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High-efficiency gene transfer to human hematopoietic cells maintained in
long-term marrow culture [see comments]
PF Hughes, CJ Eaves, DE Hogge and RK Humphries
Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver, Canada.
We used a helper-free recombinant retrovirus carrying the neomycin
resistance (neor) gene to investigate methods for improving gene transfer
efficiencies to clonogenic hematopoietic progenitor cells of human origin
and to assess the possibility of gene transfer to the more primitive cells
from which clonogenic cells are derived after several weeks in long-term
human marrow cultures. The proportion of neor CFU-GM in methylcellulose
assays of infected fresh marrow was increased by six- to eightfold (mean
37.4%) by the addition of extra GM colony- stimulating factor and
interleukin-1 beta or medium conditioned by a human marrow "stromal" cell
line to medium conditioned by agar- stimulated human leukocytes both during
the infection and the colony growth period. Similar increases were also
noted in the proportion of neor BFU-E, although the efficiencies overall
were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term
cultures with marrow exposed to virus under the same growth
factor-supplemented conditions but without any immediate selection step
resulted in sustained production of a high proportion of neor CFU-GM and
BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular
analysis was used to confirm the presence of the neo gene after culture.
These results demonstrate that stable, high-efficiency gene transfer can be
accomplished to the most primitive class of human hematopoietic cells
currently detectable that may also have in vivo reconstituting potential.
Further use of this approach should provide new insights into human
hematopoietic stem cell regulation and allow continued development and
assessment of gene therapy procedures.
Volume 74,
Issue 6,
pp. 1915-1922,
11/01/1989
Copyright © 1989 by The American Society of Hematology
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