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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3085-3093
Efficient transduction of human hematopoietic repopulating cells
generating stable engraftment of transgene-expressing cells in
NOD/SCID mice
Jordi Barquinero,
José Carlos Segovia,
Manuel Ramírez,
Ana Limón,
Guillermo Güenechea,
Teresa Puig,
Javier Briones,
Juan García, and
Juan Antonio Bueren
From the Department of Cell Therapy, Institut de Recerca
Oncològica, Barcelona, Spain; Department of Molecular and
Cellular Biology, CIEMAT, Madrid, Spain.
In an attempt to develop efficient procedures of human hematopoietic
gene therapy, retrovirally transduced CD34+ cord blood
cells were transplanted into NOD/SCID mice to evaluate the repopulating
potential of transduced grafts. Samples were prestimulated on
Retronectin-coated dishes and infected with gibbon ape leukemia virus
(GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent
protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted
recipients revealed a sustained engraftment of human hematopoietic
cells expressing the EGFP transgene. On average, 33.6% of human
CD45+ cells expressed the transgene 90 to120 days after
transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of
human CD45+ cells expressing the EGFP transgene at this
time. The transplantation of purified EGFP+ cells
increased the proportion of CD45+ cells positive for EGFP
expression to 57.7% at 90 to 120 days after transplantation. At this
time, 18.9% and 4.3% of NOD/SCID BM consisted of
CD45+/EGFP+ and
CD34+/EGFP+ cells, respectively.
Interestingly, the transplantation of EGFP cells
purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and
CD34+/EGFP+ cells, suggesting that a number
of transduced repopulating cells did not express the transgene at that
time. Molecular analysis of NOD/SCID BM confirmed the high levels of
engraftment of human transduced cells deduced from FACS analysis.
Finally, the analysis of the provirus insertion sites by conventional
Southern blotting indicated that the human hematopoiesis in the
NOD/SCID BM was predominantly oligoclonal.
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