Hemopoietic stem cells perform dual functions; self-renewal and commitment to various cell lineages. Recently, we identified in clonal culture, a new class of murine hemopoietic stem cells (S-cells) with extensive capacity for self-renewal and ability to produce committed progenitors. These cells are identified by their ability to form colonies (stem cell colonies) consisting of only blast cells after 16 days of culture. Replating of these colonies suggested that S-cells are the most primitive of all progenitors assayable in culture. Using this stem cell colony assay, we will examine the mechanisms controlling stem cell renewal and commitment. By use of a micromanipulator, doublets produced by single cells obtained from stem cell colonies will be transferred to marked areas of dishes and colony formation from the individual cells will be observed. By use fo statistical analysis, we will estimate the probabilities of commitment to specific hemopoietic pathways and we will carry out Monte Carlo simulation of the commitment processes. We will also examine the interactions between hemopoietic stem cells and microenvironment by plating individual stem cell colonies over dexamethasone-treated preadipocyte cell line or Dexter's primary marrow monolayer. We will purify and characterize humoral factors which modulate the stem cell functions by use of conventional chromatographic methods as well as high performance liquid chromatography of serum-free conditioned medium prepared with concanavalin A-stimulated spleen cells. We will also determine the hierarchical stages of S-cells and mixed hemopoietic colony-forming cells by detailed correlation analysis of in vivo and in vitro colony formation by cells prepared from individual spleen colonies and from individual stem cell colonies. Our preliminary observations on the human equivalent of mouse stem cell colonies will be extended in order to establish a quantitative assay for the most primitive human hemopoietic stem cells. Finally, as part of our analysis of the stem cell hierarchy, we will examine the lymphopoietic potentials of the stem cell colonies in order to characterize the mechanisms by which lymphopoietic progenitors depart from hemopoietic lineages.
Showing the most recent 10 out of 51 publications
