Culture And Hepatic Differentiation Of Bone Marrow-derived Mesenchymal Stem Cells In Vitro

Objectives: To establish a feasible method which separate and purify bone marrow mesenchymal stem cells (MSCs), and explore how hepatocyte growth factor (HGF),Acid fibroblast growth factor(aFGF)and oncostatin M (OSM) induce MSCs to be hepatocyte-like cells that have the hepatocellular features of phenotype and function and their effects in differentiation under certain circumstance. In a word, the experiment is to explore the best way to induce and differentiate MSCs to provide cell source for cell engineering science. Methods: 1. Separation and culture of MSCs: Myeloid cells of limbs bone were collected in Wistar male rat at the age of 3~4 weeks in asepsis condition. MSCs were separated by splitting red cells with adherent culture. The adherent difference between MSCs and other adherent cells was used to purify MSCs by strictly controlling the quantity of enzyme and the time of digestion during the subculture. 2. Identification of MSCs: Passage 4 MSCs were detected surface antigen CD44 ,CD45 and CD90 by immunofluorescence stainting. 3. Induction and differentiation of MSCs into hepatocyte-like cells: (1)Experiment groups: A.MSCs group: HGM+MSCs; B.HGF+aFGF group:HGM+20ng/mlHGF+20ng/mlaFGF+MSCs;C.HGF+OSMgroup:HGM+20ng/mlHGF+20ng/mlOSM+ MSCs; D.aFGF+OSM group:HGM+20ng/mlaFGF+20ng/mlOSM+MSCs; E.HGF+aFGF+OSMgroup:HGM+20ng/mlaFGF+20ng/mlHGF+20ng/mlOSM+MSCs;F.HGF+aFGF+OSM(divide)group:HGM+20ng/mlaFGF(1-9d)+20ng/mlHGF(9-18d )+20ng/ml OSM(9-21d) +MSCs;(2) Culture for induction and differentiation: Passage 4 MSCs were selected, digested with 0.25% trypsin solution, and inoculated 5×104/ml in 6-well-culture plate disposed by 50μg/ml FN, then were cultured with medium and inducible factors as well as above groups', at 37℃in humidified atmosphere chamber containing 5% CO2 During those days the medium was changed every 2~3 days, put cell supernatant at -20℃every time and cell differentiation was induced for 21 days; 4. Observation and identification of bone marrow-derived hepatocytes: (1) Morphologics: The form, size, nuclear number and plasma ratio change of the induced cells were observed under phase-contrast microscope; (2) Growth condition: MTT assay was used to measure cell growth curve in each group to explore the effect of inducible factors on cell growth; (3) The expression of hepatocyte markers: A. cell supernatant on the 0, 7th, 14th, 21st day of the induction period were taken to detect the expression of albumin(ALB) and alpha-fetoprotein (AFP) by bromocresol green method and chemi-luminescent technique;B. Cells on the 0, 7th, 14th, 21st day of the induction period were taken to detect the expression of albumin(ALB) alpha-fetoprotein (AFP) and cytokeratin 18(CK18) by RT-PCR method. (4) Metabolic function of hepatocyte: Culture supernatant on the 0, 3th, 6th, 9th,12th, 15th, 18th, 21th day during the induction period were taken to detect urea determination by glutamate dehydrogenases method; 5. Statistical analysis: Measurement data of variance analysis for repeated measurement data, and enumeration data of the rank-sum test by the check level ofα=0.05 and the software SPSS 13.0. Results: 1. The bone marrow monocytes separated by splitting red cells with adherent culture had more shape with activity 98%~100%. After 4~6 hours culture some cells become adherent with the round shape or oval shape. After 24 hours more and more adherent cells appeared with the shape of shuttle, triangle, polygon. And after 5~7 days, 80% or so of the adjacent colonies turned into flakes. The primary cells had different shapes, nonuniform distribution, in which there were also many mixed cells among them. After many times of replacing the liquid and passage, the cellular purity was increased, the cells had identical shape, uniform distribution, and the floating cells and mixed cells were rarely observed after passage 4; 2. The result of immunofluorescence stainting show that positive expression emerged on CD44 and CD90, and negative expression on CD45. 3. The result of MSCs induction and differentiation into hepatocyte: (1) After induction, the speed of breaking up and proliferation in MSCs was slowed down, gradually heteromorphical cells and binuclear cells were taken into being in the groups but the shapes of cells in MSCs group did not have obvious change. (2) The cell growth curve demonstrated that if induction culture medium was added, the proliferation of cells in each group was slower than in the MSCs group (P<0.05), there was no evident difference of the cell growth among the other induction groups(P>0.05). (3)chemi-luminescent technique demonstrated on the 7th day, AFP positive expression emerged in the H+a group,H+O group,a+O group,H+a+O group and H+a+O(divide)group, with the extension of induction time, the expression amount heightened gradually then slowed down except a+O group,on the 7th~14th day the expression amount in the H+a+O group was higher than in the other groups (P<0.05). positive expression emerged in the a+O group on the 21st day (P<0.05).Negative expression emerged in the MSCs group during the induction process ; bromocresol green method demonstrated on the 14th day,ALB positive expression emerged in the group except MSCs group and a+O group, the expression amount heightened gradually(P<0.05). At the same period, the expression amount was higher in the H+a+O group than in the other groups (P<0.05); Detection of the urea in the culture supernatant: the expression amount heightened gradually except MSCs group and a+O group (P<0.05). At the same period, the expression amount was higher in the H+a+O group than in the other groups (P<0.05). (4) RT-PCR demonstrated on the 0~7th day,ALB,CK18 mRNA negative expression emerged in every group and on the7th ~ 14th day AFP positive expression emerged in every group except in the a+O group.on the 21st day, AFP mRNA negative expression emerged in the groups except in the a+O group and the expression amount of AFP heightened gradually then slowed down except a+O group by contrasting gray scale , but the expression amount of AFP mRNA heightened straight in the a+O group. on the 14th~21st day, ALB and CK18 mRNA positive expression emerged in the groups except MSCs group and a+O group and the expression amount heightened gradually. on the 0~21st day ALB and CK18 mRNA negative expression emerged in MSCs group and a+O group. Conclusion: 1. split red cells culture combined with adherent culture method to separate and purify MSCs can lead to MSCs with preferable activity and purity; 2. The moderately purified MSCs by subculture can maintain their multiple differentiation ability; 3. Under certain microenvironment, HGF+aFGF, HGF+OSM, HGF+aFGF+OSM, HGF+aFGF+OSM(divide)can induce and differentiate MSCs to hepatic cells expressing AFP, ALB, CK18, having the ability of excreting urea; 4. aFGF+OSM cannot induce MSCs to differentiate into ripe hepatocyte without HGF, but they can be combined with HGF to promote the differentiation of MSCs, obviously increase the differentiation ratio.