CELL-CYCLE ANALYSIS AND SYNCHRONIZATION OF PLURIPOTENT HEMATOPOIETIC PROGENITOR STEM-CELLS

Hematopoietic stem cells purified from mouse bone marrow are quiescent with less than 2% of Lin(-) Hoechst(low)/Rhodamine(low) (Lin(-) Ho-lo w/Rho(low)) and 10% to 15% of Lin(-)/Sca(+) cells in S phase. These ce lls enter proliferative cycle and progress through G(1) and into S pha se in the presence of cytokines and 5% heat-inactivated fetal calf ser um (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 e ach of 5 subsequent divisions, These cells require 16 to 18 hours to t ransit through G(0)/G(1) 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 cel ls are high-proliferative potential (7 factor-responsive) colony-formi ng 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-de privation of Lin(-) Ho-low/Rho(low) cells in S phase of first cycle re versibly blocked them from entering into second cycle. After the relea se from isoleucine-block, these cells exhibited a G(1) period of less than 2 hours and entered into mid-S phase by 12 hours, Thus, the durat ion of G(1) 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(+) ce lls to enter into proliferative cycle, Aphidicolin treatment reversibl y blocked cytokine-stimulated Lin(-)/Sca(+) cells at G(1)/S boundary, allowing their tight synchrony as they progress through first S phase and enter into second G(1). For these cells also, SCF alone is suffici ent for their progression through S phase. These studies indicate a ve ry short G(1) phase for stem cells induced to proliferate and offer ex perimental approaches to synchronize murine hematopoietic stem cells. (C) 1997 by The American Society of Hematology.