A Novel Technique For Hepatic Progenitor Cell Isolation From Normal Adult Rat Livers

BackgroundAcute liver failure is a rare but life-threatening condition that can be caused by various kinds of chronic liver disease or that can arise with no previous history of liver disease. Liver transplantation is thought to be the most important therapeutic option for patients with end-stage liver disease. However, the feasibility of liver transplantation is limited by its high cost, a shortage of donor organs, the demand of high surgical technic and the need for immunosuppression. As many diseases result from hepatocyte dysfunction, and liver cells or hepatocyte-like cells may play an important function of liver after transplantation, transplantation of hepatocytes or hepatocyte-like cells is a promising alternative treatment for acute and chronic liver failure. However, this approach is also restricted by a shortage of donor cells and by immunological rejection. Cell transplantation with stem cells which own the ability of powerful proliferation and lower immunogenicity, could potentially replace liver transplantation in patients with end-stage liver diseases. Thus, a good deal of research has turned to cell transplantation, such as the transplantation of mature cells or stem/progenitor cells and the potential transplantation of xenogeneic organs and cells.Hepatic stem/progenitor cells have been shown to exist in adult rodent and human livers, as demonstrated by the coexpression of stem cell and hepatic lineage markers. However, in adult normal livers, stem/progenitor cells are quiescent, existing only in low numbers around the periportal region, and they only proliferate following severe, prolonged liver trauma. There are a variety of methods to isolate these stem cells. Animal models. such as diethylnitrosamine (DEN)-treated rats, the 2-acetylaminofluorene/partial hepatectomy model and the retrorsine/partial hepatectomy model, have been used. The reagents used in these models are different, but the purpose of these methods are the same:that's to cause severe, prolonged liver trauma. Although a great deal of stem/progenitor cells can be isolated from these disease model animals, the cells acquired through these methods are taken from abnormal organisms. Another hypothesis is that stem cells work throughout the development of hepatocellular carcinoma and might play important roles in this process. So even if a large number of cells can be isolated from the liver cells, but the quality is not guaranteed. So far, no experimental method can obtain a good number of liver stem/progenitor cells with high purity and good activity. The technology about isolating, predicating and identifying liver stem/progenitor cells need to be perfected. These factors will be the bottleneck of clinical applications of liver stem/progenitor cells.ObjectiveUsing non-injuried liver, explore to establish a method to isolate, culture and identify liver progenitor cells.MethodsHPCs were isolated from normal adult rat livers using a novel four-step collagenase perfusion method followed by density gradient centrifugation. The morphous and phenotypic properties of HPCs were characterized by morphological observation. RT-PCR and immunocytochemistry.ResultsAdopting the four-step collagenase perfusion combinding with density gradient centrifugation technology established by our research group. liver progenitor cells were isolated from normal rat liver, the quantity was about 2.5±0.5×104/100g, the survival rate was 95%±2%. The Isolated cells suspended with DMEM added with 10% fetal bovine serum, were inoculated into the culture bottle. The results showed that HPCs formed loose colonies and possessed a round or oval shape at culture day-3. These cells proliferated slowly and exhibited progenitor-like characteristics during the 30-day culture period. RT-PCR analysis indicated that the cultured cells were positive for several hepatocyte-specific genes, such as ALB,AFP,CK18,CK19,HNF-1a,HNF-4a, and also positive for specific genes of hematopoietic stem cell, such as Thy-1 and CD45. Immunocytochemical staining showed that these cells were consistently positive for ALB, AFP, CK18, CK19, Thy-1 and OV-6.ConclusionHPCs can be isolated from normal adult rat livers using a simple and effective technique involving the four-step collagenase perfusion combinding with density gradient centrifugation technology. Analysis showed that these cells were consistently positive for several stem cell-specific genes and liver-specific genes. This technique provide a good cells source for liver stem/progenitor cells transplantation.