Individual clones of hemopoietic cells in murine long-term bone marrow culture

Forty-seven individual hemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation clones appeared at different times, from 1 to 12 weeks after explantation, survived during 1-10 more weeks, and were characterized by marked variability in size. Usually, the number of metaphases peculiar to an individual clone rapidly increased, achieved maximum, and then underwent a decline. Cells of reliably disappearing clones were never seen again. The experimental results provide further evidence for the model of hemopoiesis by clonal succession.     

Early disappearance of murine plasmocytoma stem cells in long-term bone marrow culture.

Long-term bone marrow culture (LTBMC) was evaluated as a purging procedure in the murine plasmocytoma MOPC-315s system. MOPC-315s cells injected in Balb-c mice rapidly proliferate both in marrow and spleen, where macroscopic tumor colonies develop. A linear relationship between the number of injected cells and spleen colonies was observed, consistent with the presence of 1 clonogenic myeloma stem cell out of 1800 cells. In vitro, MOPC-315s cells are easily identifiable as rosette-forming cells (RFC+) with trinitrophenil acid (TNP) coated sheep red blood cells. When bone marrow (BM) cells containing 20-40% RFC+ were seeded in LTBMC, RFC+ rapidly decreased and were no longer detectable by day 14 of culture. Clonal Ig gene rearrangement was evident at time 0, but it was no more detectable later on. In addition, cells taken at days 14 and 21 of culture were no more tumorigenic when injected in vivo. The results suggest the efficacy of the LTBMC for the in vitro elimination of myeloma cells, including the neoplastic stem cells.     

Differentiation induction of HL60 cells in a long-term bone marrow culture of acute myeloid leukemia

Bone marrow stromal cells are critical for the proliferation and differentiation of hemopoietic stem cells. The hemopoietic microenvironment is reproduced in long term bone marrow culture (LTBMC). Normal LTBMC versus leukemic LTBMC and their stroma conditioned medium were compared with respect to their proliferative and differentiation-inducing capacities. Myeloid leukemic cells (HL60) were layered onto LTBMCs derived from normal volunteers and patients with AML. Differentiation was measured with a comprehensive panel of maturation parameters, i.e. morphology, cytochemistry, quantitative enzyme determination, NBT test and immunophenotyping. Inhibition of proliferation occurred in all cocultures. Clear maturation in monocytic direction was obvious in one culture of HL60 cells layered onto a leukemic stroma. As stroma-derived conditioned medium has no effect, a cellular interaction seems involved. These observations support not only the concept that normal stroma influences leukemic cell growth but also that leukemic stroma can modulate cell growth and maturation.     

Behaviour of heat-treated bone marrow cells in long-term bone marrow cultures

The effect of prior heat treatment on the ability of bone marrow cells to form long-term bone marrow culture has been studied. Bone marrow cells were heated for various times in the temperature range of 39-43 C and then cultured in the modified Dexter type suspension culture. At weekly intervals, the behaviour of the cultures in terms of stroma formation and confluency, cellular viability, and myelopoiesis were evaluated. The results show that there was a dose-dependent decrease in the number of viable cells in the non-adherent fraction of the cultures. Cytological analysis of these cells showed a strong shift towards macrophage population in the successive weeks of the cultures and also as a function of heat dose delivered to these cultures. The stroma formation was delayed or inhibited as a function of the heat dose. The number of granulocyte-macrophage colony forming cells (CFU-GM) in both adherent and non-adherent fraction of the cultures were decreased substantially after hyperthermia treatment. At 41 C and higher temperatures, the CFU-GM were severely diminished in both fractions. The dose response experiments showed that the decrease in the number of CFU-GM was dependent on the heat dose. The results suggest that CFU-GM is an extremely sensitive target in the hyperthermia treatment of bone marrow cells and heat-treated bone marrow cells lose their ability to maintain long-term cultures.     

Behaviour of heat-treated bone marrow cells in long-term bone marrow cultures.

The effect of prior heat treatment on the ability of bone marrow cells to form long-term bone marrow culture has been studied. Bone marrow cells were heated for various times in the temperature range of 39-43 degrees C and then cultured in the modified Dexter type suspension culture. At weekly intervals, the behaviour of the cultures in terms of stroma formation and confluency, cellular viability, and myelopoiesis were evaluated. The results show that there was a dose-dependent decrease in the number of viable cells in the non-adherent fraction of the cultures. Cytological analysis of these cells showed a strong shift towards macrophage population in the successive weeks of the cultures and also as a function of heat dose delivered to these cultures. The stroma formation was delayed or inhibited as a function of the heat dose. The number of granulocyte-macrophage colony forming cells (CFU-GM) in both adherent and non-adherent fraction of the cultures were decreased substantially after hyperthermia treatment. At 41 degrees C and higher temperatures, the CFU-GM were severely diminished in both fractions. The dose response experiments showed that the decrease in the number of CFU-GM was dependent on the heat dose. The results suggest that CFU-GM is an extremely sensitive target in the hyperthermia treatment of bone marrow cells and heat-treated bone marrow cells lose their ability to maintain long-term cultures.     

Toxic effect of polyriboinosinic-polyribocytidylic acid on mouse hemopoietic stem cells

Within a few hours following a single intraperitoneal injection of polyriboinosinic-polyribocytidylic acid (poly I:poly C) into C3H/He mice, numbers of bone-marrow and spleen-colony-forming cells are significantly decreased. The dose-response curve obtained shows an exponential decrease of the surviving CFU with increasing amounts of poly I:poly C, up to 200 μg; with higher doses of poly I:poly C, no further decrease of CFU is observed. This suggests that poly I:poly C kills colony-forming cells in replicating phase, and is devoid of activity on cells in resting phase. The lack of additive toxic effects observed when poly I:poly C and vinblastine sulfate are administred simultaneously is consistent with this hypothesis. The cytotoxic effect of the polynucleotide is related to its double-strandedness and is not mediated by interferon present in the serum.     

Colony promoting activity (CPA) produced in long-term culture of murine bone marrow cells

The differences between colony promoting activity(CPA) and colony stimulating activity(CSA) in the culture media of murine long-term bone marrow cultures(LTBMC) were demonstrated and the role of adherent cells and nonadherent cells in the production of CPA was studied in this culture system. Supernatant harvested from intact continuous marrow cultures showed high CPA but contained no CSA. Assayable CSA was detected in concentrated supernatant. However, there was no significant relationship between levels of CPA and CSA in the supernatant. When adherent cells and nonadherent cells from LTBMC were separately cultured, CPA was detected in the conditioned medium of adherent cells but not in that of nonadherent cells. The CPA level in LTBMC was related inversely to the number of nonadherent cells and addition of nonadherent cells to adherent cell cultures reduced the level of CPA. Conditioned medium of nonadherent cells showed no inhibitory activity to CPA. These results indicate that CPA is produced by bone marrow adherent cells and that it may be consumed by myeloid progenitor cells in nonadherent cells.     

Growth of normal hemopoietic cells in cultures of bone marrow from leukemic mice

Bone marrow from mice with spontaneous leukemia was studied in long-term culture in the presence of 10⁻⁷M hydrocortisone. These conditions resulted in a complete loss of leukemia cells from marrows which initially showed 95% or greater replacement with lymphoblasts. The culture conditions were also found to favor the growth of hemopoietic stem cells and the surviving cultured cells produced sufficient numbers of these cells to protect lethally irradiated (950 rads) synegeneic mice. The irradiated recipients of the cultured marrow did not develop leukemia during the 180 days of observation post-irradiation, indicating the absence of leukemia cells in the inocula. A loss of leukemia cells in vitro occurred in cultures with and without the addition of hydrocortisone to the medium. Hydrocortisone was shown to have a stimulating effect on maintenance of hemopoietic stem cells, granulocyte progenitors and granulopoiesis in these cultures.     

Radiobiological features of human pluripotent bone marrow progenitor cells (CFU-GEMM)

The purpose of this study was to evaluate the radiobiologic features of human pluripotent bone marrow progenitor cells (CFU-GEMM; colony forming unit-granulocyte-erythroid-macrophage/monocyte-megakaryocyte). Experiments were performed using fresh bone marrow cells as well as bone marrow cells stimulated with recombinant granulocyte-macrophage-colony stimulating factor (rGM-CSF) to increase the CFU-GEMM pool. The D0 values for CFU-GEMM in normal bone marrow samples (n = 9) ranged from 30.9 cGy to 85.7 cGy (mean +/- SE = 54.4 cGy +/- 6.2 cGy) and the D0 value of the composite radiation survival curve was 56.9 cGy, indicating that CFU-GEMM were acutely sensitive to the lethal effects of ionizing radiation. There was no distinct shoulder on the single dose radiation survival curves with Dq values ranging from -29.6 cGy to 4.4 cGy, and no increase in CFU-GEMM survival was observed when the radiation was fractionated. Hence, CFU-GEMM were unable to repair sublethal radiation damage. These findings confirm and extend previous studies on the radiobiologic features of human hematopoietic progenitor cell populations.     

Cells responsible for restoration of haemopoiesis in long-term murine bone marrow culture

Long-term haemopoiesis in bone marrow culture is sustained by the progeny of haemopoietic progenitor cells (HPC), which differ from CFUs by very low sensitivity to repeated hydroxyurea (HU) injections. The transit time of a haemopoietic clone from HPC to cells proliferating in culture is 6-7 weeks. The results suggest that the stem cell continuum is an expansion type compartment, members of which gradually lose their proliferative potential during differentiation.     

The effects of alpha interferon on human long-term bone marrow culture

The effect of alpha IFN on normal long term bone marrow culture (LTBMC) was assessed by measuring haemopoietic progenitor formation and stromal cell number and composition over the course of 5 weeks. When alpha IFN was added at the initiation of LTBMC, there was a marked inhibition of CFU-GEMM, BFU-E and CFU-GM formation from both adherent and non-adherent compartments of culture. There was a profound inhibition of stromal layer formation, especially the reticulo-fibroblast component, and this was not a result of TNF release from macrophages. When alpha IFN was added to established normal LTBMC, although haemopoietic progenitor formation was inhibited, there was little effect on the stromal layer in terms of number or composition. This suggests that the cytostatic effects of alpha IFN when added at commencement of LTBMC result from the anti-proliferative effects of alpha IFN on these actively dividing cells. It is concluded that in addition to the established inhibitory effects of alpha IFN on haemopoietic progenitors as previously demonstrated in semi-solid culture systems, alpha IFN has profound effects upon the marrow microenvironment. This is of particular relevance to bone marrow transplantation where such cytokines may be considered for clinical use.     

Myelopoiesis following phorbol ester exposure in human long-term bone marrow cell culture

Phorbol esters have two opposing effects on bone marrow granulocyte/macrophage colony forming cells (CFC-GM): they reduce the number of CFC-GM which respond to colony-stimulating activity (CSA), and they also induce the release of CSA from accessory marrow cells. We questioned whether the direct inhibitory effect of phorbol esters on CFC-GM was reversible and whether phorbol ester-treated accessory bone marrow cells could increase the in vitro recovery of cultured CFC-GM. Normal human bone marrow cells were exposed to phorbol-12,13-dibutyrate (PDB) for 5 days in liquid cultures. Total nucleated cell counts decreased in a dose-dependent fashion to 42 ± 6% of control at 5 × 10^?8 M PDB, and CFC-GM decreased to 20 ± 8% of control. The cultures were then washed to remove PDB and continued for up to 8 weeks. Total cell counts and CFC-GM/ml remained reduced in PDB-pretreated cultures compared to control. To determine whether this was a direct effect of PDB on nonadherent, proliferating cells or whether PDB acted indirectly by affecting adherent, accessory cells, nonadherent bone marrow cells from control and PDB-preincubated cultures were co-cultured for 8 weeks with either control or PDB-preincubated adherent bone marrow cells. Nonadherent cells preincubated with PDB had a similar growth pattern whether or not the co-cultured adherent cells had been preincubated with PDB. In contrast, nonadherent cells preincubated in control medium and co-cultured with PDB-preincubated adherent cells displayed increased numbers of nucleated cells, CFC-GM and adherent hematopoietic islands compared to control. This was associated with increased amounts of CSA present in the culture supernatants. Thus PDB causes an irreversible decrease in CFC-GM in long term marrow cultures but enhances the ability of adherent stromal cells to support normal CFC-GM growth in culture.     

Pool size of pluripotential hematopoietic stem cells increased in continuous bone marrow culture by Friend spleen focus-forming virus

Continuous mouse bone marrow cultures were infected with Friend murine leukemia virus. Production of nonadherent (NA) and adherent cells, granulocyte-macrophage colony-forming unit(s) of progenitor cells (GM-CFUc), pluripotential hematopoietic stem cells (CFUs), the self-renewal potential (Rs) of CFUs, and generation of factor-dependent (FD) multipotential and committed permanent stem cell cloned lines were measured. Uninfected marrow cultures from C57BL/6J, C57BL/6JUt, B6.S, C3H/HeJ, (C57BL/6J x DBA/2J)F1, CD- 1 Swiss, or N:NIH(S) mice generated NA cells, GM-CFUc, and CFUs for 20-41 weeks; cultures infected with Rauscher or other helper viruses generated them for 35-45 weeks. GM-CFUc and CFUs production in SFFV-positive cultures persisted for over 65 weeks and exceeded control levels by twentyfold to fiftyfold. The Rs of CFUs in SFFV-positive cultures was not detectably increased above control cultures. Multipotential (erythroid-neutrophil-mast cell-basophil-eosinophil) permanent FD cell clones were derived from control and SFFV-positive cultures. Thus SFFV amplifies the stem cell pool in vitro without detectably increasing the Rs capacity of CFUs.     

Role of stromal and hematopoietic stem cells in Friend spleen focus forming virus effects in continuous bone marrow culture

Replication of the Friend spleen focus forming virus (SFFV) in C3H/HeJ or C57BL/6J mouse continuous bone marrow cultures is associated with an increased cumulative production of pluripotential/hematopoietic stem cells (CFUs), granulocyte-macrophage progenitor cells (GM-CFUc), and total granulocytes, compared to uninfected or helper virus infected control cultures. The site(s) of action of the virus are not known. To determine whether viral effect(s) occurred in adherent stromal and/or non-adherent hematopoietic stem cells, purified cell populations, comprised exclusively of cells from each compartment, were separated from C57BL/6J (Fv-1^bb) or C3H/HeJ (Fv-1ⁿⁿ) marrow cultures and were left uninfected or were infected with host range replication restricted B-tropic or N-tropic helper pseudotype viruses of SFFV respectively. Cell populations were then mixed to establish continuous hematopoiesis in allogeneic or syngeneic combinations. Virus host range restriction between compartments was maintained in allogeneic reconstituted cultures which showed active hematopoiesis for 16–17 weeks and no significant differences between: (1) virus infected and uninfected groups or (2) groups comprised of stromal cells from one or the other strain. Thus, these data indicate that the prolongation of hematopoiesis in undisturbed long-term marrow cultures by SFFV occurs through an interaction of adherent hematopoietic stem cells with the marrow stroma.     

Recovery of blood and bone marrow stem cells following intense chemotherapy and autologous bone marrow transplantation

Sixteen patients with advanced (stage III) malignant melanoma were treated with escalating doses of intravenous BCNU and melphalan starting at 400 and 35 mg/m2, respectively, and escalating to 1,000 and 110 mg/m2, respectively, combined with autologous marrow transplantation. The duration of granulocytopenia and time to granulocyte recovery was similar in all groups regardless of chemotherapy dose. Platelet recovery was delayed in patients receiving the highest doses of chemotherapy. This study showed that bone marrow colony-forming units in culture took as long as 6 months to recover. This was adequate to bring peripheral blood counts to normal but not to pretreatment levels. These studies indicate that autologous bone marrow transplantation is beneficial in enhancing short-term recovery, but may not be beneficial in the long-term hematopoietic recovery.     

Some effects of chemotherapeutic drugs on bone marrow stem cells

Summary The non-Hodgkin lymphoma chemotherapy protocol used at the Gustave-Roussy Institute was adapted, in terms of drug doses and interval between doses, to normal CBA mice. The numbers of pluripotential stem cells (CFU-S), unipotential stem cells (CFC), differentiated bone marrow cells, and circulating white cells were determined.Eight hours after each drug of the first chemotherapy cycle the number of pluripotent stem cells decreased while the proportion of these cells in DNA synthesis increased.Six hours after the end of each complete cycle, the stem cell compartments were found to be considerably depleted, and they were not completely restored when the next cycle was begun, while the other hematologic compartments were completely restored at this time.