Jl. Chertkov et al., THE HEMATOPOIETIC STROMAL MICROENVIRONMENT PROMOTES RETROVIRUS-MEDIATED GENE-TRANSFER INTO HEMATOPOIETIC STEM-CELLS, Stem cells, 11(3), 1993, pp. 218-227
In this study we report on the establishment of novel conditions which
permit efficient retrovirus-mediated gene transfer of human adenosine
deaminase (ADA) into murine hematopoietic progenitors. Using Southern
blot analysis and an ADA probe, we demonstrated that prestimulation o
f bone marrow cells over an in vitro culture of adherent stromal cell
layers (ACLs) for two days provides favorable conditions for gene tran
sfer in the absence of exogenous growth factors. In bone marrow transp
lant recipients reconstituted with retrovirally-marked cells, ADA was
detected in spleen, thymus and bone marrow cells of the recipients eig
ht months after transplantation. These observations were also seen in
transplants of embryonal hematopoietic stem cells. By using different
incubation protocols, it was found that the developmental fate of hema
topoietic stem cells varied with the presence of exogenous growth fact
ors or an ACL in the prestimulation phase. Polyclonal hematopoiesis wi
th multiple clones appearing simultaneously was revealed in mice recon
stituted with growth factor-stimulated cells four months after transpl
antation. This was detected by multiple integration patterns of ADA in
tegration into the genomes of individual colony forming units-spleen (
CFU-S) in transplantation recipient mice. In contrast, two to five mon
ths after transplantation, polyclonal hematopoiesis was not observed i
n mice reconstituted with cells infected in the absence of growth fact
ors. It appears that utilization of the bone marrow microenvironment t
hrough the use of an ACI, results in a narrower spectrum of integratio
n patterns, suggesting that a type of oligoclonal or monoclonal hemato
poiesis is occurring. These studies demonstrate that an ACL provides n
ovel conditions for successful gene transfer and stable integration of
the vector into the genome. Use of an ACI, may be advantageous for su
ccessful hematopoietic stem cell gene therapy.