Ex vivo culture of hematopoietic stem/progenitor cells could potentially im
prove the efficacy of human placental/umbilical cord blood (CB) in clinical
hematopoietic stem cell (HSC) transplantation and allow gene transduction
using conventional retroviral vectors. Therefore, we first examined the eff
ects of a 7-day period of ex vivo culture on the hematopoietic capacity of
CB CD34(+) cells. Medium for the ex vivo cultures contained either serum an
d six recombinant human hematopoietic growth factors (GFs), including Flt-3
ligand (FL), Kit ligand (KL = stem cell factor), thrombopoietin (Tpo), int
erleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and inter
leukin 6 (IL-6), or a serum-free medium containing only FL, KL, and Tpo. Af
ter culture under both ex vivo conditions, the total numbers of viable cell
s, CD34(+) cells, colony-forming cells (CFCs), and long-term culture initia
ting cells (LTC-ICs) were increased. In contrast, the severe combined immun
odeficiency (SCID) mouse engrafting potential (SEP) of cultured cells was s
lightly decreased, as compared with fresh cells. Nevertheless, cultured hum
an: CB CD34(+) cells were able to generate engraftment, shown to persist fo
r up to 20 weeks after transplantation. We next tested the efficacy of retr
oviral transduction of cultured cells. Transduced cultured human cells were
able to engraft in NOD/SCID mice, as tested 4 weeks after transplantation,
and EGFP(+)CD34(+) cells and EGFP(+) CFCs were isolated from the chimeras.
Thus, although additional improvements in ex vivo culture are still needed
to expand the numbers and function of human HSCs, the current conditions a
ppear to allow gene transduction into hematopoietic SCID engrafting cells,
while at least qualitatively preserving their in vivo engraftment potential
.