Differentiation Potential Of Human Placenta Derived Mesenchymal Stem Cells Into Vascular Endothelial Cells

ObjectivePromoting angiogenesis, microcirculation is the key of treating ischemic diseases such as coronary heart disease, cerebrovascular disease and peripheral arterial occlusive disease. Therapeutic angiogenesis is the proposed currently new concept, which differentiate from conventional drugs and surgical intervention, which simulate in vivo angiogenesis mechanism to improve the angiogenesis and collateral circulation. The source of seed cells is the bottleneck of vascular tissue engineering, then the endothelial cells (ECs) is one of the need to be resolved seed cells in tissue-engineered blood vessels. So far some researchers have maken several kinds of stem cells differentiate into ECs, Such as embryonic stem cells, angioblast, endothelial progenitor cells (EPCs) and multi-potential adult stem cells. However, the contain of these cell lines in the body is few, which are not readily available because they are subject to various constraints. In 2004, Oswald et al successfully induced bone marrow mesenchymal stem cells(MSCs) into the vascular endothelial cell for the first time in vitro. Therefore, MSCs may be an ideal source of seed cells which can differentiate into endothelial cells. But the number of human bone marrow MSCs were very low, approximately 1/1 x 1041 x 105 single-nucleated cells. Furthermore, in some circumstances of severe infections, bone marrow malignancy, or age increased, the number of bone marrow MSCs proliferation and differentiation were significantly decreased. In 2004, Fukuchi et al obtained MSCs from the mature placenta lobule. Subsequently, placenta derived mesenchymal stem cells(PDMSCs) were proved they have a similar morphology, immune phenotype and functional characteristics with bone marrow-derived MSCs. Our lab has also proved PDMSCs can be induced to differentiate into fat, cartilage, bone and neural cells. As a postpartum "waste", the placenta researchment does not involve ethical issues, and has a rich source. So far, we have not found that PDMSCs were differentiated into the vascular endothelial cell at home and abroad. Therefore, this experiment isolated and cultured PDMSCs in vitro, then the obtained PDMSCs were induced by VEGF and bFGF differentiate into vascular endothelial cells in vitro. In the induction process at different time points, we detected the expression of endothelial-specific antigen and the ability of differentiated ECs forming blood vessels in vitro. Therefore, the results will provide experimental data for the clinical application PDMSCs for the treatment of ischemic disease and vascular tissue engineering.Method1. Isolation, purification and expansion of PDMSCs Primary was extracted by planting tissue. In the case of sterile, placental tissue was cut into small pieces, which were evenly inoculated 10cm dish. Adding with 10% FBS in DMEM 8ml, cells culturing at 37℃with water-saturated atmosphere and 5% CO2, changing fluid every 3d time. The climbed cells were passaged after removal of tissue until growth to 70%-80% of the bottom area of dish. After passaging 2nd-3rd generation, the red blood cells and fibroblast cells disappeared. In this study, we selected 3rd generation PDMSCs for inducing differentiation and detection.2. Flow cytometry Collection 3rd generation PDMSCs, subpackaged 1×106counting cells/tube after counting cells. For detecting cell surface antigen phenotype, cells were immunolabeled respectively with the mouse anti-human monoclonal antibody CD105-PE, CD34-PE and CD166-FITC, CD31-FITC, CD45-FITC, dark incubation 30 min at 4℃. Meanwhile, setting up PE, FITC, blank control group. Washed with ice PBS a time, supernatant, added 200ul PBS and blending cells, cells were detected surface antigen phenotype by flow cytometry.3. ECs differentiation in vitro Choosing 3rd generation PDMSCs, the concentration of 3×104 cells/ml and 1.5×105 cells/ml were respectively seeded onto 35mm and 60mm dishes. Adding with endothelial cell-induced differentiation medium, cells were maintained at 37℃with a water-saturated atmosphere and 5% CO2, changing fresh-induced fluid for every 4d time.4. Morphological changes of PDMSCs Observing morphological changes, proliferation and arrangement of induced PDMSCs through light microscope, we contrast with the changes of 0d,4d,8d and 12d at every other day.5. Immunocytochemistry PDMSCs were seeded in 35mm dishes with the concentration of 3×104 cells/ml,added with endothelial cell-induced differentiation medium. The induced cells were detected endothelial specific markers CD31, CD144/VE-cadherin, KDR, vWF protein expression at 0d,4d,8d,12d by immunohistochemical staining.6. Observation the induced cell ultrastructure by TEM The induced cells were digested with 0.25% Trypsin-EDTA, fixed with 1% osmium tetroxide, washed with 0.1mol/LPBS three times, and dehydrated by gradient alcohol and acetone.7. Angiogenesis in vitro The induced 7d cells were seeded onto semi-solid matrix medium and observed the ability of formation of capillary-like network structure with in vitro angiogenesis kit.Results1. The characterization of the primary PDMSCs After 7 to 10 days inoculation, a large number of adherent cells transferred from placenta tissue. The placenta-derived cells were classified into two groups according to the growth and morphologic characteristics. One group had a small and homogeneous morphology; and the other one displayed fibroblast-like cells or spindle-shaped morphology. As the cell density increases, the cell gradually became spindle-shaped monolayer cells, showing the arrangement of swirling similar to the fibroblast. The climbed cells were passaged after removal of tissue until growth to 70%-80% of the bottom area of dish.2. PDMSCs surface markers detected by flow cytometry Isolated PDMSCs typically expressed the MSCs relatively specific antigens CD 105(92.87%) and CD 166(72.40%), and were negative for hemopoietic system specific marker and leukocyte common antigen CD34 and CD45, and no expression of endothelial cell-specific surface markers CD31.3. The morphology and arrangement of the ECs ECs derived from PDMSCs displayed cobblestone-like shape. The arrangement of cell has a certain direction, showing the lumen shape, spoke-like, striped growth, and the cells were connected end to end.4. Immunocytochemical staining(1) Expression of CD31 Trace expression of CD31 appeared on the 4 day, but no significant difference compared with 0d(P>0.05). As ECs differentiating, at the 8th day, expression of CD31 was significantly enhanced, more stronger than the 0d,4d,12d (P<0.05).(2) Expression of VE-cadherin/CD 144 With the extension of induction time, VE-cadherin/CD144 expression increased gradually. After the 8th day noticeable brown granules, CD 144 expression significantly enhanced and positive cells were obviously increased(P<0.05).(3) Expression of vWF vWF slightly expressed in the 0 day. The expression gradually enhanced after 4 day and reached the peak in the 8th day, followed by stable expression. Image analysis showed that, on the 8th day and 12th day, the integral optical density(IOD) of cells was significantly stronger than the Od and 4d (P<0.05), but no difference between 8d and 12d(P>0.05).(4) Expression of Flk-1/KDR Flk-1/KDR as an early marker of endothelial cells in the 0 day that was expressed, and amounted to the summit at the 4th day, after that its expression was significantly weaken. The expression Flk-1/KDR in the 4th day was significantly stronger than the 0d,8d and 12d (P＜.05), but the three groups was no significant difference(P> 0.05).5. The ultrastructure of the differentiated ECs Differentiated ECs showed typical endothelial cells with numerous caveolae(CA) and pinocytic vesicle. The typical endothelial cell-specific structure-Weibel-Palade bodies was observed in the cytoplasmic of differentiated cells.6. Angiogenesis of differentiated ECs in vitro The cells stretched out adjacent synapses and connected with each other after seeded onto Matrigel for 2h. The differentiated cells showed capillary cavity-like structure and complex network of anastomosing cells. The undifferentiated cells displayed scattered cells and can not form tube-like structure.Conclusion1. Placenta could isolate a large number of mesenchymal stem cells.2. PDMSCs could be induced by VEGF and bFGF to differentiate into vascular endothelial cells in vitro, and the differentiated ECs had the ability of migration and angiogenesis in vitro. 3. During the differentiation of PDMSCs to ECs,the expression peak of endothelial markers Flk-1/KDR, CD31, vWF and VE-cadherin/CD144 showed time sequential.