| Deciphering a precise and tractable phenotype for stem cells has remained an elusive goal of biologists for decades. Investigation of the molecular physiology behind development and regeneration is critical, however, the first step in understanding stem cell function lies in their identification. The major focus of this work is the development of novel approaches to recognize unique cellular properties inherent to hematopoietic stem cells in the bone marrow. The first of these strategies involves the use of highly complex, synthetic antibody libraries to probe the surface of bone marrow cells. This technique and the corresponding experiments propose a means by which to obtain a complete inventory of cell surface antigens. Furthermore, this work presents an approach to the differential screening of cells in order to find those molecules that may be distinctly a function of cells with regenerative potential.; The second method discussed for the enrichment of hematopoietic stem cells involves measurements of membrane transport. This approach does not rely on the identification of any surface molecules, but on the differential kinetics of fluorescent dye uptake by cells. Membrane transport-based isolation of cells with stem cell potential is quite appealing because it does not rely on the prior knowledge of any particular surface markers.; Lastly, the study of hematopoiesis would not be possible without flow cytometry. This technology, a cornerstone for our laboratory, offers immediate insight to the makeup of a heterogeneous cell sample, and cell sorting adds the indispensable capability of being able to select any desired population from this mix for further study. Some of the technical aspects of this instrument are also discussed as well as emerging applications in molecular biology and medicine. |
