Optimization of retroviral-mediated gene transfer to human NOD SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables
B. Hennemann et al., Optimization of retroviral-mediated gene transfer to human NOD SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables, EXP HEMATOL, 27(5), 1999, pp. 817-825
Retroviral transduction of human hematopoietic stem cells is still limited
by lack of information about conditions that will maximize stem cell self-r
enewal divisions in vitro. To address this, we first compared the kinetics
of entry into division of single human CD34(+)CD38(-) cord blood (CB) cells
exposed in vitro to three different flt3-ligand (FL)-containing cytokine c
ombinations. Of the three combinations tested, FL; hyper-interleukin 6 (HIL
-6) yielded the least clones and these developed at a slow rate. With eithe
r FL + Steel factor (SF) + HIL-6 + thrombopoietin (TPO) or FL + SF + interl
eukin 3 (IL-3) + IL-6 + granulocyte-colony-stimulating factor (G-CSF), > 90
% of the cells that formed clones within 6 days undertook their first divis
ion within 4 days, although not until after 24 hours. These latter two, mor
e stimulatory, cytokine combinations then were used to assess the effect of
duration of cytokine exposure on the efficiency of transducing primitive C
B cells with a gibbon ape leukemia virus-pseudotyped murine retroviral vect
or containing the enhanced green fluorescent protein (GFP) cDNA and the neo
mycin resistance gene. Fresh lin(-) CB cells exposed once to medium contain
ing this virus plus cytokines on fibronectin-coated dishes yielded 23% GFP(
+) CD34(+) cells and 52-57% G418-resistant CFC when assessed after 2 days.
Prestimulation of the target cells (before exposing them to virus) with eit
her the four or five cytokine combination increased their susceptibility. I
n both cases, the effect of prestimulation assessed using the same infectio
n protocol was maximal with 2 days of prestimulation and resulted in 47-54%
GFP(+) CD34(+) cells and 67-69% G418-resistant CFC, Repeated daily additio
n of new virus (up to three times), with assessment of the cells 2 days aft
er the last addition of fresh virus, gave only a marginal improvement in th
e proportion of transduced CD34(+) cells and CFC, but greatly increased the
proportion of transduced LTC-IC (from 40% to >99%). Transplantation of lin
(-) CB cells transduced using this latter 6-day protocol into NOD/SCID mice
yielded readily detectable GFP(+) cells in 10 of 11 mice that were engraft
ed with human cells. The proportion of the regenerated human cells that wer
e GFP(+) ranged from 0.2-72% in individual mice and included both human lym
phoid and myeloid cells in all cases. High-level reconstitution with transd
uced human cells was confirmed by Southern blot analysis. These findings de
monstrate that transplantable hematopoietic stem cells in human CB can be r
eproducibly transduced at high efficiency using a 6-day period of culture i
n a retrovirus-containing medium with either FL + SF + HIL-6 + TPO or FL SF + IL-3 + IL-6 + G-CSF in which virus is added on the third, fourth, and
fifth day. (C) 1999 International Society for Experimental Hematology, Publ
ished by Elsevier Science Inc.