The Late Dividing Population of Gamma-Retroviral Vector Transduced Human Mobilized Peripheral Blood Progenitor Cells Contributes Most to Gene-marked Cell Engraftment in NOD/SCID Mice

¹ Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; USA; Department of Pediatrics, University Clinic Carl Gustav Carus Dresden, Germany
² Department of Pediatrics, University Clinic Carl Gustav Carus Dresden, Germany
³ Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; USA

^* To whom correspondence should be addressed. E-mail: sebastian.brenner{at}uniklinikum-dresden.de.

	Abstract

We used the 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 gamma-retrovirus vector RD114-MFGS-CFP - that requires cell division for successful transduction - at 24hrs, 48hrs and 72hrs to achieve 96% CFP-positive cells. Cells were sorted 12hrs after the last transduction into CFP-positive (divided cells) and CFP-negative populations. CFP-positive cells were transplanted postsort, while the CFP-negative cells were re-transduced and injected at 120hrs. The CFP-negative sorted and re-transduced cells contained markedly fewer vector copies and resulted in a 32fold higher overall engraftment and in an 13fold higher number of engrafted transgene positive cells. To assess cell proliferation as underlying cause for the different engraftment levels, CFSE-labeling of untransduced PBPC was performed to track the number of cell divisions. At 72hrs after initiation of culture when 95% of all cells have divided, PBPC were sorted into non-divided and divided fractions and transplanted into NOD/SCID mice. Non-divided 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, while rapidly proliferating cells lose CD133 in correlation to the number of cell divisions. Our studies demonstrate that late dividing progenitors transduced with gamma-retroviral vectors contribute most to NOD/SCID engraftment and transgene marking. Confining the gamma-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.