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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 102-110
Expansion of human cord blood CD34+CD38
cells in ex vivo culture during retroviral transduction without a
corresponding increase in SCID repopulating cell (SRC) frequency:
dissociation of SRC phenotype and function
Craig Dorrell,
Olga I. Gan,
Daniel S. Pereira,
Robert G. Hawley, and
John E. Dick
From Programs in Cancer/Blood and Developmental Biology, Hospital
for Sick Children; Department of Molecular and Medical Genetics,
University of Toronto; and The Toronto Hospital, Toronto, Ontario,
Canada.
Current procedures for the genetic manipulation of hematopoietic
stem cells are relatively inefficient due, in part, to a poor
understanding of the conditions for ex vivo maintenance or expansion of
stem cells. We report improvements in the retroviral transduction of
human stem cells based on the SCID-repopulating cell (SRC) assay and
analysis of Lin CD34+CD38
cells as a surrogate measure of stem cell function. Based on our
earlier study of the conditions required for ex vivo expansion of
LinCD34+ CD38 cells and
SRC, CD34+-enriched lineage-depleted umbilical cord
blood cells were cultured for 2 to 6 days on fibronectin fragment in
MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal
bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both
CD34+CD38 cells (20.8%) and CFC (26.3%)
were efficiently marked. When the bone marrow of engrafted NOD/SCID
mice was examined, 75% (12/16) contained multilineage (myeloid and B
lymphoid) EGFP+ human cells composing as much as 59% of
the graft. Half of these mice received a limiting dose of SRC,
suggesting that the marked cells were derived from a single transduced
SRC. Surprisingly, these culture conditions produced a large
expansion (166-fold) of cells with the
CD34+CD38 phenotype (n = 20). However,
there was no increase in SRC numbers, indicating dissociation between
the CD34+CD38 phenotype and SRC function.
The underlying mechanism involved apparent downregulation of CD38
expression within a population of cultured
CD34+CD38+ cells that no longer contained
any SRC function. These results suggest that the relationship between
stem cell function and cell surface phenotype may not be reliable for
cultured cells. (Blood. 2000;95:102-110)
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