THE HEMATOPOIETIC STROMAL MICROENVIRONMENT PROMOTES RETROVIRUS-MEDIATED GENE-TRANSFER INTO HEMATOPOIETIC STEM-CELLS

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.