Early Blood Cell Formation "in sickness and health, ´till death do us part"

Abstract: Hematopoietic stem cells (HSC), the ancestors of all blood cells, have the ability to give rise to mature effector cells through a series of developmental steps. This is a tightly regulated, though a highly dynamic process: on the one hand, dysregulation in steady-state could lead to under- or overgrowth of certain blood cell lineages, whereas, on the other hand, demands for certain mature blood cells can change dramatically in cases like bleeding and infections. In addition to the production of differentiated offspring, HSC possess the ability to undergo self-sustaining (or self-renewing) cell divisions to ascertain life-long replenishment of the hematopoietic system. High resolution profiling of the mechanisms that guide these cellular fates does not only allow to further increase our knowledge in normal cell biology, it can also provide with clues on possible pathogenetic events in cases of altered hematopoiesis like leukemia or in the aging individual. In Article I, we have investigated the role of tumor necrosis factor (TNF) in the regulation of HSC homeostasis. We found TNF to negatively regulate HSC activity in a cell cycle dependent manner, with required signaling through both TNF receptors to elicit full inhibitory actions. Thereby, this study identifies one single, cell extrinsic factor to negatively regulate HSC activity. Blood cell production in elderly is characterized by myeloid skewing at the expense of lymphoid output, in addition to increased HSC frequencies in aged mice. However, HSC replicating activity was suggested to be decreased through increased p16INK4A signaling. To determine regulating factors underlying these observations, we assayed HSC in aged mice (Article II). We found evidence for decreased proliferative kinetics of old versus young HSC, though without proof of replicative senescence. In agreement with this, we found no evidence for increased p16INK4A signaling in aged HSC, nor for a permissive epigenetic state at the p16INK4A control region. Differentiation from a multipotent HSC to mature blood cells occurs through a series of commitment steps, generating progenitors with increasing lineage restricted potentials. The earliest lineage restricting events have been subject to debate and in Article III and IV we describe our efforts to create increased insights in the developmental steps of early myeloerythroid development. By means of flow cytometric isolation, we have identified the early granulocyte/monocyte, erythroid and megakaryocytic lineage restriction steps within the cKit+Lineage-Sca1- myeloerythroid progenitor compartment. Subsequently, functional and molecular analysis of these cellular subsets has allowed us to place these cells in a hierarchical order and understand some of the genetic events that occur during lineage specification.