Haematopoietic stem/progenitor cell interactions with the bone marrow vascular niche

Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.This thesis is not currently available via ORA