In this proposal, a novel murine lymphohematopoietic stem cell line MER and its derivative will be used to isolate genes controlling neutrophil commitment and differentiation. The MER cell line is dependent on stem cell factor (SCF; c-kit ligand) and has-both lymphoid and hematopoietic (""""""""myeloid"""""""") potentials. From this stem cell line we established a neutrophilic derivative (MER-PRO) blocked at the promyelocyte stage. This block can be overcome with high concentrations of retinoic acid. This complementary pair of cell lines represent hematopoietic cells """"""""frozen"""""""" reversibly at two developmental stages and are particularly suitable as a model system for studying stem cell commitment and lineage-restricted differentiation.
The specific aims of this proposal are: (I) Isolation and characterization of neutrophil progenitor-specific genes, including those involved in lineage commitment. The technique of differential hybridization will be used to screen the cDNA library of MER-PRO (neutrophilic promyelocytes) using subtracted cDNA probes prepared from the MER-PRO and MER cell lines; (II) Isolation of genes activated or inactivated by retinoic acid during the terminal differentiation of neutrophilic promyelocytes. Either the cDNA library differential hybridization technique or the polymerase chain reaction-based """"""""differential display"""""""" of mRNAs can be used. These genes are more downstream to those in Specific Aim I but are important in the later stages of neutrophil differentiation; (III) In vivo characterization of the lymphohematopoietic stem cell line MER and establishment of a human equivalent of the MER cell line. The capacity of the MER cell line to function as stem cells will be examined in irradiated syngeneic mice. Attempts will be made to create a similar cell line from human bone marrow. The MER cell line may be the first stem cell factor-dependent cell line with both lymphoid and hematopoietic potentials and should greatly facilitate stem cell research. The knowledge gained from this unique experimental system is likely to contribute to our understanding of the biology of human lymphohematopoietic stem cells. The long-term objective of this proposal is to identify molecules directly mediating the self-renewal, commitment and differentiation of stem cells and how these molecules are in turn modulated by hormones and extracellular matrix. It is hoped that this new knowledge will enable us to control stem cell behavior for therapeutic purposes such as gene therapy in the future.
