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 G0/G1 phase of the cell cycle. By simultaneous DNA/RNA staining it is possible to distinguish G0 and G1 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 G0 phase, because transplantation of CD34(+) cells residing in G0 (G0CD34(+)) resulted on average in a 16.6- +/- 3.2-fold higher BM chimerism than infusion of equal numbers of CD34(+) cells isolated in G1. We then investigated the effect of ex vivo cell cycle progression, in the absence of cell division, on engraftment capacity. Freshly isolated G0CD34(+) 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 G0 into G1 but did not complete a cell cycle. The repopulating capacity of stimulated cells was markedly diminished compared with that of unmanipulated G0CD34(+) cells. Cells that remained in G0 during the 36-hour incubation period and those that traversed into G1 were sorted and assayed separately in NOD/SCID recipients. The repopulating ability of cells remaining in G0 was insignificantly reduced compared with that of unstimulated G0CD34(+) cells. On the contrary, CD34(+) cells traversing from G0 into G1 were largely depleted of repopulating capacity. Similar results were obtained when G0CD34(+) 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 G0-G1 progression in vitro is associated with a decrease in engraftment capacity.
Division of Hematology/Oncology, Department of Medicine, and Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana School of Medicine, Indianapolis, IN, USA.
A
A
Gothot
J C
JC
van der Loo
D W
DW
Clapp
E F
EF
Srour
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001853:Bone Marrow / Q000473:pathology; D051927:CD24 Antigen; D002453:Cell Cycle; D002455:Cell Division; D002469:Cell Separation; D002478:Cells, Cultured / Q000637:transplantation; D003114:Colony-Forming Units Assay; D006085:Graft Survival; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011828:Radiation Chimera; D016192:Resting Phase, Cell Cycle; D019089:Stem Cell Factor / Q000494:pharmacology; D014183:Transplantation, Heterologous
D015703:Antigens, CD; D051927:CD24 Antigen; C497889:CD24 protein, human; C497890:Cd24a protein, mouse; D007377:Interleukin-3; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein