Residual damage and discontinuity of recovery in the hematopoietic system of mice following gamma-irradiation.

Recovery of the hematopoietic system up to 35 days following whole body irradiation of mice was investigated by measuring the repopulation ability of femoral bone marrow in irradiated recipients. The assay was performed five days after transfusion by testing for incorporation of 125iodo-deoxyuridine (125IUdR) into DNA of femoral marrow and spleen. The DNA incorporating cells are progeny of transfused stem cells or early precursors. For non-irradiated donors 125IUdR incorporation in the recipient spleens was proportional to the number of transfused cells between 0.05-1.0 x 10(6)/mouse but decreased with larger grafts (5-30 x 10(6). Yet, in the femur 125IUdR incorporation remained proportional over the range of 0.5-30 x 10(6) transfused cells. Recovery was discontinuous and not complete 35 days after irradiation. A possible explanation is injury to stem cells that still permits these cells to produce progeny within a milieu of high proliferation stimulus.

Repopulation ability and proliferation stimulus in the hematopoietic system of mice following gamma-irradiation.

The repopulation ability of bone marrow was measured following whole body gamma irradiation of mice. Immediately after irradiation (100--500 rad) bone marrow suspensions were transfused into lethally irradiated syngeneic recipients. At various time intervals after transfusion (1 hour--20 days) DNA synthetic activity in femur and spleen was measured by incorporation of 125iodo-deoxyuridine (125IUdR). The cellularity of femoral bone marrow and the spleen weight was determined. A dose dependent retardation of repopulation was found to be correlated with an increasing duration of an elevated proliferation index (125IUdR-incorporation per unit bone marrow cells of per unit spleen weight, resp.). This suggests that the organism exerted a stimulus on the hematopoietic system to promote regeneration. This proliferation stimulus was kept at a high level unit a certain degree of hematopoietic function was restored either by regeneration of the bone marrow or, by compensating proliferation, in the spleen. The repopulation ability apparently was not only impared by the loss of stem cells but also by the induction of genetic damage in stem cells whose survival and partial functioning may have been enabled by the elevated proliferation pressure.

Proliferation kinetics of early hemopoietic precursor cells with self sustaining capacity in the mouse, studied with 125-I-labeled iodo-deoxyuridine.

With a new labeling technique in radiation chimeras, an attempt was made to determine the duration of the phases of the stem cell cycle including shortest and mean generation time and to estimate the number of hemopoietic stem cells per unit of bone marrow cellularity. The DNA of bone marrow cells in DNA synthesis was labeled with 5-125I-2'-deoxyuridine. The labeled cells were followed after being transfused into fatally irradiated mice. The stem cells were found to have a half-time of about 4.3 days in the donor mice. The average time in the population, i.e. the turnover time of the stem cells, was 6.2 days. The half-time did not change significantly even after transfusion of bone marrow into lethally irradiated recipient mice. Tritiated thymidine (3H-TdR) suicide technique revealed that bone marrow stem cells seeding to the spleens and to the femurs of lethally irradiated recipients behaved differently--S-phase in cells seeding to femurs being shorter. The radiosensitivity of stem cells in S-phase had a D0 of 80 rad whereas stem cells distributed throughout the whole cell cycle had a D0 of 185 rad. The respective extrapolation numbers were 1.23 and 1.14. It is calculated that 2--7% of all nucleated bone marrow cells belong to self renewing stem cell populations. The method described provides a new approach for the study of hematological stem cells.

Relative number and proliferation kinetics of hemopoietic stem cells in the mouse.

A model calculation of the hemopoiesis of the mouse based on known hematologic data leads to the conclusion that approximately 3% of all nucleated bone marrow cells are stem cells (pluripotent plus committed stem cells). By a new 125IUdR labeling technique on radiation chimeras, a relative number of 2%-7% stem cells was determined. In previous studies with test systems for stem cells using colony formation in vivo or in vitro, a relative number of stem cells of at least one order of magnitude lower has been estimated. In this study the stem cells are found to have a turnover time of about 4.3 days in the donor mice. This turnover time remained unchanged even after transfusion of marrow cells into lethally irradiated recipient mice. Radiosensitivity determinations yielded a D0 of 80 rad for stem cells in S-phase and D0 of 185 rad for stem cells distributed throughout the entire cell cycle. The respective extrapolation numbers were 1.23 and 1.14. Experiments using an 3H-TdR suicide technique revealed different cell cycle parameters for bone marrow stem cells seeding to the spleens and to the femurs of lethally irradiated recipients, primarily a shortening of S-phase in cells seeding to femurs. The method described here provides a new approach to hematologic stem cell research.

Determination of rates of DNA synthesis in cultured mammalian cell populations.

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [(3)H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 microM) in combination with hypoxanthine and glycine. If [(3)H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [(3)H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10(6) cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.