Highly efficient retroviral gene transfer into immortalized CD34(-) cells and organ distribution after transplantation into NOD/SCID mice.

BACKGROUND: CD34(-) stem cells are apparently the earliest progenitors of hematopoiesis and mesenchymal tissues. The majority of those progeny rests in the BM as fibroblast-like cells, but can also circulate the peripheral blood. Nevertheless, CD34(-), fibroblast-like cells can be isolated from BM aspirates and PBMC, mediated by their ability to adhere to the plastic surface of tissue culture flasks. In standard colony assays, CD34(-), fibroblast-like cells produce a significant number of colony-forming-units (CFUs), mainly CFU-F (fibroblast).

METHODS: Despite advanced cell-culture techniques and the application of various growth factors, the life span of those multipotent stem cells is limited. Therefore, we immortalized and cloned fibroblast-like, CD34(-) stem cells and used retroviral constructs containing the green-fluorescence protein (GFP) to determine the gene-transfer efficiency and their use for gene marking prior to transplantation into NOD/SCID mice.

RESULTS: We could demonstrate a highly efficient retroviral gene transfer into those immortalized CD34(-), fibroblast-like hematopoietic cells (up to 95% transduced cells), maintaining their ability to produce CFUs, as well as a distinct organ distribution after transplantation into the recipient animals, functioning as SCID-repopulating cells (SRC). Transplanted cells could be detected in the BM, as well as other parenchymal organs, such as the lung, liver, skin, small intestine and brain.

DISCUSSION: CD34(-), fibroblast-like progenitor cells can give rise to hematopoietic progeny, but also home to mesenchymal organ sites in recipient animals. There is increasing evidence that pluripotent CD34(-) stem cells can be isolated from various sources and still maintain their capabilities to generate progeny of different tissues. This could be a promising approach to using peripheral-blood derived stem cells for cellreplacement therapy and tissue engineering.