Hematopoiesis is an ongoing developmental process in adult mammals. The details of the early stages of this process remain obscure, mainly because the very low frequency of hematopoietic stem cells (HSC) in bone marrow limits the ability to study these cells. The goal of this project is to define phenotypic characteristics which will help to identify HSC as they normally exist in bone marrow tissue. Further, this project aims to document growth and differentiation characteristics of normal HSC by transplantation studies and by in vitro culture techniques. We are also interested in defining optimal conditions for infecting enriched populations of HSC with retrovirus constructs. We have developed an enrichment scheme that reproducibly recovers a full spectrum of hematopoietic progenitors from mouse bone marrow. Within this population we can identify a subset of cells that can repopulate irradiated animals at near-single cell levels. We have performed transplantation studies using very small numbers of purified HSC to demonstrate that the cell population we have identified can contribute to hematopoiesis over the life-span of the recipient animals. Further, we have followed the progeny of those cells as they contribute to the peripheral blood and have shown two general patterns of repopulation, one in which significant repopulation is seen within 5 weeks of the transplant, and another in which little repopulation is seen until later times. In both cases, contribution to the peripheral blood is maintained over long periods of time. It is unclear whether the two types of long-term repopulation are due to two distinct stem cell types, or rather are due to differential seeding to specific microenvironments in the host animal. These experiments demonstrate the potential of individual HSC to function over extended periods of time. We have obtained quantitative information on the ability of HSC to self-renew. This has been approached by re- isolating HSC progeny from transplanted animals many months after the original transplant and testing their repopulating potential. Although we have seen expansion of cells expressing the HSC antigen phenotype, we do not see a parallel expansion of HSC function. This demonstrates that phenotype alone can not be used to indicate HSC function. We have also investigated expression of the multi-drug resistance phenotype in these cells, their cell cycle status, and the ability of Friend virus-derived vectors to introduce and express foreign genes in the hematopoietic system.
