| Liver transplantation is the only established treatment for end-stage liver disease. However, donor shortage and need for life-long immunosuppression with its known side effects are 2 major limitations of this therapy. Recently, to identify new potential treatments for end-stage liver disease, attention has been focused on stem cell transplantation therapeutic strategies. Stem cells are thought to be pluripotent cells that can be differentiated into a variety of cell or tissue types and can be ideal resources of transplantation therapy.Recently accumulating evidence indicates that bone marrow includes a large variety of progenitor cells that can differentiate into specific cell types. Transplantation of bone marrow cells in vivo resulted in the appearance of differentiated cells of donor origin in various recipient tissues, including skeletal myoblasts, cardiac myoblasts, endothelium, lung, gut, and skin epithelia, and neuroectodermal cells. Transplanted bone marrow cells can also repopulate hepatic and biliary duct epithelia under certain circumstances. However, little is known about the mechanism of bone marrow cell differentiation into hepatocytes. It is important to determine suitable culture conditions in which bone marrow cells will be differentiated into hepatocytes not only for understanding differentiation mechanisms but also for efficient amplification of hepatocyte-progenitor cells of bone marrow origin, this being a prerequisite for potential therapeutic use.Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, shows mitogenic, motogenic and morphogenic activities for a wide variety of cells that express the HGF receptor c-Met, a transmembrane protein possessing an intracellular tyrosine kinase domain. Moreover, HGF plays an essential role in the development and regeneration of the liver. Several studies have shown that plasma concentrations of HGF rise substantially in humans when hepatic mass is decreased. In the rat, plasma concentrations of HGF rise more than 20-fold within 1hour after partial hepatectomy (PHx). For this reason, in the present study, we investigate the effects of subtotal hepatectomized rat serum and/or HGF on the transdifferentiation of adult rat bone marrow stem cells (ABMSCs) in vivo and in vitro. Objectives1. The aim of the present study was to investigate the effects of subtotal hepatectomized rat serum and/or HGF on the transdifferentiation of ABMSCs and to establish a culture method in vitro.2. To address the issue of the identity of the Bone marrow derived liver progenitor cell that have the potential to generate hepatocytes in vivo, we transplanted differentiated hepatic progenitor cells into subtotal hepatectomized rat and determined whether these cells could give rise to hepatocytes.3. To explore the molecular mechanism of differentiation of bone marrow cells into hepatocytes, we analyzed gene expression profiles of the cultured bone marrow cells in the presence or absence of subtotal hepatectomized rat serum and HGF using a cDNA microarray consisting of 9234 genes. Comparison of gene expression patterns of the cultured bone marrow cells between in the presence or absence of subtotal hepatectomized rat serum and HGF enabled us to identify genes that were commonly up-regulated or down-regulated. Investigation of these genes should help to disclose the molecular mechanisms of differentiation of bone marrow cells into hepatocytes.Methods1. To determine whether the cultured ABMSCs can differentiate into hepatocyte lineage, partial hepatectomy (PHx) was performed in SD rats and the serum was collected at 24 hours after operation. ABMSCs, obtained from the femurs, was cultured with DMEM/F 12(1:1) containing subtotal hepatectomized rat serum and/or HGF in vitro. And RT-PCR, Western Blot and imrnunocytochemistry were used to identify the differentiation of ABMSCs2. The hepatic progenitor cells derived from ABMSCs were labeled with CM-Dil and transplanted into the isogeneic subtotal hepatectomized rats. Liverrepopulation was assessed using irnmunohistochemistry and confocal microscopy.3. The gene expression profiles of the cultured ABMSCs in the presence or absence of subtotal hepatectomized rat serum and HGF were analyzed by using a cDNA microarray consisting of 9234 genes and enabled us to identify genes that were commonly up- or down- regulated. Hence, the up-regulation or down-regulation genes may help to disclose the molecular mechanisms of differentiation of bone marrow cells into hepatocytes.Results1. Adherent cell colonies were found in the experiment group cultured with DMEM/F12(1:1) containing subtotal hepatectomized rat serum and HGF after 2-3 day's culture. After 5 day's culture, the cells of the colony were spreading and the cell numbers were increasing. And after 18 day's culture, the cells emerged the hepatocyte-like appearance. CD34+ or CD34~ cells, derived from the human cord blood, was cultured using the similar methods and the cell growth process and the cell appearance of both these two groups were similar to the cultured ABMSCs.2. By immunocytochemistry analysis, ABMSCs cultured with DMEM/F12(1:1) containing subtotal hepatectomized rat serum and/or HGF expressed albumin after 7 day's culture and the control group did not expressed; the results were also confirmed by RT-PCR and Western Blots. Albumin positive cells also appeared in cultured CD34+ or CD34" cells derived from the human cord blood after 7 days.3. The differentiated cells induced with subtotal hepatectmized rat serum were labeled with CM-Dil and transplanted into the isogeneic hepatectomized rats by different methods (intravenous, intrahepatic and intrasplenic administration). Some cells labeled with CM-Dil expressed albumin in liver and in spleen after one week transplantation and expressing CK18 cells appeared in spleen. The cell numbers of albumin-expressing and CM-Dil positive cells in spleen were more than in the liver. By intrahepatic injection, the cell numbers of albumin-expressing and CM-Dil positive cells in liver were more than by intrasplenic or intravenous...
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