Directional Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Structural Cells For Tissue Engineering Heart Valves

Part 1 DIRECTIONAL DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS INTO ENDOTHELIAL CELLSObjective: To establish a simple and stable method for obtaining large quantity of highly purified endothelial cells in vitro, and identify the morphology and surface markers of the cells.Methods: 1.BMSCs were isolated from SD rats which were healthy, 4 weeks old and were ignored their sex, then cultured, proliferated and purified by passage culture .The P3-BMSCs were identified by flow cytometry with CD29,CD34. 2.The P3-BMSCs were divided into treated group and control group. The P3-BMSCs in treated group were cultured in the induction medium of L-DMEM ( 20%FBS+10ng/ml VEGF ) for differentiation into endothelial cells; the P3-BMSCs in control group were cultured in L-DMEM+10%FBS.All of cells were detected under inverted microscope to examine the cell's morphology every day. The cells both in treated group and control group were characterized by immunohistochemisty with Anti-Factorâ…§after being cultured for 7 days and 14 days.Results:1.We could get enough and pure BMSCs by full-marrow culture method,more than 96% of the P3-BMSCs were positive expression of CD29 and negative expression of CD34;the BMSCs had the typical feature of a long-fusform shape and whirpool arrangement ,and had high capability of proliferation in Vitro.2.The induced cells in treated group with L-DMEM(20%FBS+10ng/ml VEGF)became bigger and larger gradually,typical feature of cobblestone-like shape was observed after being cultured for 10 days.While the cells in control group maintained the typical feature of a long-fusform shape and whirpool arrangement and had no obvious change. 82% of the cells in treated group were positive expression of Anti-Factorâ…§at 7d,and 93% of the cells in treated group were positive expression of Anti-Factorâ…§at 14d.The cells in control group were negative expression of Anti-Factorâ…§.Conclusions: BMSCs could be directional differentiated into endothelial cells in the induction medium of L-DMEM(20%FBS+10ng/ml VEGF).We could obtain large quantity of highly purified endothelial cells in vitro by this way.Part 2 DIRECTIONAL DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS INTO SMOOTH MUSCLE CELLSObjective: To establish a simple and stable method for obtaining large quantity of highly purified smooth muscle cells in vitro, and identify the morphology and surface markers of the cells.Methods:The P3-BMSCs in treated group were cultured in the induction medium of L-DMEM(20%FBS+2ng/ml PDGF-BB)for differentiation into smooth muscle cells. The P3-BMSCs in control group were cultured in L-DMEM+10%FBS.All of the cells were detected under inverted microscope to examine the cell's morphology every day.The cells both in treated group and control group were characterized by immunohistochemisty with Anti-Actin alpha and Anti-Vimentin after being cultured for 7 days and 14 days.Results : The induced cells in treated group with L-DMEM(20%FBS+2ng/ml PDGF-BB)became bigger,irregularly fusiform-shaped gradually,and possessed"peak and vally"characterisrics under inverted microscope. While the cells in control group maintained the typical feature of a long-fusform shape and whirpool arrangement and had no obvious change. 78% of the cells in treated group were positive expression of Anti-Actin alpha at 7d,and 87% of the cells in treated group were positive expression of Anti-Actin alpha at 14d.The cells in treated group were negative expression of Anti-Vimentin.The cells in control group were negative expression of Anti-Actin alpha or Anti-Vimentin.Conclusions: BMSCs could be directional differentiated into smooth muscle cells in the induction medium of L-DMEM(20%FBS+2ng/ml PDGF-BB).We could obtain large quantity of highly purified smooth muscle cells in vitro by this way. Part 3 DIRECTIONAL DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS INTO FIBROBLASTSObjective: To establish a simple and stable method for obtaining large quantity of highly purified fibroblasts in vitro, and identify the morphology and surface markers of the cells.Methods: The P3-BMSCs in treated group were cultured in the induction medium of L-DMEM ( 20%FBS+20ng/ml b-FGF ) for differentiation into fibroblasts;the P3-BMSCs in the other group were cultured in L-DMEM+10%FBS as control.All of the cells were detected under inverted microscope to examine the cell's morphology every day.The cells both in treated group and control group were characterized by immunocytochemistry with Anti-Actin alpha and Anti-Vimentin after being cultured for 7days and 14 days.Results : The induced cells in treated group with L-DMEM(20%FBS+20ng/ml b-FGF) became slender,spindle-shaped morphology with long cell process gradually.While the cells in control group maintained the typical feature of a long-fusform shape and whirpool arrangement and had no obvious change. The cells in treated group were positive expression both of Anti-Actin alpha and Anti-Vimentin.72% of the cells in treated group were positive expression of Anti-Actin alpha at 7d,and the percentage climbed up to 82% at 14d. 75% of the induced cells were positive expression of Anti-Vimentin at 7d, and the percentage climbed up to 85% at 14d.The cells in control group were negative expression of Anti-Actin alpha or Anti-Vimentin .Conclusions: BMSCs could be directional differentiated into fibroblasts in the induction medium of L-DMEM(20%FBS+20ng/ml b-FGF). We could obtain large quantity of highly purified fibroblasts in vitro by this way.