The Biology of Hematopoietic Stem Cells: transgenic approaches to Dissect Native and Perturbed Hematopoiesis

Abstract: Hematopoiesis is defined as the ongoing production of blood cells. As most mature blood cells are relatively short-lived and require continuous replacement, hematopoiesis is characterized by an extraordinary turnover rate with production of trillions of new blood cells every day. To cope with the enormous proliferation required to generate sufficient numbers of blood cells to maintain homeostasis, the hematopoietic system is hierarchically organized within the bone marrow. Scarce hematopoietic stem cells (HCSs) reside at the top of this hierarchy. More abundant and increasingly developmentally restricted and proliferating progenitor cells, that massively amplify hematopoietic cell generation, reside further down in the hierarchy. HSC function is typically evaluated using transplantation experiments, which offers quantitative and qualitative information on their self-renewal and multilineage differentiation potential. In this setting, potent long-term multilineage contribution can be observed from even single HSCs. After transplantation of myeloablated hosts, HSCs are forced to proliferate extensively to rebuild the hematopoietic system. In sharp contrast, native HSCs display very low proliferation rates. Emerging data has highlighted fundamental differences between hematopoiesis as seen after transplantation compared to that in steady state. Therefore, analysis of native hematopoiesis in models that allows for evaluation in unperturbed settings is necessary. In article 1 we characterize HSC and progenitor proliferation dynamics in the steady state and following several types of induced stress. Whereas transplantation promoted sustained, long-term proliferation of HSCs, both cytokine-induced mobilization and acute depletion of selected blood cell lineages elicited very limited recruitment of HSCs to the proliferative pool. In addition, coupling of proliferation history with gene expression analysis on single cells led to identification of subtypes of HSCs that have distinct molecular signatures and differ drastically in their reconstitution potentials.The Mx1-Cre mouse strain is the most commonly used conditional gene-knockout strain in experimental hematology. The Mx1 promoter is activated by endogenous interferon release that is induced by injection of polyinosinic:polycytidylic acid (poly I:C). However, interferon is also released as a part of the inflammatory response. In Article 2, we highlight pitfalls associated with the Mx1-Cre system. Transplantation of cells where Mx1-Cre activation is required for gene knockout resulted in high rates of spontaneous gene deletion. In addition, poly I:C administration introduced alterations to the hematopoietic stem and progenitor cell (HSPC) compartment. Collectively, this study emphasize that proper controls are crucial when modeling gene deletion with the Mx1-Cre system.A model with limited HSC contribution to native hematopoiesis has been proposed. This is in sharp contrast to the continuous contribution of HSCs to hematopoiesis after transplantation. In the work leading up to article 3 we set out to explore HSC contribution to native hematopoiesis by evaluation of blood cell generation from HSCs in native adult hematopoiesis. For this we used Fgd5-CreERT2 mediated lineage tracing, a model that can label close to 100 % of adult HSCs in a highly specific manner. We show that apart from blood cells with a known fetal origin, HSC contribution to all blood cell lineages is robust and occurs via a hierarchy of defined intermediate progenitor cells. Our experiments reveal that the time course of regeneration for distinct blood lineages varied substantially. Myeloerythroid cells were generated from HSCs more rapidly than lymphoid cells, with platelets and their corresponding progenitor cells emerging first. Therefore, adult HSCs are active contributors to all lineages of adult hematopoiesis in the steady state.In summary, we have highlighted features with experimental systems/procedures that are used in experimental hematology and have explored hematopoiesis and HSC biology in models that allows evaluation of unperturbed hematopoiesis.