Enhanced growth of canine bone marrow stromal cell cultures in the presence of acidic fibroblast growth factor and heparin |
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Authors: | Shahin Emami Wilma Merrill Van Cherington Gisela G Chiang Michael Kirchgesser Joseph M Appel Michael Hansen Peter H Levine Joel S Greenberger David R Hurwitz |
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Institution: | 1. ALG Company, 374 Forest Street, 01752, Marlboro, Massachusetts 4. The Medical Center of Central Massachusetts, 119 Belmont Street, 01605, Worcester, Massachusetts
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Abstract: | Summary The ex vivo establishment, expansion, transduction, and reintroduction of autologous bone marrow stromal cells offers a potential efficacious
system for somatic cell gene therapy. It is likely that any ex vivo system will require the use of large numbers of cells which express high levels of transgene products. We present a method
for routine expansion of canine bone marrow stromal cells, established from initial 10–20 ml marrow aspirates, to greater
than 109 cells. This high level expansion of cell cultures uses the stimulatory effect of acidic fibroblast growth factor (aFGF) and
heparin. In the absence of these factors, stromal cell cultures grow actively for only 1 to 2 passages, become flattened in
morphology, and expand to only 108 cells. In the presence of heparin (5 U/ml), aFGF exerts its effect over a wide range of concentrations (0.1–10 ng/ml) in
a dose-dependent manner. The stimulatory effect is dependent on the presence of both aFGF and heparin. Immunocytochemical
and cytochemical analyses phenotypically characterize these stromal cells as bone marrow stromal myofibroblasts. Stromal cells
grown in the presence of aFGF and heparin grow actively and maintain a fibroblast-like morphology for a number of passages,
transduce efficiently with a human growth hormone (hGH) expression vector, and express and secrete high levels of hGH. Human
marrow stromal cells were also established and expanded by the same culture method. This culture method should be of great
value in somatic cell gene therapy for the delivery of secreted gene products to the plasma of large mammals. |
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Keywords: | gene expression gene therapy growth factors human growth hormone |
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