Molecular Analysis of Primitive Hematopoietic Cell Proliferation Control Mechanisms

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Abstract
Cells at two distinct early stages in the development of mature human blood cells from primitive totipotent hematopoietic stem cells can now be defined and quantitated by separate in vitro assays. Current evidence suggests that most, if not all, colony-forming cells--that is, cells that give rise to colonies of mature progeny within one to three weeks in semisolid culture systems, represent an intermediate stage of hematopoietic progenitor. These cells are not self-sustaining; if they are used to initiate hematopoiesis on competent marrow stromal layers, they rapidly disappear as they differentiate or die. However, clonogenic cells can be generated in such cultures from another cell type over a period of four to eight weeks. We have, therefore, assigned the term long-term culture initiating cell (LTC-IC) to this latter type of clonogenic precursor cell. The production and differentiation of cells in both of these compartments in LTC are dependent on, and regulated by, nonhematopoietic "stromal" cells that form a heterogeneous adherent layer in which close-range interactions with hematopoietic cells take place. The use of separate endpoints to monitor the maintenance, differentiation, and reversible activation or arrest of cycling of these cells has recently revealed different molecular mechanisms regulating their respective functions. However, an important common feature appears to be the relative local concentration of positive and negative regulators to which the target hematopoietic cell is exposed. Both gene expression and growth factor release measurements as well as results obtained using genetically engineered stroma and repeated soluble growth factor addition implicate G-CSF as an endogenous positive regulator of primitive hematopoietic cells. Similarly, gene expression, factor production, factor addition, and neutralizing antibody experiments implicate TGF-beta as an endogenous inhibitor of primitive hematopoietic cells.