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. In contrast, the presence of essentially noncycling CFU-s found in cultures fed 8-10 days previously correlates with high levels of the hemopoietic stem cell inhibitor fraction IV. These results suggest that a certain balance between these factors determines CFU-s proliferation in the long-term cultures. In support of this, DNA synthesis in actively cycling CFU-s in the long-term cultures is inhibited for at least 3 days by the addition of excess NBME fraction IV (inhibitor). Furthermore, DNA synthesis in noncycling cultured CFU-s is stimulated for at least 5 days by the addition of RBME fraction III (stimulator).

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. Analysis of the interactions between environmental and haemopoietic cells has been facilitated by the development of vitro systems reproducing the naturally occurring genetic environmental defects and other systems where the development of a competent inductive environment shows a dependency upon corticosteroid hormones. Investigations have shown that stem cell proliferation may be controlled by production of opposing activities, one stimulatory for DNA synthesis, the other inhibitory. A model is proposed whereby modulation in the production of these factors is determined by the physical presence of stem cells in a proposed cellular milieu, within the adherent layer. The adherent layer, apart from acting at the level of stem cell proliferation, can also modify the response of differentiating cells (eg, GM-CFC) to exogenous stimulatory activities. Addition of GM-CSF or of CSF-antiserum has no effect on haemopoiesis in long-term cultures.

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. In addition, a long-term bone marrow culture system has been shown to provide an in vitro model of stem cell control. Fractionation of cell populations from haemopoietic tissues reveals marked concentration differences of the CFU-S proliferation modifying activities depending on the proliferative state of the CFU-S. However, irrespective of whether the tissue contains stem cells that are actively or minimally proliferating, both stimulatory and inhibitory activities are detected. From dose-response studies it is concluded that stem cell proliferation is controlled by an appropriate balance of stimulatory and inhibitory factors which, however, are not produced by the stem cells themselves.

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. After the initial cell loss,the net growth of CFU-S was inversely related to both total cell number and CFU-S number in DC. Diffusible factors rather than cell-to-cell contact appeared to be involved in the inhibition, even though we were not able to demonstrate inhibition of net growth of CFU-S with the double chamber approach.

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. One day after re-feeding the cultures (by removal of half the growth medium and addition of fresh medium) both the "non-adherent" and the "adherent" CFU-S are in a high cycling state (greater than 40% kill with 3HTdR). This proportion decreases with time of re-feeding and 5-7 days later the majority of "adherent" and "non-adherent" CFU-S are in a low cycling state ( less than 10% 3HTdR kill). Following a further re-feeding, CFU-S again enter a high cycling state.

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. Other circulating blood cell counts were not diminished after platelet homogenates, and three intravenous doses of homogenates prepared from 0.7 to 1 X 10(5) syngeneic buffy coat cells failed to influence circulating platelet count or 75 selenomethionine incorporation.