The Effects Of Shear Stress And VEGF On The Differentiation Of Human Bone Marrow-Derived Mesenchymal Stem Cells Into Endothelial Cells In Vitro

BackgroundA novel strategy for the treatment of ischemia diseases, termed therapeutic neovascularization, is proposed in recent years. It relates to using recombinant formulations of angiogenic cells or/and growth factors to expedite and/or augment collateral artery development in ischemic tissues.. Most of the current research focus on which kind of angiogenic cells to choose, which mainly come from stem cells, and how to obtain sufficient cells.Bone marrow mesenchymal stromal cells (MSCs) belong to a kind of non-hematopoietic stem cells-derived cell subgroup. MSCs, with the characteristics of easy separation, multiplex differentiation capacity and weak immune rejection, have already become a kind of very important seeded cells for tissue engineering construction. The biochemistry environment and complex biomechanics environment in which MSCs grow have important effects for its differentiation and phenotype expression. In vitro under different inducing conditions, MSCs can differentiate into endothelial cells (ECs), osteoblasts and cartilage cells, fat cells, muscle cells, nerve cells, etc. Since ECs contribute to the formation of new vessels, MSCs can serve as the source of angiogenic cells for therapeutic neovascularization. Besides, proliferation capacity of MSCs is supposed to increase the number of angiogenic cells in vitro. However, it is expensive to utilize cytokines in the differentiation of MSCs into ECs, and there are also a few potential safety issues in the application of biological agents. Compared with chemical conditions including cytokines, the effect of biomechanical condition on the differentiation of MSCs into ECs remains to be resolved. Studies indicated that shear stress could induce the differentiation of ESCs and EPCs into ECs, which implies the same potential effect of shear stress on MSCs.AimsThe present study aims to explore the effects of VEGF and shear stress on the differentiation of human bone marrow derived MSCs into ECs. MethodsPartⅠ: Isolation and Culture of MSCs: 1. Bone marrow from healthy adult was collected, and MSCs were isolated by density gradient centrifugation and adherent culture. 2. Cells morphology was viewed with an inverted microscope. 3. MSCs were identified to express CD44 by using immunohistochemistry staining.PartⅡ: The effects of VEGF on the differentiation of MSCs into ECs. Isolated MSCs were divided into control and VEGF induction group, in which cells were administered with 10μg/L VEGF. Cells morphology was observed after induction for 24 h and 7 d. Immunofluorescence was used to identify the endothelial cell-specific marker, Von Willebrand factor (vWF). Uptake of Dil labled acetylated low-density lipoproteins (Dil-Ac-LDL) was used to examine the function of endothelial cells.PartⅢ: The effects of shear stress on the differentiation of MSCs into ECs Isolated MSCs were divided into control group and shear stress induction group, in which MSCs were subjected to different levels of shear stress, 8 or 15 dyn/cm2 respectively, for 24 h. Cells morphology was observed after induction for 24 h and 7 d. Immunofluorescence was used to identify the endothelial cell-specific marker, Von Willebrand factor (vWF). Uptake of Dil labled acetylated low-density lipoproteins (Dil-Ac-LDL) was used to examine the function of endothelial cells.PartⅣ: The effects of sheer stress combined with VEGF on the differentiation of MSCs into ECs Isolated MSCs were divided into control group and VEGF plus shear stress group, in which MSCs were subjected to different levels of shear stress, 8 or 15 dyn/cm2 respectively, combined with VEGF for 24 h. Cells morphology was observed after induction for 24 h and 7 d. Immunofluorescence was used to identify the endothelial cell-specific marker, Von Willebrand factor (vWF). Uptake of Dil labled acetylated low-density lipoproteins (Dil-Ac-LDL) was used to examine the function of endothelial cells.Results1. Isolated MSCs exhibited spindle-shaped morphology. MSCs were qualified as adherent cells positive for CD44.2. MSCs subjectied to VEGF for 24 h exhibited the same morphology with control group. Cells were negative for both vWF and Dil-Ac-LDL. However, compared with control, after stimulation with VEGF for 7 d, the shape of MSCs turn to be round and polygon, just like endothelial cells. Cells were positive for both vWF and Dil-Ac-LDL.3. Compared with control, MSCs, subjected to shear stress (8dyn/cm2) for 24 h, exhibited endothelial cell -like morphology, and were positive for both vWF and Dil-Ac-LDL. However, MSCs, subjected to shear stress of 15 dyn/cm2, remained to be the same as control in morphology and staining.4. Compared with sheer stress, shear stress plus VEGF significantly increased cell density, indicating the proliferation of cells. Cells were potently positive for vWF and positive for Dil-Ac-LDL.Conclusion1. MSCs can be isolated by density gradient centrifugation and adherent culture in vitro.2. VEGF induced the differentiation of MSCs into ECs.3. Shear stress (8dyn/cm2) induced the differentiation of MSCs into ECs, while Shear stress (15dyn/cm2) failed in this effect.4. Shear stress combined with VEGF not only induced the differentiation of MSCs into ECs, but also caused cell proliferation, which seems to have a better effect than only shear stress...