monash_80110.pdf (12.36 MB)
The role of mature megakaryocytes in the haematopoietic stem cell niche
thesisposted on 2017-01-31, 04:17 authored by Neaves, Rebecca Joy
Haematopoiesis is the tightly regulated process within the bone marrow (BM) responsible for the maintenance of all haematopoietic cells and is sustained by a rare population of haematopoietic stem cells (HSC). HSC have the capacity for quiescence and self-renewal and the ability to differentiate into all cells of myeloid and lymphoid lineages. Interestingly, it has been shown that the regulation and fate of HSC in the BM is governed by the unique surrounding microenvironment, termed the HSC niche. Elements of the BM, such as osteoblasts (OB), the sympathetic nervous system (SNS) and endothelial cells (EC) have been shown to influence haematopoietic stem and progenitor cell (HSPC) function, however, the precise interactions within the HSC niche remains unclear. In this thesis, we have established that mature megakaryocytes (MM), which are large polyploid cells of the myeloid lineage with the primary function of generating platelets, have a functional role within the HSC niche. We have shown that although MM are randomly distributed throughout the BM, a significant majority of HSPC transplanted into non-ablated recipients are found within close spatial location to MM. Additionally, in vitro studies using purified, functionally viable populations of polyploid MM, isolated by a novel multiparameter flow cytometric sort strategy, have demonstrated that co-culture with HSPC significantly increases the growth of endosteal HSC. Furthermore, co-culture of HSC with freshly isolated MM significantly increases the incidence of long-term (LT) repopulating cells, compared to HSC cultured alone. However, HSC co-cultured with MM were outcompeted by HSC cultured alone, suggesting that, in vitro, MM support the expansion of a more mature progenitor cell that is still capable of LT repopulation. Subsequent analysis of culture supernatants using cytokine arrays identified Insulin-like Growth Factor -1 (IGF-1) and Insulin-like Growth Factor Binding Protein -3 (IGFBP-3) as candidate proteins secreted by MM which may be responsible for regulation of HSC growth in vitro. The addition of these two proteins to HSC cultures resulted in a significant increase in cell growth, which was equivalent to co-culture with MM. Furthermore, the use of perturbation models with a higher proportion of MM in the BM microenvironment, there is an increase in homing of transplanted HSC. Conversely, in mice with a BM microenvironment devoid of MM there is a decrease in homing of transplanted HSC and an inability to facilitate normal haematopoietic reconstitution post-BM-ablation. In summary, these findings establish a previously unrecognised role of MM in regulating HSC function within the HSC niche.