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Nonviral delivery of basic fibroblast growth factor gene to bone marrow stromal cells.

ABSTRACT: Basic fibroblast growth factor (bFGF) is capable of stimulating osteogenic differentiation of preosteoblast cells in vitro and new bone tissue deposition in vivo. Delivering the gene for the protein, rather than the protein itself, is considered advantageous for bone repair since gene delivery obviates the need to produce the protein in pharmaceutical quantities. To explore the feasibility of bFGF gene delivery by nonviral methods, we transfected primary rat bone marrow stromal cells (BMSC) using cationic polymers (polyethylenimine and poly(L-lysine)-palmitic acid) in vitro. After delivering a bFGF-expression plasmid (pFGF2-IRES-AcGFP) to BMSC, the presence of bFGF in culture supernatants was detected by a commercial ELISA. As much as 0.3 ng bFGF/10(6) cells/day was obtained from the BMSC under optimal conditions. This secretion rate was approximately 100-fold lower than the secretion obtained from immortal, and easy-to-transfect, human 293T cells. These data suggest the feasibility of modifying BMSC with nonviral delivery systems for bFGF expression, but also highlight the need for substantial improvement in transfection rate for an effective therapy.

SUBMITTER: Clements BA

PROVIDER: S-EPMC2772933 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Nonviral delivery of basic fibroblast growth factor gene to bone marrow stromal cells.

Clements Başak Açan BA Hsu Charlie Y M CY Kucharski Cezary C Lin Xiaoyue X Rose Laura L Uludağ Hasan H

Clinical orthopaedics and related research 20090604 12

Basic fibroblast growth factor (bFGF) is capable of stimulating osteogenic differentiation of preosteoblast cells in vitro and new bone tissue deposition in vivo. Delivering the gene for the protein, rather than the protein itself, is considered advantageous for bone repair since gene delivery obviates the need to produce the protein in pharmaceutical quantities. To explore the feasibility of bFGF gene delivery by nonviral methods, we transfected primary rat bone marrow stromal cells (BMSC) usin ...[more]

PMID: 19495899

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Project description:Fibroblast growth factor 23 (FGF23) is responsible for phosphate wasting and the phenotypic changes observed in human diseases such as X-linked hypophosphatemia (XLH). Targeted overexpression of nuclear high-molecular weight fibroblast growth factor 2 isoforms (HMW isoforms) in osteoblasts resulted in a transgenic mouse with phenotypic changes similar to XLH, including increased FGF23, hypophosphatemia, and rickets/osteomalacia. The goal of this study was to assess whether HMW isoforms also reduced mineralized bone formation via phosphate-independent effects in bone marrow stromal cells (BMSCs) by modulating FGF23/FGF receptor (FGFR)/extracellular signal-regulated kinase (ERK) signaling. To determine if decreased bone formation in BMSC cultures from HMW transgenic mice could be rescued by blocking this pathway, an FGF23 neutralizing antibody, the FGFR tyrosine kinase inhibitor SU5402 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059 were used. FGF23 levels in the conditioned medium of HMW BMSC cultures were dramatically increased compared to BMSC from control (Vector) mice. Mineralized nodule formation was significantly decreased in HMW BMSC cultures compared with control cultures. The decreased nodule formation in HMW cultures was partially rescued by the FGF23 neutralizing antibody, SU5402 and PD98059. mRNA levels for the osteoblast-related genes, osteocalcin, Runt-related transcription factor 2 (Runx2), and osterix, and the osteocyte-related gene dentin matrix acidic phosphoprotein 1 (Dmp1) were significantly decreased in HMW cultures compared with control cultures, and the decreases were partially rescued by SU5402 or PD98059 treatment. Matrix-gla-protein (Mgp) mRNA was significantly higher in HMW cultures compared with control cultures, reduced by SU5402, but further increased by PD98059. Our results suggest that phosphate-independent effects of HMW isoforms in vitro may be directly mediated in part via FGF23 and that HMW isoforms signal via FGF23/FGFR/MAPK to inhibit bone formation in vitro.

Project description:Treatment of rodents after stroke with bone marrow stromal cells (BMSCs) improves functional outcome. However, the mechanisms underlying this benefit have not been ascertained. This study focused on the contribution of neurotrophic and growth factors produced by BMSCs to therapeutic benefit. Rats were subjected to middle cerebral artery occlusion and the ischemic brain extract supernatant was collected to prepare the conditioned medium. The counterpart normal brain extract from non-ischemic rats was employed as the experimental control. Using microarray assay, we measured the changes of the neurotrophin associated gene expression profile in BMSCs cultured in different media. Furthermore, real-time RT-PCR and fluorescent immunocytochemistry were utilized to validate the gene changes. The morphology of BMSCs, cultured in the ischemic brain-conditioned medium for 12 h, was dramatically altered from a polygonal and flat appearance to a fibroblast-like long and thin cell appearance, compared to those in the normal brain-conditioned medium and the serum replacement medium. Forty-four neurotrophin-associated genes in BMSCs were identified by microarray assay under all three culture media. Twelve out of the 44 genes (7 neurotrophic and growth factor genes, 5 receptor genes) increased in BMSCs cultured in the ischemic brain-conditioned medium compared to the normal brain-conditioned medium. Real time RT-PCR and immunocytochemistry validated that the ischemic brain-conditioned medium significantly increased 6/7 neurotrophic and growth factor genes, compared with the normal brain-conditioned medium. These six genes consisted of fibroblast growth factor 2, insulin-like growth factor 1, vascular endothelial growth factor A, nerve growth factor beta, brain-derived neurotrophic factor and epidermal growth factor. Our results indicate that transplanted BMSCs may work as 'small molecular factories' by secreting neurotrophins, growth factors and other supportive substances after stroke, which may produce therapeutic benefits in the ischemic brain.

Dataset Information

Nonviral delivery of basic fibroblast growth factor gene to bone marrow stromal cells.

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Nonviral delivery of basic fibroblast growth factor gene to bone marrow stromal cells.

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