Origin of stroma cells in long-term bone marrow cultures from patients with acute myeloid leukemia.

Bone marrow stroma cells from patients with acute myeloid leukemia (AML) display a variety of functional abnormalities. In order to determine whether this is related to an imbalance in the proportion of different stroma cell types or to integration of leukemic progeny into the regulatory cell network, stroma layers were established in mycophenolic acid-treated long-term marrow cultures from 16 patients with AML and 42 controls and analyzed by means of simultaneous membrane immunofluorescence and interphase cytogenetics. Macrophages were identified by CD14 expression, fibroblasts by staining with the AS02 antibody, and malignant cells by leukemia-specific numerical chromosome aberrations, including monosomy 7 and trisomy 8. Compared with normal controls, there was a slight decrease in the proportion of stroma fibroblasts (52+/-27% versus 77+/-5%) in 10-week-old cultures from patients with AML. Two of five AML patients with trisomy 8 and both patients with monosomy 7 had evidence of leukemic stroma cells. Most malignant cells were CD14+ macrophages (3.8-98.1% of all CD14+ cells), but some were AS02+ (2.8-5.2%). AML stroma layers showed a reduced capacity to support the growth of normal hematopoietic cells in standard two-stage long-term cultures, but this was unrelated to the presence or absence of leukemic stroma elements. In conclusion, AML populations vary with respect to their ability to produce a malignant microenvironment. Functional defects in the hematopoietic microenvironment, however, are not limited to AML patients with cytogenetically abnormal stroma cells, but extend to cases without evidence of malignant stroma cells.

Chimaeric cultures of human marrow stroma and murine leukaemia cells: evidence for abnormalities in the haemopoietic microenvironment in myeloid malignancies and other infiltrating marrow disorders.

PGM-1 is a transplantable C3H/HeJ leukaemia which is not viable in unstimulated in vitro culture, differentiates into mature granulocytes and macrophages in response to soluble cytokines, and undergoes self-renewing cell divisions in coculture with selected human bone marrow stromal cell lines. When PGM-1 cells were cultured on pre-established adherent layers from primary human marrow samples, their fate depended on the source of the human marrow. Adherent layers from healthy marrow donors or patients with reactive marrow alterations had no or very little capacity to maintain PGM-1 cells in an immature colony-forming state. However, in coculture with adherent layers from patients with myeloid leukaemia or, to a lesser extent, lymphoblastic leukaemia or marrow-infiltrating lymphoma the colony-forming potential was retained. There was no correlation between the remission status of the patient and the PGM-1 activity of the adherent layer. Consistent morphological differences between active and inactive stromal layers were not observed. The PGM-1 coculture system enables the detection of a hitherto undescribed regulatory abnormality in bone marrow malignancies. Whether the PGM-1 supporting activity is mediated through differences in the production of a cytokine with close homology to complement factor Bb which has recently been shown to induce self-renewal in immature PGM-1 cells, requires further investigation.

Role of mature leukemic cells in the amplification of leukemic stem cells in a murine model.

The recently described PGM-2 leukemia displays a hierarchical structure with bipotential stem cells, B-lymphocyte and macrophage progenitor cells, and post-mitotic end cells. Because the different cell types can easily be identified in vitro by clonal culture assays and simple staining procedures, this leukemia is a useful model for the study of the interactions between different cell compartments in a leukemic clone. Our analysis of the impact of mature leukemic macrophages on the proliferation of stem cells was facilitated by the establishment of long-term cultures producing new stem cells over prolonged periods of time. A prerequisite was the development of an adherent layer of fibroblasts and leukemic macrophages. Enumeration of adherent cells revealed a good correlation between the number of macrophages and the number of stem cells generated, and expansion of the macrophage population by treatment with interleukin 3 (IL-3) resulted in a significant improvement of the culture conditions. Leukemic macrophages were also able to induce the formation of stem-cell colonies in agar culture, suggesting a role for humoral mediators. Antibody neutralization experiments and bioassays identified IL-7 and IL-6 as factors cooperating in the stimulation of stem-cell self-renewal. Feed-back stimulation of leukemic stem cells by mature leukemic cells may also be relevant to human leukemias and have implications for differentiation therapy.

Self-renewal and differentiation of stem cells in a biopotential murine leukemia: an in vitro model for differentiation therapy.

PGM-2 is a variant of the transplantable PGM-1 leukemia of strain C3H/HeJ. Freshly explanted cells had lymphoid morphology with a CD5+ CD45R (B220)- IgM- phenotype. They were not viable in unstimulated cultures, but formed IgM+ lymphoid colonies in response to interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-7, and Steel factor, and macrophage colonies in response to IL-3. IL-3-stimulated colonies had no recloning potential, but colonies from IL-7 cultures gave rise to large numbers of secondary macrophage colonies in IL-3-stimulated cultures and secondary lymphoid colonies in IL-7-stimulated cultures. The latter ones could be serially transferred in vitro for several months, and formed typical PGM-2 tumors in vivo. IL-7-stimulated colonies could therefore be used to measure leukemic stem cells in vitro. Supramaximal IL-3 stimulation (2,500 U/mL) of suspension cultures was followed by an increase in overall cell numbers and a disappearance of leukemic stem cells, compatible with differentiation induction. This could not be counteracted by simultaneous stimulation with IL-7. However, lower IL-3 concentrations (500 U/mL) induced an expansion of the stem cell pool, possibly by facilitating density-dependent autostimulatory mechanisms involving endogenous production of IL-7. The system described is a simple in vitro model for differentiation therapy. It shows that leukemic stem cells can be induced by hematopoietic growth factors to undergo terminal differentiation, but the concentrations required for differentiation induction in stem cells are much higher than those required for other biologic effects. Submaximal stimulation may favor expansion rather than repression of the leukemic cell population.