Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4080-4089
Enhanced Retroviral Transduction of 5-Fluorouracil-Resistant Human
Bone Marrow (Stem) Cells Using a Genetically Modified Packaging Cell
Line
Joanna Povey,
Nishanthi Weeratunge,
Chloe Marden,
Amita Sehgal,
Adrian Thrasher, and
Colin Casimir
From the Department of Haematology, Imperial College School of
Medicine, St Mary's Campus, Norfolk Place, London; and the Molecular
Immunology Unit, Institute of Child Health, Guilford St, London,
UK.
Pluripotent hematopoietic stem cells (PHSC) are rare cells capable
of multilineage differentiation, long-term reconstituting activity and
extensive self-renewal. Such cells are the logical targets for many
forms of corrective gene therapy, but are poor targets for retroviral
mediated gene transfer owing to their quiescence, as retroviral
transduction requires that the target cells be cycling. To try and
surmount this problem we have constructed a retroviral producer line
that expresses the membrane-bound form of human stem cell factor (SCF)
on its cell surface. These cells are capable, therefore, of delivering
a growth signal concomitant with recombinant retroviral vector
particles. In this report we describe the use of this cell line to
transduce a highly quiescent population of cells isolated from adult
human bone marrow using the 5-fluorouracil (FU) resistance technique of
Berardi et al. Quiescent cells selected using this technique were
transduced by cocultivation with retroviral producers expressing
surface bound SCF or with the parent cell line that does not. Following
coculture, the cells were plated in long-term bone marrow culture for a
further 5 weeks, before plating the nonadherent cells in semisolid
media. Colonies forming in the semisolid media over the next 14 days
were analyzed by polymerase chain reaction for the presence of the
retroviral vector genome. Over six experiments, the transduction
frequency of the quiescent 5-FU resistant cells using the
SCF-expressing producer line averaged about 20%, whereas those
transduced using the parent producer line showed evidence of reduced
levels or no transduction.
