Study Of Differentiation Of Human Peripheral Blood Mononuclear Cells Into Hepatocyte-like Cells In Vitro

A stem cell is defined by two properties. First, it is a cell that can divide indefinitely, producing a population of identical offspring. Second, stem cells can, on cue, undergo an asymmetric division to produce two dissimilar daughter cells. There exist stem cells during different ontogenic stages and in adult tissues.Adult stem cells possess plasticity,that means they can differentiate into other cells in adequate microenvironment.Human peripheral blood mononuclear cells include monocytes, stem cells and lymphocytes. Human peripheral blood monocytes isolated from healthy donors can be isolated by density gradient centrifugation and further purified by adherence separation,because of monocytes can adhere to tissue culture plastics, nonadherent cells can be removed by gentle washing.Much is known about the actions of macrophage colony stimulating factor in promoting the proliferation and differentiation of monocytes and also about the effects of interleukin-3 on the differentiation of monocytes and self-renewal of multipotent stem cell.Their synergistic effect on the phenotypic plasticity of monocytes is a novel finding.In response to combined treatment, peripheral blood monocytes underwent partial dedifferentiation in that they resumed cell division and down-regulated factors known to promote their terminal differentiation.This discovery allows us to generate neohepatocytes of monocytic origin.Numerous cytokines and growth factors have been shown to have potent effects on hepatic growth and differentiation under in vitro culture conditions. HGF,EGF,TGF,and FGF are the most commonly used in the majority of studies.One study shows the hMAPCs have the better potential to differentiate into hepatocyte-like cells in the presence of HGF and FGF-4 than in the presence of LIF,OSM,EGF.Now, the best solution of liver function failure caused by acute or advanced stage of liver disease is liver transplantation.However, the inadequate supply of donor organs restricts orthotopic liver transplantation. Many patients can not get effective treatment or wait for suitable liver up to death.Cell transplantation can be envisioned as a promising alternative to liver transplantation if it can help patients to live through dangerous stages or direct repair failure livers.So,finding suitable cells capable of differentiating into hepatocytes are of particular interest.Because peripheral blood can be conveniently collected, donors need not to be anesthetic or suffer bone marrow aspiration,patients can easily accept this method,if hepatocytes can be"produced"by autologous peripheral blood stem cell , this makes them an attractive source in clinical application.Objectives:To investigate the possibility of differentiation of human peripheral blood mononuclear cells into hepatocyte-like cells in the presence of HGF and FGF-4 in vitro.Methods:1.Human peripheral blood mononuclear cells were isolated from healthy donors by density gradient centrifugation.2.Cells were cultured in RPMI1640,after 2 hours,nonadherent cells were removed by gentle washing.3.Cells were then cultured for 6 days in a RPMI1640-based medium with 140uM ?- mercaptoethanol, 5ng/ml M-CSF and 0.4ng/ml IL-3.4.Then the cells were cultured for another 20 days with 20 ng/ml HGF,10 ng/ml FGF-4.5.Morphology was observed at different time.The expression of ALB, AFP was detected with immunofluorescence to identify the characters of the differentiated cells on day6,10,14,and 20 after the cells were cultured with 20 ng/ml HGF,10 ng/ml FGF-4 and before HGF,FGF-4 added.Results:1.Spherical morphology was observed when peripheral blood mononuclear cells were just isolated,survival rate > 95 %.Crude spherical morphology was observed when nonadherent cells were removed after 2 hours.2. Adherent cells cultured in the presence of M-CSF and IL-3 for 6 days acquired a colony morphology,cells became confluent.Cells grown in the absence of M-CSF and IL-3 or cultured in M-CSF alone grew as single cells.Cells cultured in dedifferentiation medium for a prolonged period (>14 days) became fibroid or fusiform shape. Spherical morphology was still observed ,but there were minor cells.3.Part of adherent cells cultured in the presence of HGF and FGF-4 for4 days increased in cell size,acquired a spherical-like morphology, cultured in differentiation medium for a prolonged period (>20 days),more cells became spherical-like,but cell population decreased.Cells cultured in dedifferentiation medium for a prolonged period (>20days) became fibroid or fusiform shape. Spherical morphology was observed ,but there were minor cells.4.The expression of AFP was detected with immunofluorescence of the differentiated cells on day6,10,14,and 20 after the cells were cultured with HGF and FGF-4.The expression of AFP was not detected of dedifferentiation cells.5.The expression of ALB was detected with immunofluorescence of the differentiated cells on day10,14,and 20 after the cells were cultured with HGF and FGF-4. The expression of ALB was not detected of dedifferentiation cells.Concusion: Human peripheral blood mononuclear cells have the potential to differentiate into hepatocyte-like cells under specific inducing conditions.This makes them a source of cell transplantation in clinical application.