Studies on myeloid differentiation: cytokine influence and identification of a novel marker gene

Abstract: Hematopoiesis is a highly complex process by which a range of specialized blood cells are generated from a small pool of multipotent stem cells in the bone marrow. The survival, proliferation, and differentiation of hematopoietic stem cells are tightly regulated by both soluble and membrane-bound cytokines produced within the bone marrow microenvironment. Kit Ligand (KL) and Flt3 Ligand (FL) are two important hematopoietic cytokines, and signal via related tyrosine kinase receptors; c-kit and flt3. The first part of this thesis is focused on the influence of KL and FL on differentiation of a stem and progenitor cell-enriched cell population (c-kit+Lin- cells), isolated from mouse bone marrow. We found that KL and FL have different effects, and favor development of granulocytic and monocytic cells, respectively. A clear discrepancy was also seen on the expansion of multilineage progenitors (pre-CFCmulti) and granulocyte/macrophage colony-forming progenitors (CFC-G/M) which was strongly favored by KL. Furthermore, FL induced development of a biphenotypic cell population coexpressing monocytic and B-cell characteristics, restricted to the macrophage lineage. The second part of this thesis, describes the identification and characterization of a novel myeloid-associated differentiation marker gene (MYADM) which is preferentially expressed in myeloid cells, but absent in B and T lymphocytes. The predicted 32-kDa protein contains eight transmembrane domains and localizes to intracellular membranes. Although, the function of MYADM is unknown, antisense oligonucleotides inhibit colony formation of c-kit+Lin- cells, suggesting an important role for MYADM in myeloid differentiation. To gain further insight into the transcriptional control of the MYADM gene, we have analyzed a 1.5-kb sequence upstream the transcription start site for promoter activity in myeloid and lymphoid cells. We conclude that the sequence analyzed in this study does not confer the promoter tissue-specificity, thus additional regulatory elements may be located outside the region upon which this study has concentrated.