High-Efficiency Transduction and Long-Term Gene Expression with a Murine Stem Cell Retroviral Vector Encoding the Green Fluorescent Protein in Human Marrow Stromal Cells

Abstract

Bone marrow stromal cells (MSCs) are unique mesenchymal cells that have been utilized as vehicles for the delivery of therapeutic proteins in gene therapy protocols. However, there are several unresolved issues regarding their potential therapeutic applications. These include low transduction efficiency, attenuation of transgene expression, and the technical problems associated with drug-based selection markers. To address these issues, we have developed a transduction protocol that yields high-level gene transfer into human MSCs, employing a murine stem cell virus-based bicistronic vector containing the green fluorescent protein (GFP) gene as a selectable marker. Transduction of MSCs plated at low density for 6 hr per day for 3 days with high-titer viral supernatant resulted in a gene transfer efficiency of 80+/-6% (n = 10) as measured by GFP fluorescence. Neither centrifugation nor phosphate depletion increased transduction efficiency. Assessment of amphotropic receptor (Pit-2) expression by RT-PCR demonstrated that all MSCs expressing the receptor were successfully transduced. Cell cycle distribution profiles measured by propidium iodide staining showed no correlation with the susceptibility of MSCs to transduction by the retroviral vector. Human MSCs sequentially transduced with an adenoviral vector encoding the ecotropic receptor and ecotropic retroviral vector encoding GFP demonstrated that all MSCs are susceptible to retroviral transduction. We further showed that both genes of bicistronic vector are expressed for at least 6 months in vitro and that transgene expression did not affect the growth or osteogenic differentiation potential of MSCs. Future studies will be directed toward the development of gene therapy protocols employing this strategy.

Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model

“…Since MSCs possess high affinity for glioma and exert organ-protective effects, utilizing MSCs as a vehicle for gene therapy in the treatment of brain cancer represents a rational clinical approach. However, the efficiency of exogenous gene transfer is relatively low using physiochemical methods, 42 and even employing viral vectors, such as retroviral 43,44 or adenoviral vectors, 15,42 the method is not efficient. Refractoriness to viral vector infection may be mainly due to a deficit of native viral receptors.…”

Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation

“…Bone marrow was flushed from the dissected femurs and tibias of FVB͞N mice (The Jackson Laboratory), and the isolated adherent marrow cells were transduced with a GFP-expressing retroviral vector (multiplicity of infection, Ϸ5) as described (17). In separate studies, nonadherent marrow cells, isolated from a 5-day ex vivo culture in which adherent cells stuck to the plastic dish, were transduced with the same GFP-expressing retroviral vector (70-80% efficiency) and transplanted into 4-to 6-week-old lethally irradiated (1,100 cGy) FVB͞N mice (18).…”

Gene Expression Profile of Human Bone Marrow Stromal Cells: High-Throughput Expressed Sequence Tag Sequencing Analysis

“…Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.Key Words: human bone marrow stromal cells, EST sequencing analysis, novel genes, gene expression profile, data mining them potentially useful for cell and gene therapy [2][3][4]. After extensive proliferation in vitro, the HBMSC population includes precursor cells for at least four types of connective tissue: bone, cartilage, hematopoiesis-supporting stroma, and associated adipocytes [5][6][7][8].…”

“…Key Words: human bone marrow stromal cells, EST sequencing analysis, novel genes, gene expression profile, data mining them potentially useful for cell and gene therapy [2][3][4]. After extensive proliferation in vitro, the HBMSC population includes precursor cells for at least four types of connective tissue: bone, cartilage, hematopoiesis-supporting stroma, and associated adipocytes [5][6][7][8].…”