Differentiation of rat mesenchymal stem cells into smooth muscle cells induced by cell-to-cell contact / 中国组织工程研究

ABSTRACT

BACKGROUND: It is conceivable that bone marrow stem cells can differentiate into smooth muscle cells (SMCs) and contribute to neointimal formation in atherogenesis. However, the mechanism remains unknown. The "milieu-induced-differentiation" hypothesis focuses on the key role of cell-to-cell contact and cytokine on the differentiation of stem cells. Bone marrow mesenchymal stem cells (MSCs) have the potential to differentiate into SMCs.OBJECTIVE: To induce MSCs into SMCs in vitro, and investigate the influence of the differentiated SMCs or cell factors on MSCs differentiation.DESIGN: Controlled experiment in vitro with repeated observation and measurement based on cells.SETTING: Department of Cardiology, First Hospital of Xi'an Jiaotong University.MATERIALS The experiment was accomplished in the Laboratory of Cardiology, First Hospital of Xi'an Jiaotong University between May 2003 and May 2004. SD rats of either gender were provided by the Animal Center of Xi'an Jiao Tong University, 60-80 g, 90-110 g. The following antibodies were used Mouse anti human SM-α-actin (NeoMarkers),Mouse anti human Calponin (NeoMarkers), TRITC-coupled goat anti mouse IgG antibody (SBA). Mouse anti rat CD34 conjugated FITC (Santa Cruz), Mouse anti rat CD71 conjugated FITC (Oxford Biotechnology), Mouse anti rat anti-CD90 conjugated PE (Oxford Biotechnology). Lipofectamine 2000 (Invitrogen). PEGFP-N3 (the laboratory).METHODS: Bone marrow mesenchymal stem cells were obtained from rat bone marrow by using percoll density gradient centrifugation. SMCs were isolated by using tissue explantation method. Flow cytometer was used to detect the immunofluorescence stain. Then MSCs and SMCs were identified. MSCs were transfected with pEGFP-N3 by Lipofectamine 2000, while untransfected MSCs were taken as controls. Conditioned culture of MSCs and SMCs ①MSCs at passage 3 were seeded on chamber slides in a 12-well culture plate. The medium was DMEM containing 0%, 5%,7.5% fetal bovine serum (FBS) and SMCs conditioned medium containing 0%, 5%, 7.5% FBS, respectively. The cells were cultured for 10-14 days and immunofluorescence analysis was performed by using monoclonal antibodies against SM-α-actin, calponin.②Indirect co-culture of MSCs with SMCs were established using a semi-permeable membrane cell culture insert. The inserts were plated into culture well. SMCs were cultured on the inside of inserts while MSCs were added to the outside of inserts, respectively. MSCs were culture alone in medium containing 3%, 7.5% FBS and immunofluorescence analysis was performed by using monoclonal antibodies against SM-α-actin, calponin.③MSCs were transfected with pEGFP-N3. After 24 hours, the MSCs were cocultivated with SMCs at an equal density for 7-14 days.As a control, MSCs were cultured alone. MSCs co-cultured were stained with antibodies against calponin, SM-α-actin. MAIN OUTCOME MEASURES: ①Identification of MSCs by floe cytometer.②cytoplasmic antigen expression of SMCs. RESULTS: ①Immunofluorescence analysis showed that MSCs expressed SM-α-actin, but did not express calponin. As a control, SMCs expressed both SM-α-actin and calponin.②Flow cytometry showed that MSCs expressed CD71 of low level, CD90 of high level and no expression of CD34. ③The MSCs transfected with green fluorescence protein continued to express for 2-3 weeks. ④MSCs grew well in SMCs conditioned medium or different concentrations of FBS. Cell growth was FBS concentration dependent in indirect co-culture system of MSCs and SMCs. Several double-positive cells in direct co-culture system were detected enhanced green fluorescence protein and antibodies against calponin, SM-α-actin. CONCLUSION: ①SMCs conditioned medium and cell factor only promote MSCs growth and cytoplasmic granules increase. But these do not induce MSCs differentiate into SMCs. ②The cell-to-cell contact is essential for MSCs differentiation to SMCs.