title: Culture and Identification of Bone Marrow Mesenchymal Stem Cells from Enhanced Green Fluorescent Protein-transgenic Rats.
abstract: OBJECTIVE
  To isolate, culture, and identify bone marrow mesenchymal stem cells (BMSCs) from enhanced green fluorescent protein (EGFP)-transgenic rats in vitro.
  
  
  METHODS
  Bone marrows were isolated from tibia and femur of healthy EGFP-transgenic rats of specific pathogen free (SPF) grade. Then,the whole bone marrow adherent method was used for isolation,culture,and purification of BMSCs. The morphological change was noted by continuous observation under inverted fluorescence microscope. The growth curve of cells was drawn through the method of CCK-8 and the proliferation compared with wild type BMSCs. The surface markers of BMSCs were detected by flow cytometry. The BMSCs were induced to differentiate into osteoblasts, adipocytes, and chondrocytes lineages. The EGFP-BMSCs were transplanted into the rats intravenously, and the expression of GFP was detected.
  
  
  RESULTS
  BMSCs stably expressing EGFP gene were obtained successfully, with the fusiform-shaped appearance and the forming of circinate cell colonies. The growth curve of EGFP-MSCs showed the characteristic of active proliferation, showing no significant difference compared with the wild-type BMSCs. The expression rates of the surface markers of BMSCs CD29, CD90, CD34, CD49d, and CD45 were 99.4%, 96.4%, 0.171%, 0.049%, and 0.038%. The GFP were detected in lung 3 days after transplantation. After osteogenic, adipogenic, and chondrogenic induction, oil red-O and alizarin red positive signals and toluidine blue positive cells were detected.
  
  
  CONCLUSIONS
  High-purity BMSCs stably expressing green fluorescent protein gene can be cultured using the whole bone marrow adherent method. EGFP does not affect the stem cell properties and expresses stably after transplantation. The cells can be used as seed cells for subsequent research.
authors.affiliation: Department of Pediatrics,Zhongda Hospital,Southeast University,Nanjing 210009,China.
authors.forename: Hui-juan
authors.initials: HJ
authors.lastname: Liu
authors.affiliation: Department of Cardiothoracic Surgery,Zhongda Hospital,Southeast University,Nanjing 210009,China.
authors.forename: Ruo-yu
authors.initials: RY
authors.lastname: Hu
authors.affiliation: Department of Pediatrics,Zhongda Hospital,Southeast University,Nanjing 210009,China.
authors.forename: Wang-juan
authors.initials: WJ
authors.lastname: Dai
authors.affiliation: Department of Cardiothoracic Surgery,Zhongda Hospital,Southeast University,Nanjing 210009,China.
authors.forename: Yuan-guo
authors.initials: YG
authors.lastname: Liu
authors.affiliation: Department of Cardiothoracic Surgery,Zhongda Hospital,Southeast University,Nanjing 210009,China.
authors.forename: Bo
authors.initials: B
authors.lastname: Min
authors.affiliation: Department of Pediatrics,Zhongda Hospital,Southeast University,Nanjing 210009,China.
authors.forename: Li
authors.initials: L
authors.lastname: Jiang
mesh_terms: D017667:Adipocytes; D000818:Animals; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D019902:Chondrocytes; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells; D059630:Mesenchymal Stem Cells; D010006:Osteoblasts; D051381:Rats; D055647:Rats, Transgenic
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins