Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells.

Gene Therapy Program, Department of Medicine, LSU Health Sciences Center, New Orleans, LA 70112, USA.
Stem Cells and Development (Impact Factor: 4.2). 07/2008; 17(3):441-50. DOI: 10.1089/scd.2007.0194
Source: PubMed

ABSTRACT Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and retain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo modification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an efficient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more difficult to transduce with lentiviral vectors than their human counterparts, and because many studies use mouse models of human disease, an improved method of transduction would facilitate studies using ex vivo-modified mouse MSCs. We have worked toward improving the production of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus (LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were readily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treatment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient transduction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved production and transduction conditions for lentiviral vectors resulted in efficient transduction of mouse MSCs, and these improvements should facilitate the application of such cells in the context of mouse models of human disease.

Download full-text


Available from: Xian-Yang Zhang, Jul 28, 2015
  • Source
    • "). Fortunately, this drawback can be overcome either by improving purification of the lentiviral particles using gradient centrifugation to eliminate unincorporated transgene particles (Ricks et al., 2008) or by using other proteins for pseudotyping. VSV-G-pseudotyped particles are convenient to use ex vivo to express a transgene in a broad spectrum of cell lines. "
  • Source
    • "Design of lentiviral vectors for targeted transgene delivery into IL-13Ra2-positive cells For targeted transgene delivery into IL-13Ra2-positive cells in vitro and in vivo, safety-improved lentiviral vectors, based on a previously described second-generation lentiviral vector packaging system (Zhang et al., 2004; Ricks et al., 2008), were developed (Fig. 1). The improved packaging system is Tat independent and includes the pCD/NL-BHD1 helper plasmid encoding the HIV-1 Gag and Pol functions; it also includes the pCMV-rev plasmid (Lewis et al., 1990) that encodes Rev (Fig. 1B). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability to selectively and efficiently target transgene delivery to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. Lentiviral vectors have several advantages that make them attractive as gene delivery vehicles and their tropism can be altered through pseudotyping, allowing transgene delivery to specific populations of cells. The human interleukin-13 receptor α2 (IL-13Rα2) is uniquely overexpressed in many different human tumors, making it an attractive target for cancer therapy. In this study, we examined whether IL-13Rα2-positive tumor cells can be specifically targeted with lentiviral vector pseudotypes containing a truncated fusion (F) protein derived from measles virus (MV) and a tail-truncated and receptor-blind MV hemagglutinin (H) protein bearing IL-13 at the C terminus. The retargeted lentiviral vector efficiently transduced cells that express high levels of IL-13Rα2, but not cells expressing low levels of IL-13Rα2 in vitro. In vivo, it specifically targeted IL-13Rα2-positive glioma cell xenografts in immunodeficient mice in the context of subcutaneous and intracranial glioma models. Similar lentiviral vectors may be developed for targeting other tumors expressing specific cell surface receptors.
    Human Gene Therapy Methods 05/2012; 23(2):137-47. DOI:10.1089/hgtb.2012.054 · 1.64 Impact Factor
  • Source
    • "The transduction protocol was modified from a method reported by Rick et al. [30]. In brief, after detachment by trypsin, MSCs at passage 3 were immediately exposed to virus at a multiplicity of infection (MOI) of 40 for 16 h in 0.5 ml complete medium in the presence of 8 ␮g/ml polybrene (Sigma), after which the medium was replaced with fresh complete medium. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mesenchymal stem cells (MSCs) possessing immunoregulatory activities have been evaluated in the treatment of graft-versus-host disease (GVHD). However, the immunomodulatory effects of MSCs are not always successfully achieved in some animal models, and this deficiency may be caused in part by poor homing of these cells to hematopoietic tissues. In this study, we assessed the immunsuppressive capacity of lentiviral vector transduced MSCs expressing CXCR4 in a major histocompatibility complex (MHC)-mismatched mouse model of bone marrow (BM) transplantation from C57BL/6 donors to BALB/c recipients. The survival, body weight and clinical score of GVHD in transplanted mice were monitored. Liver, intestine and skin from mice in each group were obtained for histological examination. Plasma concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α and IL-17A were also determined using a Cytometric Bead Array. CXCR4 over-expressing MSCs maintained their immunsuppressive capacity and showed enhanced migration capacity in vitro. In the mouse GVHD model, treatment with CXCR4 over-expressing MSCs decreased the mortality rate and attenuated clinical and pathological GVHD scores. Moreover, compared with control groups, the plasma IL-2, IL-6, IFN-γ and TNF-α levels in recipients infused with CXCR4 over-expressing MSCs were significantly decreased, while those of IL-4 and IL-10 were increased. In conclusion, our report reveals that CXCR4-transduced MSCs effectively controlled the occurrence of mouse GVHD following allogeneic BM transplantation.
    Immunology letters 02/2012; 143(2):161-9. DOI:10.1016/j.imlet.2012.01.015 · 2.37 Impact Factor
Show more