Chronic myelocytic leukaemia: cytogenetical studies on haemopoietic colonies and diffusion chamber cultures.

In 20 patients with chronic myelocytic leukaemia in the chronic phase or in blast crisis, several in vitro (CFU-C, BFU-E) and in vivo (diffusion chamber) culture techniques were used to demonstrate the existence of a still diploid cell clone. In 4 out of 12 patients in the chronic phase, Ph1-negative metaphases were found in the various cultures for haemopoietic progenitor cells but not in the standard suspension cultures used for cytogenetics. However, all the 8 patients investigated during blast crisis had only Ph1-positive and other abnormal clones, indicating the loss of the diploid clone during progression of the disease.

Proliferative state of normal in vitro colony-forming cells during development of L5222 rat leukemia and their reaction to chemotherapy.

In the experimental rat leukemia, L5222, the decrease of normal in vitro colony-forming cells (CFU-C) after chemotherapy with daunomycin is much less than in nonleukemic controls. The leukemia is therefore used here to test the hypothesis that in leukemia the CFU-C are expelled from the active cell cycle to a resting state and are thereby less sensitive to cycle-dependent chemotherapeutic agents. The L5222 leukemia has the advantage that the leukemic blast cells do not form colonies in agar culture so that normal CFU-C can be assessed under leukemic conditions. To compare the proportions of CFU-C in the S-phase in normal and leukemic rats, two S-phase-specific agents, 3H-thymidine and hydroxyurea, were used to kill proliferating bone marrow cells. Following treatment with 3H-thymidine in vitro, about 41% of the CFU-C were killed in normal and about 25% in leukemic bone marrow. Hydroxyurea administered in vivo resulted in the death of about 33% and 26%, respectively. The results indicate that fewer normal CFU-C are in S-phase in the L5222 leukemia, which might help to explain how enough normal stem cells survive chemotherapy to regenerate the bone marrow.

Evidence for differentiation of human leukemic blood cells in diffusion chamber culture.

Peripheral blood cells of 21 patients with different forms of acute leukemia were cultured in diffusion chambers (5 x 10(5) cells/chamber) implanted intraperitoneally in 650 R preirradiated host mice over a period of up to 21 days. In patients with acute myeloid leukemia (AML), acute erythroleukemia (AEL), or acute myelomonocytic leukemia (AMMoL), the total number of cells which developed during this culture period exceeded the implanted value and also the values for normal peripheral blood cells from ten controls. In acute undifferentiated leukemia (AUL), two out of six patients showed considerable growth whereas the others, and also two patients with acute lymphoid leukemia (ALL), had poor growth. Differential counts revealed that the rise in total cells was due mainly to proliferation of blast cells and formation of granulopoietic cells. The latter exceeded the numbers from normal peripheral blood cells in 9 out of 13 patients with AML, AEL, or AMMoL and in 2 out of 6 patients with ALL. The production of granulopoiesis was not restricted to proliferating cells, but included mature cells which were of abnormal morphology in some cases. From the amount of granulopoiesis and the time of its development it was assumed that they were at least partly derived from leukemic blast cells. Chromosome analyses to decide whether the granulopoietic cells were of leukemic or normal cell origin are in progress.

Growth and differentiation of rat bone marrow stem cells in diffusion chamber culture.

The growth of granulopoietic progenitors (CFU-C) and morphologically identifiable haemopoietic cells from rat bone marrow was studied in intraperitoneal diffusion chambers over a 6-day period. The aim of this study was to investigate whether stem cells preceding the granulopoietic progenitors are able to restore and to maintain depleted compartments of CFU-C and immature granulopoietic cells under the conditions of diffusion chamber culture. After treatment of rats with repeated doses of hydroxyurea (HU) a bone marrow cell suspension could be obtained wherein proliferating haemopoietic cells were reduced to a very low level and CFU-C to undetectable levels. During culture in diffusion chambers, growth of CFU-C, immature granulopoietic cells and macrophages could be observed from this HU-treated bone marrow. It is concluded that in diffusion chamber culture stem cells preceding CFU-C are able to rebuild functioning compartments of granulopoietic progenitors and morphologically identifiable haemopoietic cells.

Growth of in vitro colony-forming cells from normal human peripheral blood leukocytes cultured in diffusion chambers.

The growth of granulopoietic progenitor cells (CFU-C) in diffusion chambers during culture of peripheral blood leukocytes from 10 normal subjects has been studied. At various times after initiation of diffusion chamber culture, cells harvested from the chambers were transferred to agar culture for measurement of CFU-C concentration. Under these conditions colonies could be grown successfully in agar culture provided pronase, necessary for the chamber harvesting procedure, was first removed by careful washing. A marked increase in the number of CFU-C, up to 25-fold the initial value, was observed in 8 out of 10 subjects. Here the growth pattern was similar, independent of the initial CFU-C throughout the diffusion chamber culture period was very poor. The growth of CFU-C from a given individual's blood was shown to be reproducible in repeated studies in 2 subjects, one of whom showed a proliferative and the other a non-proliferative pattern. Evidence suggests that the increase in CFU-C in diffusion chambers is the result of both self-renewal of these cells and influx from a more primitive compartment, although the present data do not allow an estimate of the relative magnitude of each.

Evidence for stem cell function of resting bone marrow lymphocytes identified by the complete 3H-thymidine labelling method.

Resting bone marrow lymphocytes, recongised as small lymphocytes by light microscopy, were labelled by the complete 3H-thymidine labelling technique, enriched by fractionation on a discontinuous albumin gradient and investigated for their stem cell properties by culture in diffusion chambers. Fraction 3, with the highest enrichment of labelled small lymphocytes and slight enrichment of other labelled cells (reticulum and endothelial cells), produced substantial growth, in contrast to fraction 4, with no enrichment of these cells. The number of labelled small lymphocytes per chamber in fraction 3 remained constant or tended to increase. This is assumed to be an indication of some degree of self-replication in the small lymphocyte population. Since, however, their labelling intensity decreased only slowly, it must be further concluded that part of the labelled small lymphocyte population probably remained resting. Some labelled transitional and blast cells appeared before the development of recognisable myelopoietic and erythropoietic precursors and of megakaryocytes, in agreement with the concept that small lymphocytes transfrom to transitional cells during the developmetn of normal haempopiesis.

Regulation of normal hemopoiesis in the acute leukemia L5222.

Normal hemopoiesis becomes markedly depressed in rats during the development of the rat leukemia L5222. Whether this could be due to the influence of a circulating humoral factor was investigated by comparing the growth of normal bone marrow cells in diffusion chambers implanted into the peritoneal cavity of normal or leukemic hosts. Similar growth in total cell numbers was observed in both groups of hosts, and no significant difference could be detected between them. In an attempt to exclude the possibility that an inhibitor either did not penetrate to the peritoneum or was inactivated by the chamber membrane, normal serum, or serum from a highly leukemic rat was mixed with the bone marrow cell suspension in the chambers. Again, no difference in growth between the 2 groups could be detected. Therefore, the influence of a circulating humoral factor in the depression of normal bone marrow hemopoiesis in this experimental leukemia seems to be ruled out, and the decrease may be attributable to local events in the bone marrow such as short-range factors or cellular interaction between the leukemic and normal hemopoietic cell populations.

Reproducibility of survival time in L 5222 rat leukaemia and its implications for chemotherapeutic tests.

The reproducibility of survival time in the L 5222 leukemia in rats was tested by transfer of varying numbers of untreated and X-irradiated cells. A linear relationship between log cell dose and survival time was established for the range of unirradiated cell doses between 10-8 to "10-0", resulting in survival times from 6 to 16 days, with very little variation between individual animals of each group. This narrow deviation of survival time makes it possible to use mean survival time as a measuring parameter instead of the cell dose required to kill 50 percent of recipient animals. From the longer survival times observed after transfer of a given number of X-irradiated cells, the number of viable cells transferred in the inoculum could be calculated and thus the degree of cell death due to X-radiation. Agains a correlation between log cell dose and survival time was found but host survival was prolonged when small numbers (similar to 10-2) of viable irradiated cells were transferred. It is suggested that this is not due to a change in proliferation kinetics but rather to host factors, such as an immunological reaction. Thus the L 5222 leukaemia seems to be a good model in its reproducibility of survival time, and may have some similarity to human acute leukaemia when low numbers of treated cells are concerned.