AIM： To investigate the hepatocytic differentiation of mesenchymal stem cells （MSCs） in co-cultures with fetal liver cells （FLC） and the possibility to expand differentiated hepatocytic cells. METHODS： MSCs were...AIM： To investigate the hepatocytic differentiation of mesenchymal stem cells （MSCs） in co-cultures with fetal liver cells （FLC） and the possibility to expand differentiated hepatocytic cells. METHODS： MSCs were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor （SCF）, hepatocyte growth factor （HGF）, epidermal growth factor （EGF）, and fibroblast growth factor 4 （FGF-4） alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction （RT-PCR） for the liver specific markers cytokeratin-18 （CK-18）, albumin, and alpha-fetoprotein （AFP） was performed in different cell populations. RESULTS- Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. CONCLUSION： The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also prorides a beneficial environment for expansion and differentiation of FLC.展开更多
AIM： The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments reveale...AIM： The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stern cell cultures. We investigated the potential of rat mesenchymal stem cells （MSC） from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS： Mesenchymal stem cells were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thyl and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS： Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression, CONCLUSION： The results indicate that （1） rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and （2） that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.展开更多
文摘AIM： To investigate the hepatocytic differentiation of mesenchymal stem cells （MSCs） in co-cultures with fetal liver cells （FLC） and the possibility to expand differentiated hepatocytic cells. METHODS： MSCs were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor （SCF）, hepatocyte growth factor （HGF）, epidermal growth factor （EGF）, and fibroblast growth factor 4 （FGF-4） alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction （RT-PCR） for the liver specific markers cytokeratin-18 （CK-18）, albumin, and alpha-fetoprotein （AFP） was performed in different cell populations. RESULTS- Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. CONCLUSION： The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also prorides a beneficial environment for expansion and differentiation of FLC.
基金Supported by the "Rudoff Bartling Foundation" and "Foerdergemeinschaft Kinder-Krebs-Zentrum Hamburg e.V."
文摘AIM： The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stern cell cultures. We investigated the potential of rat mesenchymal stem cells （MSC） from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS： Mesenchymal stem cells were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thyl and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS： Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression, CONCLUSION： The results indicate that （1） rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and （2） that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.
