Residual radiation injury exhibited in long-term bone marrow cultures.

Residual radiation injury was demonstrated in long-term primary cultures of mouse bone marrow. Control cultures underwent three phases of hematopoietic activity as distinguished by initial establishment, steady high (plateau) production of granulocytes, and gradual decline. Irradiation with 50, 300, or 550 rads, given at the end of the initial phase, did not prevent any culture flasks from entering the plateau phase. However, actual production levels and the time they were maintained varied inversely with the radiation dose so that the accumulated postradiation cell production corresponded to an exponential dose-response relationship at any time after treatment. The accumulated cell productions were found to be similar in all groups when expressed by the number of stem cell doublings necessary to produce them. The findings cannot be explained by reproductive cell death and are consistent with the notion of a limited division capacity in hematopoietic stem cells.

Proliferative capacity of murine hematopoietic stem cells in vitro.

Large numbers of granulocytes can be collected repeatedly from the supernatant medium of long-term cultures of mouse bone marrow cells. A constant relationship was found between the number of adherent hematopoietic stem cells and the lifetime cell production per culture. The data indicate that there is a limit to the proliferative capacity of normal and of irradiated stem cells. A similar limitation was found in the production of marked granulocytes from clonal cultures of "beige" C57 (bg/bgJ) stem cells placed in limiting dilutions into stromal culture layers.

Differential effects of cytotoxic agents on hematopoietic progenitors.

In order to predict the effect of chemotherapeutic agents on the hematopoietic progenitor compartment, it is necessary to have a hypothesis concerning the dynamics of the cells within the compartment. By viewing this compartment as a continuum of cells with varying self-renewal capacity, one can assess the significance of the stem cell damage incurred following a single drug dose. Vinblastine, 5-fluorouracil, cyclophosphamide, busulfan, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were investigated. The acute toxicity of the drug-exposed marrow was studied by the colony-forming units-spleen (CFU-S) and agar diffusion chamber assay. Busulfan and BCNU were found to kill CFU-S preferentially. By following CFU-S recovery for 14 days post drug, different recovery patterns are noted. Busulfan and BCNU produce prolonged depression in CFU-S, whereas cyclophosphamide and 5-fluorouracil show relatively rapid recovery. If one determines the Rs (a measure of proliferative capacity) after drug, busulfan and, to a lesser extent, BCNU produce a prolonged depression without return to normal even 650 days post drug. No such depression is noted with the other drugs. The data from the recovery curves and Rs support the notion of stem cells being heterogeneous with regard to self-renewal capacity. The agar diffusion chamber and CFU-S acute survival curves would not have predicted which drugs cause significant proliferative damage. Only with the use of CFU-S recovery and ratio of CFU-S can prolonged marrow damage be ascertained.

Nature of the hemopoietic stem cell compartment and its proliferative potential.

The nature of the hemopoietic stem cell compartment has been the subject of much controversy. Data are presented to support the concept of a 'continuum' model of the stem cell compartment. The important characteristics of this model are that within the continuum there are cells with varying proliferative capacities. As cells move through the compartment, their proliferative capacity becomes more limited, their likelihood to be in cycle increases, and their commitment to a specific differentiated pathway increases. Experiments with busulfan, cyclophosphamide, 5-flurouracil, and BCNU demonstrate defects in proliferative potential of the surviving CFU-S population. These defects persist throughout the life of the animal without any evidence of recovery. The clinical implications of late stem cell failure may be important as a consideration in the use of cytotoxic agents.

Proliferative capacity of murine hematopoietic stem cells.

The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferative quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cell progress in the continuum in one direction and such progression is not reversible.