title: Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells.
abstract: 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.
authors.affiliation: Cancer Center and Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01605, USA.
authors.forename: G P
authors.initials: GP
authors.lastname: Reddy
authors.forename: C Y
authors.initials: CY
authors.lastname: Tiarks
authors.forename: L
authors.initials: L
authors.lastname: Pang
authors.forename: J
authors.initials: J
authors.lastname: Wuu
authors.forename: C C
authors.initials: CC
authors.lastname: Hsieh
authors.forename: P J
authors.initials: PJ
authors.lastname: Quesenberry
mesh_terms: D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001562:Benzimidazoles; D015415:Biomarkers; D001854:Bone Marrow Cells; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D003470:Culture Media; D005434:Flow Cytometry; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D017370:Interleukin-11 / Q000494:pharmacology*; D007377:Interleukin-3 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000494:pharmacology*; D051379:Mice; D012235:Rhodamines; D016196:S Phase / Q000187:drug effects; D019089:Stem Cell Factor / Q000494:pharmacology*
chemical_list: D001562:Benzimidazoles; D015415:Biomarkers; D003470:Culture Media; D016298:Hematopoietic Cell Growth Factors; D017370:Interleukin-11; D007377:Interleukin-3; D015850:Interleukin-6; D012235:Rhodamines; D019089:Stem Cell Factor; D016590:Aphidicolin; C017807:bisbenzimide ethoxide trihydrochloride