ENHANCED GROWTH OF CANINE BONE-MARROW STROMAL CELL-CULTURES IN THE PRESENCE OF ACIDIC FIBROBLAST GROWTH AND HEPARIN

The ex vivo establishment, expansion, transduction, and reintroduction of autologous bone marrow stromal cells offers a potential efficaciou s 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 hi gh levels of transgene products. We present a method for routine expan sion of canine bone marrow stromal cells, established from initial 10- 20 ml marrow aspirates, to greater. than 10(9) cells, This high level expansion of cell cultures uses the stimulatory effect of acidic fibro blast growth factor (aFGF) and heparin. In the absence of these factor s, stromal cell cultures grow actively for only 1 to 2 passages, becom e flattened in morphology, and expand to only 10(8) cells. In the pres ence 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 stimulat ory effect is dependent on the presence of both aFGF and heparin. Immu nocytochemical and cytochemical analyses phenotypically characterize t hese stromal cells as bone marrow stromal myofibroblasts. Stromal cell s grown in the presence of aFGF and heparin grow actively and maintain a fibroblast-like morphology for a number of passages, transduce effi ciently with a human growth hormone (hGH) expression vector, and expre ss and secrete high levels of hGH. Human marrow stromal cells were als o established and expanded by the same culture method. This culture me thod should he of great value in somatic cell gene therapy for the del ivery of secreted gene products to the plasma of large mammals.