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Efficient retroviral gene transfer to purified long-term repopulating
hematopoietic stem cells
SJ Szilvassy and S Cory
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne
Hospital, Victoria, Australia.
Efficient gene delivery to multipotential hematopoietic stem cells would
greatly facilitate the development of effective gene therapy for certain
hematopoietic disorders. We have recently described a rapid multiparameter
sorting procedure for significantly enriching stem cells with competitive
long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil
(5-FU)-treated mouse bone marrow. The sorted cells have now been tested as
targets for retrovirus-mediated delivery of a marker gene, NeoR. They were
cocultured for 4 days with fibroblasts producing a high titer of retrovirus
in medium containing combinations of the hematopoietic growth factors
interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory
factor (LIF) and then injected into lethally irradiated recipients,
together with sufficient "compromised" bone marrow cells to provide
short-term support. Over 80% of the transplanted mice displayed high levels
(> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months
later. Proviral DNA was detected in 87% of these animals and, in half of
them, the majority of the hematopoietic cells were marked. Thus, infection
of the stem cells was most effective. The tissue and cellular distribution
of greater than 100 unique clones in 55 mice showed that most sorted stem
cells had lymphoid as well as myeloid repopulating potential. Secondary
transplantation provided strong evidence for infection of very primitive
stem cells because, in several instances, different secondary recipients
displayed in their marrow, spleen, thymus and day 14 spleen colony-forming
cells the same proviral integration pattern as the primary recipient.
Neither primary engraftment nor marking efficiency varied for stem cells
cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four
factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower
secondary engraftment potential. Provirus expression was detected in 72% of
the strongly marked mice, albeit often at low levels. Highly efficient
retroviral marking of purified lymphomyeloid repopulating stem cells should
enhance studies of stem cell biology and facilitate analysis of genes
controlling hematopoietic differentiation and transformation.
Volume 84,
Issue 1,
pp. 74-83,
07/01/1994
Copyright © 1994 by The American Society of Hematology
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