AbstractMammalian blood cell production is maintained by a heterogenous hematopoietic stem cell (HSC) population with remarkable capacity for self-renewal and pluripotency. While bone marrow is the major site for HSC residence, the red pulp region of the spleen can also act as a niche for HSC, despite the lack of osteoblasts. HSC differentiation in bone marrow is influenced by signals imposed by the stromal niche. It is therefore important to consider how the splenic niche might signal hematopoiesis and how spleen-resident HSC might contribute to hematopoiesis in the steady-state and also across the life span of the animal. Hematopoiesis in spleen is commonly linked to stress and disease, a phenomenon termed extramedullary hematopoiesis. However, no studies have yet addressed lineage output in steady-state spleen. Evidence from in vitro studies and in vivo models of extramedullary hematopoiesis suggest that spleen-resident HSC might be primed for myeloid cell production.
Transplantation studies performed to address the precise hematopoietic potential of spleen HSC under physiological conditions have been hampered by the limitation of using traditional conditioning methods to ablate HSC niches. Clearance of HSC niches is required for donor engraftment to occur. With success with dietary-based HSC niche clearance techniques which are non-inflammatory, the study of steady-state hematopoiesis specific to the spleen is now possible. Here we attempt to address spleen-specific steady-state hematopoiesis using valine-restricted dietary conditioning, so removing the need for irradiation therapy prior to HSC transplantation. Following optimization of a dietary-based conditioning regime to clear HSC niches, donor chimerism was achieved within both the spleen and bone marrow, so demonstrating the potential value of this model particularly in regard to steady-state hematopoiesis involving the spleen.
Published lineage commitment models describing hematopoiesis are controversial and still incomplete in predicting how fate-decision occurs. Current models fall short of describing the symmetry by which HSC progress through lineage commitment, and whether there are defining genes or imprinted epigenetic programs driving the various differentiation pathways at particular periods during life. For example, myeloid-biased CD150hi HSC appear increasingly with age, evident by an imbalance in myeloid and lymphoid cell development. Given that HSC localized in spleen reflect cells with myeloid-restricted potential, this study considers how the spleen contributes to the age-associated decline in balanced lineage output potential.
The overall hypothesis of this thesis is that HSC differentiation in spleen under steady-state conditions is biased towards myelopoiesis, and that throughout adult aging, the spleen microenvironment continually imprints myeloid bias into HSC programming, therefore contributing to a rise in myeloid-biased HSC numbers. The first aim of this study is to address these unknown gaps in research by optimizing a new transplant pre-conditioning method that maintains the host steady-state condition in order to study the spleen-specific contribution to hematopoiesis. These results then complement the second aim which is to investigate the role of spleen in the age-associated dominance of myeloid-biased HSC.
Optimization of valine-restriction preconditioning found that mice fed a valine-deficient diet for three weeks tolerated immediate complete diet reintroduction. By allowing 2-days of valine reintroduction prior to transplantation it was found that donor cell engraftment could be achieved. We suggest that cell-dose titration is necessary to enhance donor engraftment since valine-restriction preconditioning does not clear hematopoietic niches entirely. Results of investigation of the second aim indicate that spleen does not play a significant role in the progressive myeloid-biased HSC shift over an animal’s lifetime. However, no firm conclusion can be drawn due to data variability found in aged mice. Overall, the work presented in this thesis does not show conclusively that spleen contributes to the accumulation of myeloid-biased HSC over age. Optimization of a dietary-based preconditioning method will assist future investigation into the hematopoietic contribution of spleen in the steady-state condition.
|Date of Award
|11 Jun 2020
|Jonathan Tan (Supervisor) & Helen O'Neill (Supervisor)