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Rapid and efficient selection of human hematopoietic cells expressing
murine heat-stable antigen as an indicator of retroviral-mediated gene
transfer
E Conneally, P Bardy, CJ Eaves, T Thomas, S Chappel, EJ Shpall and RK Humphries
Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, Canada.
Recombinant retroviruses offer many advantages for the genetic modification
of human hematopoietic cells, although their use in clinical protocols has
thus far given disappointing results. There is therefore an important need
to develop new strategies that will allow effectively transduced primitive
hematopoietic target populations to be both rapidly characterized and
isolated free of residual nontransduced but biologically equivalent cells.
To address this need, we constructed a murine stem cell virus (MSCV)-based
retroviral vector containing the 228-bp coding sequence of the murine
heat-stable antigen (HSA) and generated helper virus-free amphotropic
MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells.
Light density and, in some cases, lineage marker-negative (lin-) normal
human marrow or mobilized peripheral blood cells preactivated by exposure
to interleukin-3 (IL- 3), IL-6, and Steel factor in vitro for 48 hours were
then infected by cocultivation with these MSCV-HSA producer cells for a
further 48 hours in the presence of the same cytokines.
Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours
later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have
acquired the ability to express HSA. The extent of gene transfer to
erythroid and granulopoietic progenitors (burst-forming unit-erythroid and
colony- forming unit-granulocyte-macrophage), as assessed by the ability of
these cells to form colonies of mature progeny in the presence of normally
toxic concentrations of G418, averaged 11% and 12%, respectively, in 6
experiments. These values could be increased to 100% and 77%, respectively,
by prior isolation of the CD34+HSA+ cell fraction and were correspondingly
decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells.
In addition, the extent of gene transfer to long-term culture-initiating
cells (LTC-IC) was assessed by G418 resistance. The average gene transfer
to LTC-IC-derived colony- forming cells in the unsorted population was <
or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells
increased this value to 86% and decreased it to 3% for the LTC-IC plated
from the CD34+HSA- cells. Transfer of HSA gene expression to a
phenotypically defined more primitive subpopulation of CD34+ cells, ie,
those expressing little or no CD38, could also be shown by FACS analysis of
infected populations 24 hours after infection. These findings underscore
the potential use of retroviral vectors encoding HSA for the specific
identification and non-toxic selection immediately after infection of
retrovirally transduced populations of primitive human hematopoietic cells.
In addition, such vectors should facilitate the subsequent tracking of
their marked progeny using multiparameter flow cytometry.
Volume 87,
Issue 2,
pp. 456-464,
01/15/1996
Copyright © 1996 by The American Society of Hematology
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