The Fluctuating Phenotype of the Lymphohematopoietic Stem Cell with Cell Cycle Transit

Abstract

The most primitive engrafting hematopoietic stem cell has been assumed to have a fixed phenotype, with changes in engraftment and renewal potential occurring in a stepwise irreversible fashion linked with differentiation. Recent work shows that in vitro cytokine stimulation of murine marrow cells induces cell cycle transit of primitive stem cells, taking 40 h for progression from G0 to mitosis and 12 h for subsequent doublings. At 48 h of culture, progenitors are expanded, but stem cell engraftment is markedly diminished. We have investigated whether this effect on engraftment was an irreversible step or a reversible plastic feature correlated with cell cycle progression. Long-term engraftment (2 and 6 mo) of male BALB/c marrow cells exposed in vitro to interleukin (IL)-3, IL-6, IL-11, and steel factor was assessed at 2–4-h intervals of culture over 24–48 h using irradiated female hosts; the engraftment phenotype showed marked fluctuations over 2–4-h intervals, with engraftment nadirs occurring in late S and early G2. These data show that early stem cell regulation is cell cycle based, and have critical implications for strategies for stem cell expansion and engraftment or gene therapy, since position in cell cycle will determine whether effective engraftment occurs in either setting.

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

“…The average magnitude of the HSC expansions achieved by overexpression of the NUP98-HOXB4 fusion gene was ~300-fold, i.e., ~4 times the effect of HOXB4 alone using the same vector. The greater than 2000-fold expansions of HSCs obtained using NUP98-HOXA10 fusion genes are unprecedented and come close to the theoretical limit in a maximum period of 7 to 8 days of gene expression, assuming no significant shortening of the reported 12-to 14-hour cell cycle time for these cells [26,27]. Even further levels of HSC expansion could be anticipated in more prolonged cultures (~10-fold expansion every 2 additional days).…”

“…Assuming a HSC cell cycle time of 12 to 14 hours [26,27], the continuous execution of symmetric self-renewal divisions, and no cell death, the maximum expansion of HSCs that would be predicted to occur over a period of 5 to 8 days is 1000-fold, i.e., greater than 10 times that found to be achieved by overexpressing HOXB4. In a related paper, we show that HSC expansions of this magnitude can be realized by combining the overexpression of HOXB4 with the suppressed expression of the Hox cofactor, Pbx1.…”

Murine allogeneic in vivo stem cell homing,

“…Evidence for this has been shown by Nilsson et al (1997) finding that homed stem cells enter into cell cycle and encounter their first division only 12 h after seeding. Additionally, there are other cell cycle-related changes that could have altered the final engraftment efficiency with variation of surface antigen expression (Becker et al, 1999), homing (Cerny et al, 2002), engraftment (Habibian et al, 1998), and gene expression profile (Lambert et al, 2003) through cell cycle position of the stem cells prior to initial cell division. Askenasy and Farkas (2003) have looked at allogeneic and syngeneic homing with whole marrow or lineage-depleted marrow labeled with PKH.…”

The chiaroscuro stem cell: a unified stem cell theory

“…Subsequent studies evaluated the engraftment capacity of BALB/c or C57BL/6J marrow cells cultured in IL-3, IL-6, IL-11, and steel factor in nonadherent Teflon bottles every 2 to 4 hours from 24 to 80 hours of culture. 66 Separate studies had determined the cell cycle status of Lin Ϫ Rho dull Ho dull marrow cells under the same cytokine and culture conditions. 42 These hematopoietic stem cells isolated on the basis of quiescence progressed in a highly synchronized fashion through the cell cycle, entering S phase at about 18 hours and completing the first population doubling at about 36 to 38 hours.…”