Cancer Center and Department of Cell Biology
Animals; Aphidicolin; Benzimidazoles; Biological Markers; Bone Marrow Cells; Cell Cycle; Cells, Cultured; Culture Media; Flow Cytometry; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Interleukin-11; Interleukin-3; Interleukin-6; Mice; Rhodamines; S Phase; Stem Cell Factor
Cancer Biology | Cell and Developmental Biology | Oncology
Hematopoietic stem cells purified from mouse bone marrow are quiescent with less than 2% of Lin- Hoechst(low)/Rhodamine(low) (Lin- Ho(low)/Rho(low)) and 10% to 15% of Lin-/Sca+ cells in S phase. These cells enter proliferative cycle and progress through G1 and into S phase in the presence of cytokines and 5% heat-inactivated fetal calf serum (HI-FCS). Cytokine-stimulated Lin- Ho(low)/Rho(low) cells took 36 to 40 hours to complete first division and only 12 hours to complete each of 5 subsequent divisions. These cells require 16 to 18 hours to transit through G0/G1 period and 28 to 30 hours to enter into mid-S phase during the first cycle. Up to 56% of Lin- Rho(low)/Ho(low) cells are high-proliferative potential (7 factor-responsive) colony-forming cells (HPP-CFC). At isolation, HPP-CFC are quiescent, but after 28 to 30 hours of culture, greater than 60% are in S phase. Isoleucine-deprivation of Lin- Ho(low)/Rho(low) cells in S phase of first cycle reversibly blocked them from entering into second cycle. After the release from isoleucine-block, these cells exhibited a G1 period of less than 2 hours and entered into mid-S phase by 12 hours. Thus, the duration of G1 phase of the cells in second cycle is 4 to 5 times shorter than that observed in their first cycle. Similar cell cycle kinetics are observed with Lin-/Sca+ population of bone marrow cells. Stem cell factor (SCF) alone, in the presence of HI-FCS, is as effective as a cocktail of 2 to 7 cytokines in inducing quiescent Lin-/Sca+ cells to enter into proliferative cycle. Aphidicolin treatment reversibly blocked cytokine-stimulated Lin-/Sca+ cells at G1/S boundary, allowing their tight synchrony as they progress through first S phase and enter into second G1. For these cells also, SCF alone is sufficient for their progression through S phase. These studies indicate a very short G1 phase for stem cells induced to proliferate and offer experimental approaches to synchronize murine hematopoietic stem cells.
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Citation: Blood. 1997 Sep 15;90(6):2293-9.
Reddy, G. Prem Veer; Tiarks, Cheryl Y.; Pang, Lizhen; Wuu, Joanne; Hsieh, Chung-Cheng; and Quesenberry, Peter J., "Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells" (1997). Open Access Articles. 285.