Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic severe combined immune-deficient mice

Most primitive hematopoietic progenitor cells reside in vivo within the G(0)/G(1) phase of the cell cycle. By simultaneous DNA/RNA staining it is possible to distinguish G(0) and G(1) states and to isolate cells in defined phases of the cell cycle. We report here the use of cell cycle fractionation to separate human mobilized peripheral blood (MPB) CD34(+) cells capable of repopulating the bone marrow (BM) of non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice. In freshly isolated MPB, repopulating cells were predominant within the G(0) phase, because transplantation of CD34(+) cells residing in G(0) (G(0)CD34(+)) resulted on average in a 16.6- +/- 3.2-fold higher BM chimerism than infusion of equal numbers of CD34(+) cells isolated in G(1). We then investigated the effect of ex vivo cell cycle progression, in the absence of cell division, on engraftment capacity. Freshly isolated G(0)CD34(+) cells were activated by interleukin-3 (IL-3), stem cell factor (SCF), and flt3-ligand (FL) for a 36-hour incubation period during which a fraction of cells progressed from G(0) into G(1) but did not complete a cell cycle. The repopulating capacity of stimulated cells was markedly diminished compared with that of unmanipulated G(0)CD34(+) cells. Cells that remained in G(0) during the 36-hour incubation period and those that traversed into G(1) were sorted and assayed separately in NOD/SCID recipients. The repopulating ability of cells remaining in G(0) was insignificantly reduced compared with that of unstimulated G(0)CD34(+) cells. On the contrary, CD34(+) cells traversing from G(0) into G(1) were largely depleted of repopulating capacity. Similar results were obtained when G(0)CD34(+) cells were activated by the combination of thrombopoietin-SCF-FL. These studies provide direct evidence of the quiescent nature of cells capable of repopulating the BM of NOD/SCID mice. Furthermore, these data also demonstrate that G(0)-G(1) progression in vitro is associated with a decrease in engraftment capacity. (C) 1998 by The American Society of Hematology.