Study On Induction Of Human Bone Marrow Mesenchymal Stem Cells To Differentiate Into Endothelial Cells In Vitro And Analysis Of Differentially Expressed Genes With Microarray

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes, chondrocytes, and adipocytes in vivo and in vitro. The aim of this study was to investigate the feasibility of human bone marrow MSCs differentiating into endothelial cells (ECs) in vitro. 1. ATERIALS AND METHODS 1.1 Isolation and Culture of Human Bone Marrow-Derived MSCsBone marrow samples were collected from a healthy donors (male, 39 years old ). 5ml bone marrow aspirated from the iliac crest and dilutes with equivalent in Dulbecco's-modified Eagle's Medium-Low Glucose (DMEM) +F12. A 5ml aliquot was layered over a Ficoll solution (d=1.077g/ml) and centrifuged at 1200rmp for 30 minutes at 4℃. Bone marrow mononuclear cells (BM-MNCs) at the interface were recovered, and washed twice in DMEM+F12. All cells were seeded into 25-cm2 flasks containing DMEM + F12 and MCBD (3:2, V/V) supplemented with penicillin 10 U/ml, streptomycin 100 μg/ml, 10% fetal calf serum (FCS), 2ng/ml basic fibroblast growth factor (bFGF), 10ng/ml epidermal growth factor (EGF), 10ng/ml insulinlike growth factor (IGF). MSCs cultures grew at 37℃ in 5% CO2. Non-adherent cells were removed after 24 hours by changing medium. The medium was changed half subsequently every 4 days, twelve later the culture reached 90% confluency. MSCs were recovered using 0.25% Trypsin-0.02%EDTA and seeded at flask as passage 1 (P1) cells.1.2 Induction of Differentiation into Endothelial CellsAt passage 4, cells were cultivated in the presence of 10 ng/ml bFGF, 10 ng/ml VEGF. Medium was changed half every 4 days.1.3 Flow Cytometry AnalysisCells were trypsinized, washed with PBS, fixed with paraformaldehyde and incubated with antibodies against CD34, CD45, CD54, CD 105, CD 106, HLA-DR, KDR, and vWF. Analysis was performed with a flow cytometer (Becton Dickinson).1.4 Absorbing ac-LDL and Binding UEA TestsCells cultured reached 90% confluency, incubated with Dil-ac-LDL in 4|j.g/ml for 4h at 37 °C, 5%CO2. Then, cells were rinsed with PBS, fixed with 1% poly formaldehyde, and incubated with FITC-labelled UEA in lOug/ml for lh at 37"C, staining was detected by fluorescence microscopye.1.5 Analysis of Differentially Expressed Genes with MicroarraymRNA was obtained from MSCs and ECs differentiated from MSCs. cDNA was synthesized and expanded with RT-PCR, labelled with fluorescent reagent. The labelled cDNA hybridized with oligonucleotide in misroarray. Scanner (PerkinElmer) read fluorescent signal value. When signal value ratio in same spot is above 2 times, this spot is differentially expressed genes. 2. RESULTS2.1 Primary culture of MSCsNearly all MSCs adhered after 24h of plating. Non-adherent cells removed with changes in medium. The adherent elongated with spindle-shaped, triangular or polygonal morphology. They grew in clusters later and reached confluent after 12d.2.2 Expansion Culture of MSCSPassaged cells attached several hours after plating. They gradually stretched tospindle-shaped or polygonal morphology. When reached confluence after 5-7d. They became uniformly shaped and densely arranged.2.3 Differentiation of MSCs Into Endothelial-Like CellsInducting differentiation of MSCs into endothelial-like cells by cultivating MSCs in the presence lOng/ml bFGF, lOng/ml VEGF for 24 days, there were no visible difference between two cells in morphologyFlow cytometry analysis showed signals of expression of CD34, CD45, KDR, and enhanced signals of CD54, CD 106 ,vWF. These cells were positively stained for Dil-ac-LDL and UEA-FITC.2.4 Differentially Expressed GenesOf all 7267 genes in microarray, there were 814 differdntially expressed genes. The newly and intensely expressed genes was 72, including some genes encoded adhence molecule, extracellularmatrix, protein associated with vascular enthelual cells function and receptor. 3. DISCUSSIONMSCs are plurpotent cells with extensive proliferative potential. They have been shown to differentiate into bone, cartiage, adipocytes, hematopoietic support stroma and sheletal myocytes. Recent investigations proved that these cells can also differentiate into hepatocytes, cardiomyocytes and epithelial cells of lung and gastrointestinal tract, as well as cells of skin and neurectoderm. These findings indicate that adult stem cells share the common potential of transdifferentiation with embryonic stem cells, namely "plasticity" .There has been no clear evidence that MSCs are capable of differentiating into ECs yet. AC133+, CD34+progenitor cells (hemangioblasts) from bone marrow are thought to be the origin of circulating ECs. Reyes et al indentified a sort of AC133+,Flk34+, CD34" multipotent adult progenitor cells (MAPCs) that can, at single-cell level, differentiate into endothelial and nurectodermal cells. Since MAPCs were coppurified with MSCs from postnatal bone marrow, they were thought to be more primitive stem cells in MSCs. because MAPCs are rare in bone marrow and their expansion requires serum-free culture, it will take longer time to get sufficient cells for experiment.MSCs have no unique surface antigens, so it is unable now to obtain purifed MSCs by sorting methods. In this study, BM-MNCs by were isolated by density gradient centrifugation. Purification of MSCs was achieved by removing non-adherent cells with replacement of medium.Our persent study showed that MSCs were cultured supplement with VEGF. MSCs presented phenotypes of ECs. Them are positive for CD34, CD45, KDR. Meantime, them express more CD54, CD 106, vWF, HLA-DR. In addition, these cells have the ability to bind UEA and to absorb ac-LDL, which are the characteristic functions of mature ECs. These findings indicated that the differentiated cells are ECs. While bFGF, EGF and IGF enhance proliferation of MSCs, VEGF plays a crucial role in inducing MSCs to differentiate into ECs.ECs differentiated from from MSCs are long spindle-shaped still, cobblestone monolayer of mature ECs do not present here. So, these cells could be diffenert from real endothelium. Further studies are needed to investigate whether the differentiated ECs from MSCs could be useful in treatment ischemic disease and tissues engineering.After induction MSCs to differentiate into ECs, many genes were expressed newly and intensely. Some genes encoded adhence molecule, extracellularmatrix, protein associated vascular enthelual cells function, receptor. These proteins play aimportant role in vasculogenesis, angiogenesis, earring out function of ECs. Moreover, Oxidised low density lipoprotein (lectin-like) receptor 1 (LOX-1) and Vascular cell adhesion molecule bear specificity of ECs.