Bone Marrow Colony-forming Units Research Articles

High-dose chemotherapy shows a dose-dependent toxicity to bone marrow osteoprogenitors: a mechanism for post-bone marrow transplantation osteopenia.

Osteoporosis is a sequela of hemopoietic cell transplantation with a complex multifactorial pathogenesis in which the relative role of chemotherapy and irradiation is not completely understood. Therefore, the authors investigated the toxicity of chemotherapy-only conditioning regimens on bone homeostasis and bone marrow osteoprogenitors, its dose dependency, and the mechanism of chemotherapy-induced osteopenia. Fifty-one patients with high-grade non-Hodgkin lymphoma or breast carcinoma who had been treated previously with high-dose + peripheral blood progenitor cell or conventional chemotherapy or who had not received any treatment (prechemotherapy) were enrolled. The authors measured the bone marrow colony-forming unit fibroblast (CFU-f) and long-term culture-initiating cell frequency, forearm bone mineral density, serum osteotropic hormones and metabolic markers of bone formation (plasma osteocalcin), and resorption (urinary collagen I C-crosslinks). Both high-dose chemotherapy regimens caused a 50% reduction in CFU-f frequency, independently of gonadal function status, whereas conventional chemotherapy and prechemotherapy groups were unaffected. Bone mineral density was measured in 26 non-Hodgkin lymphoma patients and again only high-dose chemotherapy caused a 10% loss in cortical bone and 20% in trabecular bone. No endocrine abnormality was found except for the secondary amenorrhea uniformly induced in the high-dose chemotherapy group. In these patients, plasma osteocalcin unexpectedly failed to increase in response to the menopausal increase in bone resorption rate, showing a selective impairment of the osteoblast compartment to cope with increased functional demand. Chemotherapy without irradiation shows a dose-dependent toxicity to bone marrow stromal osteoprogenitors and can cause osteopenia by direct damage of the osteoblastic compartment, as a mechanism distinct from and summable to hypogonadism.

Influence of age and K, Mg aspartate (Cardilan) on murine haemopoiesis

We have studied the effect of age as well as the effect of short-term and long-term intake of K and Mg salts of aspartic acid (Cardilan) on haemopoiesis in ICR mice strain. The cellularity of the bone marrow does not change with aging, but the number of granulocyte-macrophage colony-forming cells (GM-CFC) and also the number of spleen colony-forming units (CFU-S) and erythroid burst-forming units (BFU-E) in two-year-old mice increased in the bone marrow. In two-year-old mice the number of leukocytes decreased in the peripheral blood with aging, mainly as a result of a decrease in mononuclear cells. Short-term drinking (STD) of Cardilan caused increased numbers of CFU-S and BFU-E in bone marrow and increased numbers of reticulocytes in the peripheral blood of one-year old animals (STD/12 months old). In the oldest mice (STD/24) increased weight and cellularity of the spleen and rapid increase of leukocytes and reticulocytes in the peripheral blood was recorded. After long-term drinking (LTD) of Cardilan the number of spleen GM-CFC rose markedly in one-year-old mice (LTD/12) and in two-year-old mice (LTD/24) the number of reticulocytes in the peripheral blood rose. Our results indicate that K and Mg salts of aspartic acid influence erythropoietic activity most widely.

Erythroid defects and increased retrovirally-induced tumor formation in Evi1 transgenic mice.

Aberrant expression of the Evi1 (ecotropic virus integration site 1) proto-oncogene has been associated with hematopoietic malignancies in both mice and man. To determine the effect of enforced expression of Evi1 in vivo, we developed a transgenic mouse model utilizing the murine Sca-1 (Ly-6E.1) promoter. Here, we describe the generation and analysis of three independent lines of Evi1 transgenic mice. Transgenic animals of two founder lines developed normally. These mice did not show any obvious hematological abnormalities but showed a significant reduction in the number of bone marrow colony-forming unit erythroid (CFU-E)-derived colonies. This implies a defect of normal erythroid hematopoiesis affecting relatively late erythroid progenitor cells. We also show that when newborn Evi1 transgenic mice of these two lines were infected with Cas-Br-M MuLV, tumor incidence was greatly enhanced in comparison with nontransgenic littermates, indicating an increased susceptibility for leukemia development. Interestingly, analysis of a third founder line revealed that all male progeny consistently displayed severely impaired erythropoiesis with major defects in the bone marrow, spleen and peripheral blood. Taken together, our results present the first evidence of Evi1 disturbing normal erythropoiesis in vivo and provides evidence for cooperative potential of Evi1 in tumor progression.

Colorimetric determination of inhibition of hematopoietic progenitor cells in soft agar

In vitro colony forming unit (CFU) assays have been used to measure the effects of compounds that regulate the growth of hematopoietic progenitor cells. These assays are time consuming and subjective and are therefore not amenable to high throughput of large numbers of compounds. Here we have shown that the traditional murine bone marrow CFU assay can be modified into a robust non-subjective colorimetric assay format. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added after colony formation in an agar based 96-well plate culture system. Optical density correlated with increasing cell input concentrations in the presence of growth factor. The linearity of this response was equivalent to the standard CFU assay. Several hematopoietic inhibitors were tested in both assays. Effects on colony number and size were compared to optical density. Compounds that reduced colony numbers with little effect on colony size had identical IC 50 values in both the colorimetric assay and CFU assay. The IC 50 values of compounds that also decreased colony size did not correlate in the two assays. These results demonstrate the utility of the colorimetric assay to rapidly screen for compounds that specifically inhibit hematopoietic progenitor cell colony formation in vitro.

17] Selection and maintenance of multidrug-resistant cells

This chapter discusses the selection and maintenance of multidrug-resistant cells, including specialized techniques, such as transfection and transduction of multidrug resistance genes and the introduction of multidrug resistance genes into bone marrow to form drug-resistant bone marrow colony-forming units (cfus). In the former approach, a cell line is carefully cloned so that all the cells in the population derive from a single cell; these cloned cells are exposed to a single cytotoxic agent in doses that kill the great majority of the cells in the population. This approach results in single-step resistance to the agent and individual surviving clones may be screened for cross-resistance to other drugs. The second approach is to expose an uncloned cell population to a selective drug in gradually increasing doses, allowing the population to die back and reexpand after each increase in the stringency of selection. This approach may result in highly multidrug-resistant populations in which the mechanisms of multidrug resistance may be relatively unique, particularly if a potent mechanism is responsible for resistance at an early stage of selection and the derivatives of the original resistant cell have overgrown the population. The advantage of this approach is that it casts a wide net for many different possible kinds of drug resistance.

4.P.30 Bone marrow colony-forming units for fibroblasts in the blood of patients with primary hyperlipidemia

4.P.30 Bone marrow colony-forming units for fibroblasts in the blood of patients with primary hyperlipidemia

The Phenomenology and Possible Mechanisms of a New Experimental Method for Accelerating Postirradiation Restoration of Hemopoietic Stem Cell Potential

Extraction of bone marrow from one or several bones immediately after irradiation, with or without subsequent incubation for a short period under suitable conditions, and subsequent reimplantation into the same organism can reduce the lethal effect of irradiation in a number of species. Extraction plus a period of incubation is referred to as the reimplantation method. The effectiveness is roughly relative to the amount of bone marrow extracted and transplanted. This effect has been demonstrated by assays of 30-45-day survival and of hemopoietic stem cell potential. The effectiveness of the reimplantation method has been tested in a dose range of 6.5 to 8.5 Gy and was found to be 1.2-1.06 in terms of dose reduction factors assayed by bone marrow row cellularity 9 days after exposure and 1.18-1.09 for survival of bone marrow colony-forming units. The favorable effect of incubating irradiated bone marrow with cycloheximide on the stem cell potential has been proven by experiments using a donor-recipient method. The positive effect of the reimplantation procedure and the partial extraction procedure on the stem cell potential in irradiated mice can be shown as soon as 2 h after exposure and the procedures. The results suggest that ther exist some reserves that can be stimulated to accelerate hemopoietic restoration in a heavily irradiated organism. The recruitment of these reserves seems to be related to the response of the structures producing cytokines after lethal irradiation. In addition, repair processes may be involved in the rescue of lethally irradiated hemopoietic stem cells.

BB-10010/MIP-1 alpha in vivo maintains haemopoietic recovery following repeated cycles of sublethal irradiation.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is an inhibitor of stem cell proliferation affording protection against damage from agents that express their cytotoxicity specifically in the DNA synthesis phase of the cell cycle. Its ability also to modify the self-renewal capacity of the regenerating cells is now shown to improve and maintain haemopoietic recovery following therapy (sublethal irradiation) whose cytotoxic damage is not limited solely to the DNA-S phase of this cycle. Such non-cell cycle-active cytotoxic agents are used clinically in repeated treatment regimens, which are often limited or terminated because of accumulating haemopoietic damage. BB-10010, a non-aggregating variant of MIP-1 alpha, was administered as a continuous dose (1600 micrograms kg-1 24 h-1) via a subcutaneously implanted pump over a period of 7 days. A dose of 4.5 Gy total-body gamma-rays was given 3-4 h after implantation. Day 8 and 12 spleen colony-forming units (CFU-S) were assayed on days 1, 7 and 14 after irradiation. This cycle of treatment was repeated four times (total 56 days), and on day 14 of the last two cycles the marrow-repopulating ability (MRA) was also measured. In the control bone marrow (no BB-10010) CFU-S fell to < 1% of normal within 1 day of irradiation and recovered to 40% at 14 days. Repeated treatments increased the level of damage, and after four cycles CFU-S recovered to only 10% of normal. BB-10010 afforded little benefit in the first treatment cycle, but by the end of the fourth cycle CFU-S still recovered to 35% of normal. MRA was reduced to 7% of normal by the irradiation protocol-about half that maintained by BB-10010 protection. We conclude that BB-10010 (MIP-1 alpha) reduces the degree of accumulated haemopoietic stem cell damage following repeated non-cell cycle-specific cytotoxic insults-a principle which should be valuable in repeated clinical cytotoxic therapy regimens.

Interleukin-12 Enhances Peripheral Hematopoiesis In Vivo

Interleukin 12(IL-12) is a cytokine that supports the proliferation and activation of cytotoxic T lymphocytes and natural killer (NK) cells. Recent evidence has suggested that IL-12 also has hematopoietic activities in vitro. We report studies that show that IL-12 has significant in vivo hematopoietic stimulating activity that includes enhancement of peripheral (splenic) hematopoiesis and mobilization of hematopoietic progenitor cells to the peripheral circulation. A single injection of recombinant murine IL-12 significantly reduced the number of bone marrow (BM) colony-forming unit granulocyte-macrophage (CFU-GM) in a time-dependent manner, while concomitantly stimulating high proliferative potential. In contrast, splenic CFU-GM and HPP were increased in a time- and dose-dependent manner. Chronic administration of IL-12 resulted in significant splenic hyperplasia with increased progenitor cells, increased circulating progenitor cells, and BM hypoplasia with decreased progenitor cells. These data show that IL-12 has significant in vivo hematopoietic effects that include the ability to mobilize progenitor cells to the peripheral circulation, which may prove to be of significant benefit for peripheral blood stem cell transplantation. Thus, IL-12 has potential to be an important agent for clinical transplantation because of its hematopoietic mobilization and its previously shown immune augmenting and therapeutic activities. This combination of hematopoietic and immune functions is unique and not achievable with currently used hematopoietic growth factors.

Enrichment of early fetal-liver hemopoietic stem cells of the rat using monoclonal antibodies against the transferrin receptor, Thy-1, and MRC-OX82.

Fetal livers from inbred rat fetuses at 14 days' gestation were dispersed into a single-cell suspension by physical disruption and collagenase digestion. Pluripotent stem cells were characterized and partially purified by a combination of monoclonal antibodies. These included CD71 (anti-transferrin receptor, MRC-Oχ26, used for rosetting), Cdw90 (anti- Thy-1, MRC-Oχ7), and the newly described MRC-Oχ82 (reacting with myeloid cells in peritoneal exudate), employed in FACS sorting. Enrichment was monitored by long-term reconstitution of lethally irradiated congenic rats genetically distinguishable from the donor by an allelomorphic variant of the CD45 cell-surface antigen. At intervals from 3 months to 1 year, lymph-node cells and peritoneal exudate cells were biopsied for analysis by two-color flow cytometry-one color to determine donor origin, the other to identify Th cell (CD4+), Tc cell (CD8+), B cell (sIg+ or CD45RC+ ), neutrophil (Oχ82 + or Oχ43- ), and macrophage (Oχ43+) compartments. The degree of chimaerism was taken as the read out of stem-cell activity. No significant differentials between lymph-node and peritoneal exudate chimaerisms were detected in any of the recipients; therefore, the enrichment procedure revealed only pluripotent cells, not stem cells of restricted potency. All recovered stem-cell activity was in the Oχ26(CD71)-negative, Oχ7(CDw90)-positive, Oχ82-positive fraction. In the optimum case, an enrichment of very roughly 200-fold in cell-for-cell activity was obtained.Rat bone-marrow colony-forming units in the spleen (CFUs-12) were found to lack the surface antigens recognized by the monoclonal antibodies CD53 (MRC-Oχ44), MRC-Oχ39, MRC-Oχ59, and 144.2.15. These would provide a strategy for their enrichment by depletion.

In vivo synergistic effect of the immunomodulator AS101 and the PKC inducer bryostatin

The immunomodulator AS101 has recently been found to have radioprotective properties when injected prior to sublethal and lethal doses of irradiation. In addition, this compound was found to protect mice from hemopoietic damage caused by sublethal doses of cyclophosphamide (CYP) and to increase the rate of survival of mice treated with lethal doses of CYP. AS101 was previously shown to exert a synergistic effect with the PKC-inducer bryostatin in cytokine secretion in vitro. The present studies were designed to evaluate the effects of in vivo combined treatment with AS101 and bryostatin on bone marrow and spleen cellularity and on the number of committed progenitors in the bone marrow at various points of time after their treatment with a sublethal dose of CYP or irradiation. In addition, the combined effect was tested on the survival of mice irradiated with a lethal dose of irradiation. Our data show the presence of synergism which greatly enhances the number of bone marrow and spleen cells 48 hr and 9 days after CYP treatment or irradiation. The combined effect was also demonstrated when bone marrow colony-forming units granulocyte-macrophage (CFU-GM) progenitor cells were evaluated. Moreover, AS101 and bryostatin synergized in their protective effects against lethal damages of irradiation. These results strongly suggest that bryostatin, which lacks tumor-promoting activity, is a particularly good candidate in combination with AS101 for treatment in vivo in counteracting chemotherapy- or radiation-induced hematopoietic suppression or in generally improving the restoration of immune response under conditions involving immune or hemopoietic damage.

Elimination of small cell carcinoma of the lung from human bone marrow by monoclonal antibodies and immunomagnetic beads : Vredenburg JJ. Ball ED. Department of Medicine, Dartmouth Medical School, Hanover, NH 03756. Cancer Res 1990;50:7216-20

The majority of patients with small cell carcinoma of the lung (SCCL) have bone marrow involvement detected by monoclonal antibodies (mAb). High dose chemotherapy followed by autologous bone marrow transplantation may improve treatment results for patients with SCCL, but the bone marrow may need to be purged of contaminating tumor cells. This study investigates the reactivity of a panel of mAb with two SCCL cell lines and normal bone marrow and the ability of the mAb and immunomagnetic beads to eliminate the SCCL cells from a mixture of 90% normal bone marrow cells and 10% SCCL cells. The mAb and immunomagnetic beads removed 4 to 5 log of SCCL cells in the model system. The immunomagnetic separation did not significantly adversely affect normal hematopoietic progenitor cells, as determined by bone marrow colony-forming units. The results suggest that the mAb and immunomagnetic beads could safely and effectively separate SCCL cells from the bone marrow for autologous bone marrow transplantation following high dose chemotherapy.

Effects of recombinant human granulocyte colony-stimulating factor on the hematologic recovery and survival of irradiated mice

We studied the effects of intraperitoneal injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) according to various administration schedules on the recovery of spleen colony- forming units (CFU-S) and peripheral blood counts, and on the survival of irradiated mice. The sooner and more frequently the mice were injected with rhG-CSF after irradiation, the more enhanced the recovery of CFU-S in bone marrow was obtained on day 7. Twice-daily injections of rhG-CSF from day 0 to day 2 significantly enhanced the recovery of platelets and hematocrit, but two injections of rhG-CSF on only day 0 did not. Twice-daily injections of rhG-CSF from day 0 to day 6 enhanced the recovery of platelets more effectively than twice-daily injections of rhG-CSF from day 1 to day 7, and increased the survival of irradiated mice more effectively than any other examined administration schedules. Twice-daily injections of rhG-CSF from day 0 to day 6 were significantly effective in enhancing the survival of mice irradiated with 8.5-, 9.0-, and 9.5-Gy x-rays, although not effective after irradiation of 10.5-Gy x-rays.

Effects of Recombinant Human Granulocyte Colony-Stimulating Factor on the Hematologic Recovery and Survival of Irradiated Mice

We studied the effects of intraperitoneal injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) according to various administration schedules on the recovery of spleen colony-forming units (CFU-S) and peripheral blood counts, and on the survival of irradiated mice. The sooner and more frequently the mice were injected with rhG-CSF after irradiation, the more enhanced the recovery of CFU-S in bone marrow was obtained on day 7. Twice-daily injections of rhG-CSF from day 0 to day 2 significantly enhanced the recovery of platelets and hematocrit, but two injections of rhG-CSF on only day 0 did not. Twice-daily injections of rhG-CSF from day 0 to day 6 enhanced the recovery of platelets more effectively than twice-daily injections of rhG-CSF from day 1 to day 7, and increased the survival of irradiated mice more effectively than any other examined administration schedules. Twice-daily injections of rhG-CSF from day 0 to day 6 were significantly effective in enhancing the survival of mice irradiated with 8.5-, 9.0-, and 9.5-Gy x-rays, although not effective after irradiation of 10.5-Gy x-rays.

A one step cell sorting procedure for the enrichment of murine bone marrow CFU-S which is independent of their proliferative activity

Murine bone marrow day 9 splenic colony-forming units (CFU-S) have been concentrated using a one step cell sorting technique. CFU-S were discriminated on the basis of their rather high forward light scatter intensity, their high affinity for the lectin WGA and the absence of a cell surface marker expressed by the majority of hematopoietic cells and recognized by the RA3-5B3 MoAb. This procedure permitted a 40-fold enrichment of quiescent or cycling CFU-S (obtained from normal and Ara-C-treated mice respectively) and did not alter their differentiation pathways towards the various myeloid lineages.

Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer.

Adenosine deaminase (ADA) deficiency, an autosomal recessive inborn error of metabolism, leads to severe combined immune deficiency in man. This enzyme, although constitutively expressed in most tissues, is expressed at high level in immature T cells, and study of the pathophysiology of the disorder indicates that increased deoxyadenosine or altered methylation capacity have toxic effects on T-cell maturation. Although bone marrow transplantation can correct the immune deficiency, this therapy is associated with graft-versus-host disease and incomplete immune restoration, and so our laboratory and others have sought to develop a method of gene replacement as a possible treatment for the disease. Moreover, characterization of the complementary DNA of the human ADA gene and some of its mutants makes it possible to design gene transfer strategies. We have now subcloned a human adenosine deaminase cDNA into the retrovirus shuttle vector pZIP-SV(B), and in this way have isolated a cell line, 4.2T, which produces high titres of replication-defective retrovirus which have been used to transfer the gene for human ADA to mouse bone marrow cells. Transfer and expression of the neomycin-resistance gene (neo) and the ADA gene in murine bone marrow colony-forming units (CFU) was demonstrated by in vitro colony formation in the presence of the antibiotic G418 or 9-xylofuranosyladenine plus deoxycoformycin, respectively. Isoenzyme analysis also showed human ADA expression in the cultured mouse bone marrow.

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.

Potentiation of differential hyperthermic sensitivity of AKR leukemia and normal bone marrow cells by lidocaine or thiopental.

Previous work has utilized spleen colony formation to evaluate the fractional survival of AKR leukemia and normal bone marrow cells after in vitro heat exposure. An inherently greater sensitivity of neoplastic cells to thermal killing, as compared to normal syngeneic stem cells, has been established both at 41.8 degrees C and 42.5 degrees C. Normal bone marrow colony-forming units were assayed in lethally irradiated (750 cGy) mice. Leukemic colony-forming units were assayed in nonirradiated mice. Using this methodology, the authors demonstrated that the differential effect of hyperthermia on AKR murine leukemia and AKR bone marrow cells can be further enhanced by the addition of lidocaine or thiopental to incubation mixtures. These findings may have application to autologous bone marrow transplantation in humans.

Functional status of cells from lymphoid and myeloid tissues in mice with severe combined immunodeficiency disease.

Cells from mice with severe combined immunodeficiency disease (SCID) were tested in assays that measure myeloid and lymphoid function. Results showed that C.B-17 scid and their normal counterparts (C.B-17) have similar levels of spleen colony-forming units. The frequency of in vitro myeloid colony-forming units in C.B-17 scid spleen is elevated, but the absolute number of colony-forming units in C.B-17 scid and C.B-17 spleen is similar. The absolute number of bone marrow colony-forming units in C.B-17 scid and C.B-17 mice is comparable. Cells from C.B-17 scid spleen are consistently negative in all tests of B and T cell function. C.B-17 scid splenocytes fail to proliferate in response to T and B cell mitogens or to allogeneic lymphocytes in a one-way MLR; C.B-17 scid cells do serve as stimulators in MLR. B lymphocyte colony-forming units are absent, as are cytotoxic lymphocyte precursors and cells that can generate T cell colonies with cytotoxic progenitors. The microenvironment of the C.B-17 scid mouse is conducive to lymphocyte differentiation, because functional B and T cells are easily detectable in mice reconstituted with normal bone marrow cells. The results of this study indicate that scid specifically impairs the differentiation of stem cells into mature lymphocytes; myeloid cell differentiation is not affected.

Regulation of granulopoiesis and distribution of granulocytes in early phase of bacterial infection.

Studies have been carried out to determine the effect of bacterial infection on CSF production, CFU-C activation, and bacterial clearance by mature granulocytes in mice infected with Escherichia coli. These studies have shown that immediately after bacterial infection (5 minutes), serum colony-stimulating factor (CSF) levels and bone marrow colony-forming units in culture (CFU-C) levels are elevated. This is followed by oscillatory rises in both of these parameters and the appearance of granulocytes in the infected site. With clearance of bacteria, CSF and CFU-C levels return to normal. These studies have indicated further that bacterial infection is a major stimulus for granulocyte production through the CSF-CFU-C system and that clearance of bacteria by mature granulocytes may serve as a negative feedback regulatory arm.