Mesenchymal stem cells transduced by PLEGFP-N1 retroviral vector maintain their biological features and differentiation
Author(s): HE Xu, LI Yu-lin, WANG Xin-rui, GUO Xin, NIU Yun
Pages: 1728-1734
Year: 2005 Issue: 20
Journal: CHINESE MEDICAL JOURNAL
Keyword: mesenchymal stem cells; bone marrow; enhanced green fluorescent protein; retroviral vector; gene therapy;
Abstract: Methods hMSCs were isolated from human bone marrow by density gradient 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 whether it could differentiate into osteoblasts or adipocytes under conditioned media was investigated. Results The rate of stably transduced hMSCs-EGFP was up to 96% after being screened by G418. hMSCs-EGFP exhibited fibroblast-like morphological features. Flow cytometric analyses showed that hMSCs-EGFP were positive for CD73, CD105, CD166, CD90 and CD44, but negative for CD34 and CD45. In addition, it could functionally be induced into 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 can be used in vivo securely because they can maintain their biological characteristics and differentiation. It is a simple and reliable way to trace the changes of hMSCs in vivo by EGFP during cell transplantation and gene therapy.
Pages: 1728-1734
Year: 2005 Issue: 20
Journal: CHINESE MEDICAL JOURNAL
Keyword: mesenchymal stem cells; bone marrow; enhanced green fluorescent protein; retroviral vector; gene therapy;
Abstract: Methods hMSCs were isolated from human bone marrow by density gradient 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 whether it could differentiate into osteoblasts or adipocytes under conditioned media was investigated. Results The rate of stably transduced hMSCs-EGFP was up to 96% after being screened by G418. hMSCs-EGFP exhibited fibroblast-like morphological features. Flow cytometric analyses showed that hMSCs-EGFP were positive for CD73, CD105, CD166, CD90 and CD44, but negative for CD34 and CD45. In addition, it could functionally be induced into 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 can be used in vivo securely because they can maintain their biological characteristics and differentiation. It is a simple and reliable way to trace the changes of hMSCs in vivo by EGFP during cell transplantation and gene therapy.
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