Background Enhanced green fluorescent protein(EGFP) has been an important reporter gene for gene therapy. Human mesenchymal stem cells (hMSCs) are ideal target cells in cell transplantation and tissue engineering. We investigated their biological characteristics and differentiation mediated b y PLEGFP-N1 retroviral transduction. Methods hMSCs were isolated from human bone marrow by density g radient fractionation and adherence to plastic flasks. Individual colonies were selected and cultured in tissue dishes. Packaging cells PT67 were transfected by PLEGFP-N1 retroviral vector , and hMSCs were transduced by viral supernatant infection. Meanwhile, hMSCs-EGFP were identified by immune phenotypes and whe ther it could differentiate into osteoblasts or adipocytes under conditioned med ia was investigated. Results The rate of stably transduced hMSCs-EGFP was up to 96% after being screened by G418. hMSCs-EGFP exhibited fibroblast-like morpholog ical features. Flow cytometric analyses showed that hMSCs-EGFP were positive f or CD 73, CD 105, CD 166, CD 90 and CD 44, but negative for CD 34 and CD 45. In addition, it could functionally be induced i nto osteocytes or adipocytes under conditioned media. These biological features of hMSCs-EGFP were consistent with those of hMSCs. Conclusions hMSCs transduced by PLEGFP-N1 retroviral vector ca n be used in vivo securely because they can maintain their biological charac teristics and differentiation. It is a simple and reliable way to trace the ch anges of hMSCs in vivo by EGFP during cell transplantation and gene therapy . Background of the Invention Green fluorescent protein (EGFP) has been an important reporter gene for gene therapy. Human mesenchymal stem cells (hMSCs) are ideal target cells in cell transplantation and tissue engineering. We investigated their biological characteristics and differentiation mediated by PLEGFP-N1 retroviral transduction . Methods hMSCs were isolated from human bone marrow by density g radient fractionation and adherence to plastic flasks. Individual colonies were selected and cultured in tissue dishes. Packaging cells PT67 were transfected by PLEGFP-N1 retroviral vector, and hMSCs were transduced by viral supernatant infection . Meanwhile, hMSCs-EGFP were identified by immune phenotypes and whe ther it could differentiate into osteoblasts or adipocytes under the conditioned med ia was investigated. HMSCs-EGFP was identified to be 96% after being screened by G418. HMSCs- EGFP fiber fibroblast-like morphological features. Flow cytometric analysis shows that hMSCs-EGFP were positive f or CD73, CD105, CD166, CD90 and CD44, but negative for CD34 and CD45. In addition, it could functionally be induced i nto osteocytes These biological features of hMSCs-EGFP were consistent with those of hMSCs. Conclusions hMSCs transduced by PLEGFP-N1 retroviral vector ca n be used in vivo vivo as they can maintain their biological charac teristics and differentiation. It is a simple and reliable way to trace the ch anges of hMSCs in vivo by EGFP during cell transplantation and gene therapy.