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TISSUE-SPECIFIC STEM CELLS

The Late Dividing Population of -Retroviral Vector Transduced Human Mobilized Peripheral Blood Progenitor Cells Contributes Most to Gene-Marked Cell Engraftment in Nonobese Diabetic/Severe Combined Immunodeficient Mice

^aLaboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA;
^bDepartment of Pediatrics, University Clinic Carl Gustav Carus, Dresden, Germany

Correspondence: Sebastian Brenner, M.D., Department of Pediatrics, University Clinic Carl Gustav Carus, Building 21, Fetscherstr. 74, 01307 Dresden, Germany. Telephone: 49 351 458-6884; Fax: 49 351 458-6333; e-mail: sebastian.brenner{at}uniklinikum-dresden.de

Received September 18, 2006; accepted for publication April 3, 2007.
First published online in STEM CELLS EXPRESS   April 26, 2007.



We used the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model to assess the repopulation potential of subpopulations of mobilized human CD34+ peripheral blood progenitor cells (PBPC). First, PBPC were transduced with -retrovirus vector RD114-MFGS-CFP, which requires cell division for successful transduction, at 24 hours, 48 hours, and 72 hours to achieve 96% cyan fluorescent protein (CFP)-positive cells. Cells were sorted 12 hours after the last transduction into CFP-positive (divided cells) and CFP-negative populations. CFP-positive cells were transplanted postsort, whereas the CFP-negative cells were retransduced and injected at 120 hours. The CFP-negative sorted and retransduced cells contained markedly fewer vector copies and resulted in a 32-fold higher overall engraftment and in a 13-fold higher number of engrafted transgene positive cells. To assess cell proliferation as an underlying cause for the different engraftment levels, carboxyfluorescein succinimidyl ester-labeling of untransduced PBPC was performed to track the number of cell divisions. At 72 hours after initiation of culture, when 95% of all cells have divided, PBPC were sorted into nondivided and divided fractions and transplanted into NOD/SCID mice. Nondivided cells demonstrated 45-fold higher engraftment than divided cells. Late dividing PBPC in ex vivo culture retain high expression of the stem cell marker CD133, whereas rapidly proliferating cells lose CD133 in correlation to the number of cell divisions. Our studies demonstrate that late dividing progenitors transduced with -retroviral vectors contribute most to NOD/SCID engraftment and transgene marking. Confining the -retroviral transduction to CD133-positive cells on days 3 and 4 could greatly reduce the number of transplanted vector copies, limiting the risk of leukemia from insertional mutagenesis.

Disclosure of potential conflicts of interest is found at the end of this article.