Biological characteristics of mouse peripheral blood derived mesenchymal stem cells after the granulocyte colony-stimulating factor and stem cell factor mobilization / 中国组织工程研究

ABSTRACT

AIM: Granulocyte colony-stimulating factor and stem cell factor are the important cell factor in the hematopoiesis. According to changes in the cell information conduction and obstruction of the expression of the stem cell adhesion molecule, they can increase the quantities of stem cells, restrain the apoptosis and alter the function of bone marrow mesenchymal stem cells. The experiment was to discover how to isolate and culture the granulocyte colony-stimulating factor and stem cell factor mobilized peripheral blood derived mesenchymal stem cells, and to explore the induction to the cartilage cells in the monolayer culture. METHODS: Experiments were performed at the Hebei Provincical Key Laboratory, Research Center, Fourth Hospital of Hebei Medical University from January 2005 to March 2006. ①The 4-6 weeks male BALB/c mice weighting 15-20 g were provided by Animal Experimental Center of Hebei Medical University. The lab procedure met the animal ethical standard. ②The density gradient centrifugation combined with the adherence method were adopted to isolate the mesenchymal stem cells from peripheral blood of BALB/C mice after granulocyte colony-stimulating factor and stem cell factor mobilization. Detections of colony forming unit-forming ability were preformed on grow well cells. Mesenchymal stem cells were identified by flow cytometry and immunohistochemical method. The 3rd passage mesenchymal stem cells were induced to differentiate into chondrocytes in high glucose DMEM medium containing transforming growth factor beta 1. The expression of type Ⅱ collagen was detected by immunohistochemical method. RESULTS: ①The peripheral blood derived mesenchymal stem cells possessed the similar phenotypes with bone marrow derived mesenchymal stem cells and could differentiate into chondrocytes. Flow cytometry showed that CD29 and CD44 were positive, while CD34 and CD45 of them were negative. ②The passage 3 mesenchymal stem cells did not express type Ⅱ collagen and laminin, expressed vimentin in immunohistochemical method. The differentiated mesenchymal stem cells expressed type Ⅱcollagen ③The passage 1,3,5 mesenchymal stem cells showed different ratios of colony forming unit. Pairwise comparison showed that with the passage increasing, the ratio of colony forming unit degraded gradually (21.32%,16.13%,9.63%,P