Differentiation Profiling of Marrow Stem Cells: A Megakaryopoietic Hotspot and the Continuum Model of Hematopoiesis.

Hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show here that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. This along with other data strongly suggest that stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum We have previously shown that hematopoietic stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Further work has shown that adhesion protein, cytokine receptor, gene expression and progenitor phenotypes also shift. Evolving data indicate the phenotype of murine marrow stem cells reversible change with cell cycle transit. Murine experiments have been performed on highly purified, quiescent G_0-1 lineage^negativerhodamine^lowHoeschtl^ow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor, they synchronously pass through cell cycle as measured by propidium iodide, cell doublings and tritiated thymidine. LRH cells enter S-phase in a synchronized fashion by 18 hours, leave S-phase at 40–42 hours and divide between 44–48 hours. The capacity of these cells to respond to a differentiation inductive signal (granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and steel factor) is altered at different points in cell cycle. Megakaryocyte production is specifically focused at early to mid S-phase, this returned to baseline before the first cell division. Population based cultures after 14-days of differentiation culture produced up to 49% megakaryocytes with stem cells sub-cultured during early-mid S-phase with little to no production with colonies cultured from stem cells in G_0-1 or G₂ phase at time of differentiation induction signaling. Cell type was confirmed by staining cells with acetylcholinesterase, antibodies to platelet glycoprotein complex IIb/IIIa and von Willebrand’s factor. Evaluation of gene expression at this hotspot showed a marked increase in expression of CD4 with up to 464.2 fold increase above baseline. Sca-1 and transcriptional factor FOG was strikingly amplified at S-phase as well as other relevant markers. While pertinent cytokine receptors were not increased, studies on a clonal level confirm the existence of a reversible megakaryocytic hotspot. Compared with other time-points relating to cell cycle position prior to differentiation sub-culture in one experiment, 33% of clonally derived colonies that grew from early S-phase cells and 10% of colonies that grew from mid S-phase cells had megakaryocytes present two weeks after initiation of culture compared with 0% for G_0-1 and G₂ cells. Granulocyte differentiation also showed specific differentiation hotspots, but presentation is outside the scope of this abstract. These data indicate that marrow hematopoiesis stem cells exist in a continuum, not in a hierarchy with continuously changing windows of transcriptional opportunity.