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Blood, 15 December 2002, Vol. 100, No. 13, pp. 4391-4400
GENE THERAPY
Lentiviral gene transfer and ex vivo expansion of human primitive
stem cells capable of primary, secondary, and tertiary multilineage
repopulation in NOD/SCID mice
Wanda Piacibello,
Stefania Bruno,
Fiorella Sanavio,
Sara Droetto,
Monica Gunetti,
Laurie Ailles,
Francesca Santoni de
Sio,
Andrea Viale,
Loretta Gammaitoni,
Angelo Lombardo,
Luigi Naldini, and
Massimo Aglietta
From the Department of Oncological Sciences, University
of Torino Medical School, Torino, Italy; Laboratory Division of
Clinical Oncology, Institute for Cancer Research and Treatment, Torino,
Italy; and Laboratory of Gene Therapy, Institute for
Cancer Research and Treatment, Torino, Italy.
The ability of advanced-generation lentiviral vectors to transfer
the green fluorescent protein (GFP) gene into human hematopoietic stem
cells (HSCs) was studied in culture conditions that allowed expansion
of transplantable human HSCs. Following 96 hours' exposure to
flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF),
and interleukin-6 (IL-6) and overnight incubation with vector
particles, cord blood (CB) CD34+ cells were further
cultured for up to 4 weeks. CD34+ cell expansion was
similar for both transduced and control cells. Transduction efficiency
of nonobese diabetic/severe combined immunodeficient (NOD/SCID)
repopulating cells (SRCs) was assessed by transplants into NOD/SCID
mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice
receiving transplants of transduced nonexpanded cells (with transplants
of 1 × 105 CD34+ cells, the percentages of
CD45+ cells were 20.5 ± 4.5 [week 1, expanded] and
27.2 ± 8.2 [week 4, expanded] vs 11.7 ± 2.5 [nonexpanded];
n = 5). The GFP+/CD45+ cell fraction
was similar in all cases (12.5% ± 2.9% and 12.2% ± 2.7% vs
12.7% ± 2.1%). Engraftment was multilineage, with
GFP+/lineage+ cells. Clonality analysis
performed on the bone marrow of mice receiving transduced and
week 4 expanded cells suggested that more than one integrant likely
contributed to the engraftment of GFP-expressing cells. Serial
transplantations were performed with transduced week 4 expanded CB
cells. Secondary engraftment levels were 10.7% ± 4.3% (n = 12);
19.7% ± 6.2% of human cells were GFP+. In tertiary
transplants the percentage of CD45+ cells was lower
(4.3% ± 1.7%; n = 10); 14.8% ± 5.9% of human cells were
GFP+, and human engraftment was multilineage. These results
show that lentiviral vectors efficiently transduce HSCs, which can
undergo expansion and maintain proliferation and self-renewal ability.
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