The overall goal of this project is to dissect the marrow microenvironment (ME) for the purpose of identifying gene products that work in concert to regulate early events in hematopoiesis. Functionally distinct stromal cell lines have been generated to address this goal: one line supports immature progenitors (HS-27a) and one line drives differentiation (HS-5). Microarray technology has been used to generate gene expression profiles from these lines to identify differentially expressed genes (Data from this comparative analysis is available at htpp://parma.fhcrc.org/M Iwata).
In Aim 1, the array data will be confirmed for select genes by Northern and Western blotting and immune cytochemistry. Once their relevance has been established by detection in primary marrow cultures and marrow biopsies, the role of candidate gene products will be investigated by determining how gain and/or loss of function effects the ability of the ME to support hematopoiesis.
Aim 2 will address how monocytes/macrophages, which are an integral part of the ME, effect stromal function. Gene expression profiles have been generated for both normal monocytes and for HS-27a cells cultured alone. These have been compared to profiles from the two cell types cultured together to identify significant changes in both monocyte-derived and stroma-derived gene products. Several gene products with significantly altered expression and potential relevance to ME function have been selected for functional studies. These include: osteopontin (OPN) which down regulates Notch on CD34 cells; CXCL7 which may influence fibroblast growth; bone morphogenetic protein antagonist -1(BMPA-1); Cadherin-6 (CDH6) which is expressed by stroma and CD34 cells and mediates homophilic, yet heterotypic cell-cell interactions; and stromal derived factor-1 (SDF-1). The knowledge gained from Aims 1 and 2 will determine how normal monocytes interact with stromal cells and contribute to ME function. This information will be used as a platform to determine in Aim 3 how abnormal monocytes may compromise the ME. Specifically, we hypothesize that abnormal monocytes in patients with myelodysplastic syndrome (MDS) adversely affect the function of an intrinsically normal stroma, giving rise to the dysplasias characteristic of this disease. Additionally, we propose that MDS monocytes, through their retention after nonmyeloablative conditioning, cause the marrow graft failure and consequent disease progression seen in a significant proportion of MDS patients given stem cell transplants. This research plan is based on historical as well as new preliminary studies indicating that monocytes, normal or abnormal, interact with stromal cells to modulate ME function. Hypothetically this can advantageously or adversely influence hematopoiesis in general and stem cell engraftment in particular. A better understanding of these effects should facilitate the development of new therapies to correct dysplasias and optimize stem cell transplantation.
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