Enhanced growth of canine bone marrow stromal cell cultures in the presence of acidic fibroblast growth factor and heparin.
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 10(9) 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 10(8) 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.
Emami
S
S
ALG Company, Marlboro, Massachusetts 01752, USA.
Merrill
W
W
Cherington
V
V
Chiang
G G
GG
Kirchgesser
M
M
Appel
J M
JM
Hansen
M
M
Levine
P H
PH
Greenberger
J S
JS
Hurwitz
D R
DR
D000818:Animals; D001769:Blood; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells; D002455:Cell Division; D002478:Cells, Cultured; D003470:Culture Media; D004285:Dogs; D016220:Fibroblast Growth Factor 1 / Q000494:pharmacology*; D015870:Gene Expression; D006493:Heparin / Q000494:pharmacology*; D006736:Horses; D019382:Human Growth Hormone / Q000235:genetics; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007150:Immunohistochemistry; D010957:Plasmids; D011994:Recombinant Proteins; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
D003470:Culture Media; D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1; D019382:Human Growth Hormone; D006493:Heparin; D006854:Hydrocortisone