumin could be detected by SDS/PAGE on the 2nd, 4th, 6th day. The rates of lidocaine-transforming ranged from 89.6% to 96.1%. The release of LDH into the culture medium increased from (23.7+/-4.6) U/L to (127.8+/-17.4) U/L (F=39.582, P<0.01) during the experiments, and the viability of pig hepatocytes in hollow fiber bioreactor reduced from 95.8%+/-0.3% to 83.8%+/-4.7% (t=5.135, P<0.01).

CONCLUSIONThe hollow fiber bioreactor for culturing pig hepatocytes can be prepared by artificial capillary cell culture system, which provides a certain liver-specific function in 1 week.

Animals ; Bioreactors ; Biotechnology ; Cells, Cultured ; Female ; Hepatocytes ; cytology ; Liver ; cytology ; physiology ; Liver, Artificial ; Male ; Swine ; Swine, Miniature

Animals ; Cell Differentiation ; Hepatocytes ; cytology ; Humans ; Liver Diseases ; therapy ; Liver Regeneration ; physiology ; Research ; trends ; Stem Cell Transplantation ; Stem Cells ; cytology

OBJECTIVETo investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepatocyte for hepatocyte replacement therapy in the treatment of liver failure.

METHODSES cells from Balb/C mice were cultured and maintained in an undifferentiated state in gelatin-coated dishes using Dulbecco's modified Eagle's medium (DMEM) containing 1000 U/ml leukemia inhibitory factor (LIF). Then, LIF was withdrawn from the DMEM to allow the ES cells to develop into embryonic bodies (EBs). EBs were plated onto tissue culture dishes, and growth factors such as acidicfibroblast growth factor (aFGF) and hepatocyte growth factor (HGF) were added to the medium to promote directional differentiation. The course of development and differentiation was observed dynamically using an inversion microscope. The expression of hepatic proteins, such as alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK8), cytokeratin 18 (CK18), in cytoplasm was analyzed by immunocytochemistry (ICC). The concentration of ALB in the medium was determined dynamically by radioimmunoassay (RIA).

RESULTSES cells replicated as clones, without differentiating, in DMEM containing LIF. They developed into EBs in medium without LIF. Our ICC assay showed that differentiating cells did not express hepatic proteins, such as AFP, ALB, CK8, and CK18 until day 7, day 9, day 11, and day 11, respectively (up to 2 days later when growth factors are not present). The concentration of AFP in the medium was first detected on day 8, at a concentration of 3.4 ng/ml, and increased to 22.8 ng/ml by day 15. The concentration of ALB in the medium was 0.2 micro g/ml on day 11, and increased to 2.2 micro g/ml by day 15. ALB-positive cells under ICC manifest morphological structures were consistent with normal mouse hepatocytes. The differentiation ratio of hepatocytes in the ES cell differentiation system was 30% on day 15 (significantly lower without the presence of growth factors).

CONCLUSIONSES cells can differentiate into mature hepatocytes. Growth factors, such as aFGF and HGF, can enhance this differentiation and produce sufficient numbers of functional hepatocytes. This method may be a reliable new way of differentiating ES cells into hepatocytes for use in replacement therapy in the treatment of liver failure.

Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Hepatocytes ; cytology ; Liver ; cytology ; Mice ; Mice, Inbred BALB C ; Stem Cells ; cytology

Orthotopic liver transplantation has proven to be effective in the treatment of a variety of life-threatening liver diseases, however, the limitations of donated organs available and long-term immunosuppression provided an impetus for developing alternative therapies. Cell replacement strategies have been one major effective approach for overcoming the obstacles of organ transplantation in recent years. The exogenous cells should be able to proliferate and differentiate into mature hepatic cells after grafting. Use of mature hepatocytes is also hampered by limited tissue source and inability to proliferate and maintain the function for a long term in vitro. Embryonic stem cells are immortal and pluripotent and may provide a novel cell source for potential cell therapy. This review summarizes the mechanisms of controlling early liver development and hepatic differentiation of visceral endoderm in embryoid bodies, and provides an overview of diverse differentiation systems in vitro and in vivo that were applied to hepatic research in recent years. Several studies have demonstrated that ES cell-derived hepatocytes can incorporate into liver tissue and function in vivo , but a few of them have shown complete restoration of liver function after transplantation into mice with liver diseases. Further studies should be made to exploit efficient methods and clinical applications of hepatocytes derived from ES cells in the future. In addition to clinical transplantation for treatment of liver diseases, ES cells can provide a valuable tool for drug discovery applications and study on of molecular basis of hepatic differentiation.
Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; transplantation ; Hepatocytes ; cytology ; Humans ; Liver Diseases ; therapy

The purpose of this study was to explore the role of epithelial-mesenchymal transition in the pathogenesis of hepatolithiasis. Thirty-one patients with primary hepatolithiasis were enrolled in this study. Expressions of E-cadherin, alpha-catenin, alpha-SMA, vimentin, S100A4, TGF-beta1 and P-smad2/3 in hepatolithiasis bile duct epithelial cells were examined by immunohistochemistry staining. The results showed that the expressions of the epithelial markers E-cadherin and alpha-catenin were frequently lost in hepatolithiasis (32.3% and 25.9% of cases, respectively), while the mesenchymal markers vimentin, alpha-SMA and S100A4 were found to be present in hepatolithiasis (35.5%, 29.0%, and 32.3% of cases, respectively). The increased mesenchymal marker expression was correlated with decreased epithelial marker expression. The expressions of TGF-beta1 and P-smad2/3 in hepatolithiasis were correlated with the expression of S100A4. These data indicate that TGF-beta1-mediated epithelial-mesenchymal transition might be involved in the formation of hepatolithiasis.
Adult ; *Bile Ducts/cytology/metabolism/pathology ; Biological Markers/*metabolism ; Cell Differentiation/*physiology ; Epithelial Cells/cytology/*physiology ; Epithelium/physiology ; Female ; *Gallstones/metabolism/pathology ; Humans ; Liver Diseases/metabolism/*pathology ; Male ; Mesoderm/cytology/*physiology ; Middle Aged

OBJECTIVETo establish a rat model for hepatic oval cell proliferation and to observe the relationship between 2-acetaminofluorene (AAF) dosage and oval cell proliferation in the rat liver.

METHODSMale Wistar rats weighing 150 g received daily oral gavage of AAF for 4 days before operation and up to 7 days after operation. Two-thirds hepatectomy was performed on the 5th day and the gavage was not performed on the day of operation. AFF was given with the dosage of 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, and 20 mg/kg body weight. Animals in control group were given saline. Three rats from each group were killed every 2~3 days after hepatectomy and liver slices were fixed and processed for routine histology and immunohistochemistry.

RESULTSHepatic oval cells were not observed in the liver of controls and only a few were detected in the liver of 2.5 mg/kg and 5 mg/kg groups. However, obvious oval cell proliferation was seen in the liver of 10 mg/kg, 15 mg/kg, and 20 mg/kg groups. Hepatic oval cells were stained positive for cytokeratin 19, OV6, vimentin and proliferating cell nuclear antigen (PCNA).

CONCLUSIONSSatisfactory rat models for hepatic oval cell proliferation can be obtained using our scheme when AAF is dosed at 10~20 mg/kg body weight.

Animals ; Cell Division ; physiology ; Cells, Cultured ; Culture Media ; Immunohistochemistry ; Liver ; cytology ; Male ; Models, Animal ; Rats ; Rats, Wistar ; Stem Cells ; physiology

Most liver diseases lead to hepatic dysfunction with organ failure. Liver transplantation is the best curative therapy, but it has some limitations such as donor shortage, possibility of rejection, and maintenance of immunosuppressant. New therapies have been actively searched for over several decades, primarily in the form of artificial liver support devices and hepatocyte transplantation, but both of these modalities remain experimental. Stem cells have recently shown promise in cell therapy because they have the capacity for self-renewal and multilineage differentiation, and are applicable to human diseases. Very recent reports of unexpected plasticity in adult bone marrow have raised hopes of stem cell therapy offering exciting therapeutic possibilities for patients with chronic liver disease. Both rodent and human embryonic stem cells, bone marrow hematopoietic stem cells, mesenchymal stem cells, umbilical cord blood cells, fetal liver progenitor cells, adult liver progenitor cells, and mature hepatocytes have been reported to be capable of self-renewal, giving rise to daughter hepatocytes both in vivo and in vitro. These cells can repopulate livers in animal models of liver injury and appear to be able to improve liver function. However, significant challenges still exist before these cells can be used in humans, such as the lack of consensus about the immunophenotype of liver progenitor cells, uncertainty of the physiological role of reported candidate stem/progenitor cells, practicality of obtaining sufficient quantity of cells for clinical use, and concerns over ethics, long-term efficacy, and safety. There have been reports of phase 1 trials using stem cell transplantation in humans for liver diseases, but more effective trials are needed. We review the use of stem cells (focusing on adult ones) and the reported human clinical trials, and highlight the challenges facing clinicians in their quest to use liver stem cells to save lives.
Bone Marrow Cells/cytology ; Cell Differentiation ; Embryonic Stem Cells/cytology/transplantation ; Hematopoietic Stem Cells/cytology ; Humans ; Immunophenotyping ; Liver/*cytology/physiology ; Liver Diseases/*therapy ; Mesenchymal Stem Cells/cytology ; *Stem Cell Transplantation