Determination of the probability of self-renewal in haemopoietic stem cells: a puzzle.

This paper calls attention to anomalous behaviour of the spleen colony forming cell system, when quantitative measurements of the self-renewal probability of the CFU-S are attempted.

Tissues and stem cells.

This brief overview of the different cell populations types contained in the various tissues of the adult organisms, with special reference to cell population hierarchies and kinetic patterns, indicates that not all cell populations have--nor do they need--stem cells; in those which have, the stem cells usually represent a minority subpopulation.

Which are the leukaemic cells?

Two features are known about acute myeloid leukaemia in man: (1) the long time-scale from identifiable leukaemogenic stimulus and onset of the clinical disease and (2) the successful induction and duration of long clinical remission. These indicate three probabilities: First, that the target cell for leukaemogenetic insult (in AML) is the pluripotent stem cell; second, that the leukaemic stem line is a small minority population within the total leukaemic cell mass; third, when the leukaemic stem line is not greatly exceeding the normal stem cell numbers, its proliferation may be still under partial control.

The regulation of hemopoiesis in long-term bone marrow cultures. II. Stimulation and inhibition of stem cell proliferation.

The isolation of a DNA synthesis inhibitor (NBME fraction IV) and stimulator (RBME fraction III) specific for the hemopoietic stem cell (CFU-s) from freshly isolated normal adult and regenerating murine bone marrow, respectively, has been well documented. We have utilized long-term liquid bone marrow cultures in a further analysis of the role of these factors in the regulation of CFU-s proliferation. Our results show that shortly after feeding, at a time when the cultured CFU-s are actively proliferating, high levels of the hemopoietic stem cell proliferation stimulator fraction III can be isolated from the culture medium.

The role of cells and their products in the regulation of in vitro stem cell proliferation and granulocyte development.

In long-term marrow cultures haemopoiesis can be maintained in vitro for up to 6 months. Critical analysis of the cell populations produced has shown that the stem cells and their committed progeny have characteristics in common with the corresponding cell types in vivo. The maintenance of haemopoiesis in vitro is associated with the development of an appropriate inductive environment provided by bone marrow derived adherent cells.

Strategic reserves.

New methodologies--particularly the combination of flow cytometry and the use of monoclonal antibodies--as adjuncts to existing techniques for the analysis of the various subpopulations of haemopoietic cells are becoming available. These enable a significant advance in understanding and monitoring haemopoietic mechanisms, both in normal and in abnormal states. With this help, a functional measurement of the proliferation and differentiating capacity of haemopoietic tissues is becoming possible.

Mechanisms of haemopoietic stem cell proliferation control.

The control of stem cell (CFU-S) proliferation is mediated by short-range acting factors which can be detected by the proliferation modifying activities present in media conditioned by haemopoietic cells. A specific inhibitor of stem cell proliferation is obtained from haemopoietic tissue containing minimally proliferating CFU-S, whilst stimulatory material is obtained from cell suspensions containing rapidly proliferating CFU-S. Used competitively, these factors, which are detected in different molecular weight range fractions, manipulate the rate of CFU-S proliferation in a manner compatible with a physiological control mechanism.

Stimulation of platelet production in vivo by a factor in cell culture conditioned medium.

Injection of medium conditioned by a murine myelomonocytic leukaemic cell-line (WEHI-CM) stimulates platelet production in irradiated, bone marrow reconstituted mice. Media conditioned by the growth of normal bone marrow cells (BM-CM) or by a lymphoid leukaemia cell line had no effect on platelet production. However, the effect of WEHI-CM on platelet production was further enhanced when injected along with BM-CM, indicating that more than one factor may play a role in the regulation of megakaryocytopoiesis.

Inhibition of haemopoietic stem cell proliferation by a diffusible product of bone marrow cells.

In a double diffusion chamber (DC) culture system bone marrow cells elaborated diffusible factor(s) that prevented spleen colony-forming cells (CFU-S), but not PHA stimulated lymphocytes, from entering cell cycle. Mature granulocytes and macrophages did not produce such factors(s). Various number of steady-state or regenerating mouse bone marrow cells were cultured in single diffusion chamber for periods up to 7 d.

Regulation of haemopoietic stem cell proliferation in long term bone marrow cultures.

The development of a suitable bone marrow derived adherent cell population appears to be essential for the prolonged maintenance of haemopoietic stem cells in vitro. When established adherent layers are inoculated with freshly isolated bone marrow cells, proliferation of stem cells (CFU-S) regularly occurs both in the adherent layer and amongst the non-adherent cells. Furthermore, CFU-S present within the adherent layer are able to regenerate both themselves and the "non-adherent" CFU-S.

Specific thrombopoietic inhibition by syngeneic platelet homogenates.

Injections of 1 to 2.5 X 10(8) syngeneic, uninjured platelets did not diminish the circulating platelet count nor the bone marrow megakaryocyte content in mice, and did not influence the incorporation of 75selenomethionine into circulating platelets. However, platelet homogenates, prepared by repeated freezing and thawing of identical amounts of syngeneic paltelets induced a dose-dependent thrombocytopenia along with a diminution on bone marrow megakaryocyte content, and a decrease in 75 selenomethionine incorporation.

Conditions controlling the proliferation of haemopoietic stem cells in vitro.

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S), production of granulocyte precursor cells (CFU-C), and extensive granulopoiesis can be maintained in vetro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells, "epithelial" cells, and "giant fat" cells.

The effects of red blood cell extracts on the proliferation of erythrocyte precursor cells, in vivo.

Saline incubation extracts of mature erythrocytes were assayed in vivo by a variety of techniques in order to study their ability to modify the proliferation of maturing erythroid cells. Using comparable extracts from granulocytes and lymphocytes, the specificity of the effect of the red cell extract for erythroid cells was confirmed by measurement of autoradiographic labelling indices, radio-iron incorporation and spleen colony growth. The erythroid cells were found to be very sensitive to the effects of the extract, as little as 10 microgram per mouse producing a maximum effect on iron incorporation.

Bone marrow culture studies in refractory cytopenia and 'smouldering leukaemia'.

As an adjunct to conventional haematological and cytogenetic data, 22 cases of refractory cytopenia, and five with chronic myelomonocytic leukaemia, (CMML) were studied by bone marrow culture. Cultures from II such patients without an excess of marrow myeloblasts usually showed low, or undetectable, numbers of cells capable of giving rise to colonies of granulocytes and/or macrophages (CFUc) but near-normal numbers of cluster-forming cells and cells capable of forming erythroid colonies (CFUE). Those with similar blood pictures, but in whom the marrow contained a slight excess of myeloblasts (II cases), showed a more profound defect in growth patterns: low or undetectable numbers of CFUC, clusters and CFUE, results similar to those found in acute myeloblastic leukaemia, into which three of this group evolved.

Inhibitor of stem cell proliferation in normal bone marrow.

A saline extract from normal bone marrow cells having a molecular weight in the range 50000-1000000 daltons has been found to protect rapidly proliferating haemopoietic spleen colony forming cells (CFUs) from the lethal effects of large doses of tritiated thymidine. This extract is non-toxic to the cells. It is not found in regenerating marrow where the CFUs population is rapidly proliferating.