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PubMed1976

Fetal hemopoiesis in diffusion chamber cultures: II. Cell proliferation and differentiation.

The growth and differenciation of hemopoietic cells from murine fetal liver (FL) and adult bone marrow (ABM) in diffusion chambers (DC) implanted into normal CF1 mice were evaluated. Initially FL suspensions were approximately 90% erythrocytic, but after 4 days in DC implanted into either normal or phenylhydrazine (anemic) pretreated hosts, growth was essentially restricted to the granulocyte-macrophage cell types. The number of in vitro colony forming cells (CFC), as assayed in a double layer soft-agar technique was determined after varying periods of growth in DC of both ABM and FL. Both groups showed a progressive decline in CFC number. FL and ABM CFC generated the same proportion of different morphologic types of soft agar colonies. After 7 days of DC culture, there was a decrease in the proportion of macrophage colonies from both groupes. During DC culture the ratio of clusters (3-50 cells to colonies progressively increased in both groups suggesting a parent-progeny relationship between CFC and cluster forming cells. The results of these studies provide further evidence that the environment rather than properties intrinsic to murine stem cell determines the pattern of proliferation.

Animals↗
PubMed1989

Leucopoiesis versus concentration of cytokines in diffusion chamber cultures of mouse bone marrow cells: clues to the physiological roles of growth factors.

Physiological mechanisms that regulate formation of neutrophil granulocytes, macrophages and their precursor cells were studied with the diffusion chamber (DC) technique. DC inoculated with mouse bone marrow cells were implanted intraperitoneally into host mice. When these in vivo cultures had been established and their marrow populations were expanding (2-day cultures), the DC were transferred to different environments: new, normal mice, lethally irradiated mice, or incubation flasks with optimal concentrations of growth factors. Culture development during the following final culture period was correlated to the concentration of some select candidate growth regulators in DC. After 3 d the cellularity of DC in irradiated hosts had increased significantly more than in the normal hosts. A difference was detectable already after 1-2 d when preculturing was omitted. The increased growth appeared to take place at several stages of cell maturation and not only at the progenitor cell level. Colony stimulating factor(s) for granulocyte and macrophage progenitors, as well as cytokines stimulating the bone marrow-derived cell line, 14 M.1, were present in DC fluid (DCF) at higher concentrations in irradiated than in normal mice throughout the final culture period. On the other hand, DCF concentrations of tumour necrosis-like factor (that may either induce CSF secretion or directly inhibit myelopoiesis), were not significantly different in irradiated compared with normal DC hosts. The cytokines detected in the DC may at least in part stem from inflammatory cells accumulating around the chambers. This animal model should be useful in further investigation of the highly complex regulatory network governing formation of white blood cells in the intact organism.

Animals↗
PubMed1984

Growth kinetics of CFU-GEMM, BFU-E, and CFU-C in diffusion-chamber cultures of normal human bone marrow cells.

The proliferation and differentiation of human pluripotent hemopoietic precursor cells CFU-GEMM as well as of the committed precursors BFU-E and CFU-C within conventional diffusion chambers (DC) was studied. Low-density human bone marrow cells from eight normal donors were cultured in DC implanted into 750-rad-preirradiated mice. Cells harvested from DC after 1-14 days were assayed for the content of CFU-GEMM, BFU-E, and CFU-C. The mean number of progenitor cells inoculated per DC on day 0 was 59 (range 12-144) for CFU-GEMM, 367 (range 165-878) for BFU-E, and 632 (range 122-1168) for CFU-C. After an initial decrease in the number of all progenitors, an increase of CFU-GEMM was observed in five out of eight experiments and in the number of BFU-E and CFU-C in seven and five experiments, respectively. The assessment of the percentage of precursor cells sensitive to ara-C demonstrated a rise in the proportion of cells in S phase after implantation into DC consistent with reentry into active proliferation. From the observed behavior of the CFU-GEMM it is concluded that the earliest detectable human precursor cells do proliferate within DC.

Bone Marrow Cells↗
PubMed1999

Ectopic haematopoiesis in the mouse: roles of stroma and cytokines in granulocytopoiesis in in vivo diffusion chamber cultures.

We have examined a possible role of two different types of irradiated stromal cells, i.e. murine bone marrow (BM) stromal cells and stromal cell line MS-5R, when cocultured with murine blood-borne progenitors or sorted Lin- Sca-1+ bone marrow cells in vivo in peritoneal diffusion chambers (DC). Retrieval and quantification of the cultured cells were performed after 4, 7, and 14 d. Granulocyte and/or macrophage colony-forming cells (G/M-CFC) were enumerated in subcultures from the DC. G/M-CFC production was not enhanced in the stroma-contact cultures, in comparison with the standard stroma-non-contact cultures, but early granulocytopoiesis was stimulated. Perturbation of the humoral environment of DC was investigated in a number of ways, for example with continuous infusion of rhG-CSF from a subcutaneous implanted minipump to DC host mice, with DC host mice carrying a transplantable leukaemia, secreting interleukin 3 (IL-3), and with injections of various cytokines. None of these interventions sustained the expansion of the G/M-CFC population. In conclusion, for ectopic haematopoiesis to take place, several requirements must be met. Relevant stromal cells apparently affect haematopoiesis both via direct cell-cell interactions and via humoral mediators (viz. cytokines) which they secrete.

Animals↗
PubMed1987

B lymphocyte precursors and myeloid progenitors survive in diffusion chamber cultures but B cell differentiation requires close association with stromal cells.

The aim of this study was to investigate the relative contribution of direct contact with stromal cells v stromal cell-derived soluble mediators to the differentiation of B lymphocytes and cells from other hematopoietic lineages. This was investigated by making a comparison between hemopoietic cells grown in direct contact with stroma to those in diffusion chambers (DCs) placed over purified populations of stroma. The source of stromal cells was adherent layers from myeloid or lymphoid long-term bone marrow cultures that had been treated with mycophenolic acid, an antibiotic that depletes hemopoietic cells from the cultures but retains a functional stroma. The cells seeded into the chambers were fresh marrow cells that had been passed through two consecutive nylon wool columns to deplete cell populations capable of forming an adherent cell layer in vitro. DCs were placed in wells in which the adherent stroma, growing under myeloid or lymphoid conditions, was present. The results indicate that progenitors of granulocytes and macrophages survived and differentiated in DCs under myeloid culture conditions, as the number of cells and absolute number of CFU-GM increased over that present in the reseed population. These levels, however, were markedly less than in parallel cultures in which the cells were seeded directly onto stroma. Hematopoiesis in DCs placed over hemopoietically active stroma was not optimal, suggesting that factors were used by those hemopoietic cells closest to the stroma. A B lymphocyte precursor survived in DCs under myeloid but not lymphoid conditions, and its differentiation into B lymphocytes was dependent on close association with stromal cells; B lymphopoiesis initiated when cells from DCs grown under myeloid conditions were harvested from the chambers and seeded directly onto stroma initiated and maintained under lymphoid bone marrow culture conditions. B lymphopoiesis did not initiate if the DC from the myeloid conditions was left intact and placed directly over a lymphoid stromal cell layer in lymphoid conditions.

Animals↗