Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments.
BACKGROUND
Retroviral transduction in the presence of fibronectin (FN) fragments has proven an efficient and clinically-applicable procedure for gene transfer into hematopoietic cells. So far, FN-based transduction protocols have been optimized primarily for transduction of stem cells, whereas for several therapeutic applications transduction of clonogenic progenitors (CFU) may be sufficient.


METHODS
Transduction protocols for CFU were optimized by evaluating the effect of growth factors, timing of retroviral transduction, CD34-selection and heparin, using a neomycin-phosphotransferase (neo(R))-expressing retroviral vector.


RESULTS
The presence of multiple growth factors during prestimulation and transduction, including the differentiating cytokines G-CSF or GM-CSF, substantially enhanced transduction of CFU. Best results were achieved when 24 h of prestimulation were followed by a 24-48 h transduction period in the presence of the CH-296 FN-fragment and IL-3, IL-11, SCF, erythropoietin (EPO), and GM-CSF. With this proto-col we observed highly efficient transduction of BM-derived CFU (90.7 +/- 8.8 % G 418-resistant colonies), even with retrovirus preparations of moderate infectious titer (5 x 10(4) - 2 x 10(5) CFU/mL). The number of CFU increased on average 2.6-fold (range 1.5-3.8) during the transduction procedure. Selection of CD34(+) cells prior to transduction did not improve transduction efficiency. Heparin, even in concentrations as low as 2.0 microg/mL, significantly inhibited transduction of CFU on FN-fragments.


CONCLUSIONS
An optimized protocol for retroviral gene transfer into human clonogenic progenitor cells that allows highly efficient transduction, even with moderate titer retroviral vectors, is presented.
Trarbach
T
T
Department of Internal Medicine, West German Cancer Center, University of Essen, Germany.
Greifenberg
S
S
Bardenheuer
W
W
Elmaagacli
A
A
Hirche
H
H
Flasshove
M
M
Seeber
S
S
Moritz
T
T
D018952:Antigens, CD34 / Q000032:analysis; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D005353:Fibronectins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005822:Genetic Vectors / Q000235:genetics; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D012190:Retroviridae / Q000235:genetics*; D033581:Stem Cell Transplantation / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods*
D018952:Antigens, CD34; D005353:Fibronectins; D016298:Hematopoietic Cell Growth Factors; D010446:Peptide Fragments; D006493:Heparin