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Analysis of optimal conditions for retroviral-mediated transduction of
primitive human hematopoietic cells
JA Nolta, EM Smogorzewska and DB Kohn
Childrens Hospital Los Angeles, University of Southern California School of
Medicine, Department of Pediatrics 90027, USA.
We sought to define optimal conditions for retroviral-mediated transduction
of long-lived human hematopoietic progenitors from bone marrow and
peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral
vectors in the presence or absence of stromal support and with or without
cytokine addition. After transduction, a portion of the cells was plated in
methylcellulose colony-forming assay, with or without G418, to assess the
extent of gene transfer into committed progenitors. The remaining cells
from each experiment were transplanted into immunodeficient mice to allow
analysis of transduction of long- lived progenitors. Human colony-forming
cells contained within the murine bone marrow were analyzed after
engraftment periods of 2 to 11 months. Cells were plated in a
human-specific colony-forming assay with and without G418 to assess the
extent of transduction of primitive progenitors. Individual human colonies
were also analyzed by polymerase chain reaction for the presence of
provirus. Bone marrow progenitors were efficiently transduced only when
stroma was present, whereas mobilized peripheral blood progenitors were
effectively transduced in the presence of either stroma or cytokines.
Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell
factor did not further augment the extent of gene transfer in the presence
of a stromal support layer. Additionally, human CD34+ progenitors from bone
marrow or mobilized peripheral blood that had been transduced for 3 days in
the absence of stroma failed to produce sustained, long-term engraftment of
bnx mice. Mice transplanted with the same pools of human progenitors that
had been transduced in the presence of stroma for 3 days had significant
levels of human cell engraftment at the same timepoints, 7 to 11 months
after transplantation. Our data show loss of long-lived human progenitors
during 3-day in vitro transduction periods in the absence of stromal
support. Therefore, the presence of bone marrow stroma has dual benefits in
that it increases gene transfer efficiency and is essential for survival of
long-lived human hematopoietic progenitors.
Volume 86,
Issue 1,
pp. 101-110,
07/01/1995
Copyright © 1995 by The American Society of Hematology
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