Stem cell continuum: Directed differentiation hotspots

Abstract

The purpose of this study was to evaluate the technique of stem cell-directed differentiation in the context of cell-cycle position. The hypothesis was that stem cells would have different sensitivities to an identical inductive signal through cell-cycle transit and that this would affect the outcome of its progeny. Differentiation of murine marrow lineage(negative)rhodamine-123(low-)Hoechst-33342(low) (LRH) stem cells was determined at different points in cell cycle under stimulation by thrombopoietin, flt3 ligand, and steel factor. LRH stem cells were subcultured in granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and steel factor at different points in cell cycle and differentiation determined 14 days later. There was a significant, reproducible, and pronounced reversible increase in differentiation to megakaryocytes in early S-phase and to nonproliferative granulocytes in mid S-phase. Megakaryocyte hotspots also were seen on a clonal basis. Elevations of the transcription factor FOG-1 were seen at the hotspot along with increases in Nfe2 and Fli1. We show that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell-cycle transit, suggesting that stem cell regulation is not based on the classic hierarchical model, but instead on a functional continuum. We propose that there is a tight linkage of commitment to a lineage and a particular phase of cell cycle. Thus, windows of vulnerability for commitment can open and close depending on the phase of cell cycle. These data indicate that stem cell differentiation occurs on a cell-cycle-related continuum with fluctuating windows of transcriptional opportunity.