A General Theory of Marrow Stem Cell Fate Determination

Abstract 4794

We propose that the long-term multipotent marrow renewal stem cell is on a continuum of change in potential tied to cell cycle stage. Furthermore our data indicate that marrow stem cells cannot be defined on a clonal basis and that purification of stem cells, as is currently carried out, leads to a biased population which provides only limited information with regard to marrow stem cells.

Previous data have clearly demonstrated continuous and reversible phenotype change when murine marrow stem cells are driven through cell cycle in vitro by cytokines (Quesenberry et al, Exp Hematol, 2010). The relevance of this to normal hematopoiesis is placed into question by numerous observations indicating that highly purified long-term hematopoietic stem cells are in the G0 phase of cell cycle. However, no studies have addressed the cell cycle status of long-term multilineage stem cells in vivo from normal unseparated marrow. Given the fact that marrow stem cell separations are not random and that over 90% of stem cells are lost in these separations (Nilsson et al, Blood, 1997), the possibility exists that the purified stem cells are not representative of stem cells in the whole population. Accordingly, we have investigated the cell cycle status of murine whole marrow stem cells employing Hoechst 33342 alone or pyronin/Hoechst 33342 staining with FACS separation into G0, G1 and S/G2/M fractions, followed by competitive engraftment of each sub fraction into lethally irradiated mice and analysis of multilineage engraftment from 28 to 52 weeks. In five separate experiments we have found that 50 ± 4 % of whole marrow long-term engrafting cells were in S/G2/M. Engraftment in every instance was multilineage as determined by analysis of B220, Gr-1. CD11b and CD3 engraftment. Given that this is an instantaneous look at cell cycle status, it implies that virtually all long-term engrafting cells are in active cell cycle. Similar studies on LT-HSC (lin-Sca+c-kit+ Flk2-) essentially confirmed previous studies (with one stochastic exception) that LT-HSC are a dormant, noncycling population in G0. In order to further confirm these studies, donor whole marrow was subjected to high-specific activity tritiated thymidine suicide for thirty minutes and long-term multilineage engraftment was determined in lethally irradiated mice. In these on-going studies we find that 52 ± 8.6 % of engrafting cells were in S/G2/M out to 6 months (5 experiments). Given the short exposure to tritiated thymidine, these data also indicate that virtually all marrow stem cells are in cell cycle. With progression through cell cycle there is a continuous change in all aspects of cellular phenotype and thus the characteristics of the stem cell will vary from moment to moment. In this setting, purification of individual cells can not define the population. Thus the marrow long-term repopulating stem cell is on a continuum of change in potential which will be realized if interrogated with appropriate stimuli. These potentials continuously change with progression through cell cycle with different fate outcomes at different points in cell cycle. The present challenge for the field is to appropriately define the stem cell population, not the “stem cell”.