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.


CONCLUSIONS
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.
Thalmeier
K
K
Institute of Pathology, University of Munich, Germany.
Huss
R
R
D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology*; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology; D002461:Cell Line, Transformed; D004285:Dogs; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000276:immunology; D018014:Gene Transfer Techniques / Q000639:trends*; D005822:Genetic Vectors / Q000627:therapeutic use*; D006085:Graft Survival / Q000276:immunology*; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology*; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008168:Lung / Q000166:cytology / Q000276:immunology; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D012190:Retroviridae / Q000235:genetics*; D013154:Spleen / Q000166:cytology / Q000276:immunology; D014161:Transduction, Genetic / Q000379:methods; D014781:Viscera / Q000166:cytology / Q000276:immunology
D018952:Antigens, CD34; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins