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To assess the drug-induced hematotoxicity to granulocyte progenitors, we established a modified colony-forming assay using rat bone marrow cells (BMCs). In the presence of various colony-stimulating factors (CSFs), rat BMCs were disseminated on methylcellulose at a concentration of 1.3 x 10(4) cells/cm(2) (5 x 10(4) cells/0.5 ml/well in a 12-well plate). Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) stimulated the formation of almost all macrophage colonies. Human granulocyte colony-stimulating factor (hG-CSF) alone or in combination with mouse interleukin-3 (mIL-3) did not significantly effect on the number of rat colony-forming units in culture (CFU-C). When BMCs were seeded at 5.2 x 10(4) cells/cm(2) (5 x 10(5) cells/1 ml/dish in a 35-mm dish), hG-CSF increased the number of the colonies in a dose-dependent manner, and resulted in about 50 colonies at 50 ng/ml. The constituent cells of the colonies were identified as neutrophils. Under these conditions, the effects of 5-fluorouracil (5-FU) on granulocyte colony-forming units (CFU-G) were examined in rats and mice. The inhibitory effect of 5-FU on rat CFU-G was similar to the effect on mouse CFU-G. These results indicate that the rat CFU-G induced by hG-CSF is capable of being used for the evaluation of drug-induced hematotoxicity.

Kell is one of the major human red blood cell groups and comprises 22 antigens. These antigens are produced by alleles located on chromosome 7, including sets of antithetical antigens such as Kell (K, K1) and cellano (k, K2), which differ in a single amino acid change (T193M). It consists of a 93-Kd transmembrane glycoprotein that is surface-exposed and shares sequence and structural homology with zinc endopeptidases, which are involved in regulating bioactive peptides. Anti-Kell antibodies have been shown to suppress fetal erythropoiesis. Recently published data indicate a similar effect on myeolopoiesis and megakaryopoiesis. Substantial thrombocytopenia in fetuses affected with HDN due to anti-K antibodies led to the discovery of the inhibitory effect of Kell-related antibodies on CFU-MK growth. In addition to its inhibitory effect on BFU-E growth, anti-Kell antibodies significantly reduced CFU-GM colony formation from haematologically normal individuals. Moreover, anti-cellano and anti-Kp(b) antibodies also inhibited the growth of CFU-GM from antigen positive MNC. These data indicate that Kell is not restricted to erythroid blood cells, but is expressed on a broader spectrum of haematopoietic cells than previously believed.

The counting of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units erythrocyte (BFU-E) provides substantial in vitro information about the graft quality after peripheral stem cell transplantation (PBSCT). By using different techniques for culturing and scoring, high inter- and intralaboratory coefficients of variation (CV) are frequently reported. We minimized the imprecision by using flow cytometry-based incorporation of constant numbers of CD34(+) cells per culture dish instead of the formerly used mononuclear cells. Our results show acceptable CVs for CFU-GM (12.3%) and for BFU-E (13.3%) based on this seeding technique, which contributes to fulfilling the demands of a quality assurance system in stem cell laboratories.

In this work, we investigated the effects of the proteic aggregated polymer of magnesium ammonium phospholi-noleate-palmitoleate anhydride (MAPA) isolated from Aspergillus oryzae on the growth and differentiation ofbone marrow granulocyte-macrophage progenitor cells (CFU-GM) inListeriamonocytogenes-infectedmice.Asignificant reduction in the CFU-GM numberwas observed in the initial phase of infection with a sublethal dose of Listeria. Treatment of mice with 0.5, 2.0 and 5.0 mg/kg MAPA for 7 days prior to infection significantly stimulated myelopoiesis in a dose-dependent manner. Moreover, treatment with 0.5 and 5.0 mg/kg MAPA resulted in 30%/6 and 40% cures of mice lethally infected with Listeria, respectively. MAPA added directly to the culture dishes hardly affected colony formation by bone marrow cells, suggesting an indirect effect ofthis compound on myelopoiesis in vivo. In summary, the data show that MAPA can modulate the CFU-GM generation and antibacterial resistance in listeriosis. As the ability of hematopoietic tissues to produce phagocytes is of particular significance to mediate resistance to Listeria, the promotion ofbone marrow CFU-GM by MAPA may contribute to a rapid restoration of phagocyte numbers in infected sites, thus mitigating the course of infection.

Although very high doses of 5-fluorouracil was used in the weekly 24-h infusion, high-dose 5-fluorouracil (2600 mg/m2/week) and leucovorin (500 mg/m2/week) protocol, myelosuppression was surprisingly low. The current study was conducted to investigate the possible mechanism underlying the low myelosuppression. To mimic the clinical situation, peripheral blood progenitor cells collected from 12 patients were used for colony forming unit-granulocyte and monocyte clonogenic assay; and 2 representative modes of 5-fluorouracil exposure (30 min. versus 24 hr) were examined for cytotoxic effects on human myeloid progenitor cells. Previous pharmacokinetic studies have estimated the concentrations of 5-fluorouracil in the bone marrow to be 200-400 microM and 1-2 microM for the 30 min. infusion (600-900 mg/m2) and the 24 hr-infusion (1000-2000 mg/m2) regimens, respectively. The results of our colony-forming unit-granulocyte and monocyte clonogenic assay showed that 24-hr exposure to 5-fluorouracil (2 microM) and 30 min. exposure to 5-fluorouracil (100 microM) resulted in 27.2% and 78.2% inhibition of the colony formation, respectively. Our data provided direct evidence which may explain why myelotoxicity is significantly less in weekly 24 hr infusion of fluorouracil than in the conventional bolus regimens.

The pig is widely used as a large animal model for biomedical research and could be an interesting experimental model for studies of the hematopoietic system and its response in physiological and pathological conditions. With the intention of using the pig as a large animal model in hematopoietic research, a clonal assay in methylcellulose was developed and standardized for detection of committed progenitors of the granulocyte-macrophage lineage from adult pig bone marrow and peripheral blood. Progenitor cells were stimulated to proliferate and differentiate in vitro by adding pig leukocyte conditioned medium (LCM) as a source of homologous growth factors. The number of CFU-GM (Colony Forming Unit -Granulocyte-Macrophage) directly depended on the concentration of LCM. The proliferative rate of CFU-GM progenitor cells was determined by the cytosine arabinoside suicide technique. The percentage of bone marrow and peripheral blood CFU-GM cells in S phase of the cell cycle was 34.7% and 22.2%, respectively. The data obtained regarding the number and characteristics of pig bone marrow and peripheral blood CFU-GM confirmed that the organization of the pig CFU-GM progenitor cell compartment is similar and comparable to that in miniature swine, other animal species and humans. Svinja je životinja koja se koristi kao model u različitim biomedicinskim istraživanjima, a mogia bi biti i interesantan model u ispitivanju fiziologije i patolofizioloških promena hematopoetskog sistema. U cilju razvoja eksperimentalnog modela svinje u istraživanju hematopoeze, stanadardizovan je esej za odredivanje i karakterizaciju opredeljenih matičnih ćelija granulocitno-monocitne loze iz kostne srži i krvi odrasle svinje. Stimulacija proliferacije i diferencijacije ovih matičnih ćelija postignuta je dodavanjem medijuma kondicioniranog leukocitima (LCM - Leukocyte conditioned medium) bogatog faktorima rasta. Broj CFU-GM (Colony forming unit- granulocyte-macrophage) je direktno zavisio od koncentracije LCM-a. Procenat CFU-GM ćelija u S fazi ćelijskog cikiusa odredjivan je tehnikom 'suicida' korišćenjem citozin arabinozida (Ara-C) i iznosio je 34.7% za CFU-GM iz kostne srži i 22.2% za CFU-GM iz periferne krvi. Podaci dobijeni za broj i karakteristike CFU-GM iz kostne srži i periferne krvi potvrđuju da je ovaj odeljak matičnih ćelija kod odraslih svinja organizovan na isti način kao i kod minijaturnih svinja, drugih vrsta životinja i ljudi.

The pig is widely used as a large animal model for biomedical research and could be an interesting experimental model for studies of the hematopoietic system and its response in physiological and pathological conditions. With the intention of using the pig as a large animal model in hematopoietic research, a clonal assay in methylcellulose was developed and standardized for detection of committed progenitors of the granulocyte-macrophage lineage from adult pig bone marrow and peripheral blood. Progenitor cells were stimulated to proliferate and differentiate in vitro by adding pig leukocyte conditioned medium (LCM) as a source of homologous growth factors. The number of CFU-GM (Colony Forming Unit -Granulocyte-Macrophage) directly depended on the concentration of LCM. The proliferative rate of CFU-GM progenitor cells was determined by the cytosine arabinoside suicide technique. The percentage of bone marrow and peripheral blood CFU-GM cells in S phase of the cell cycle was 34.7% and 22.2%, respectively. The data obtained regarding the number and characteristics of pig bone marrow and peripheral blood CFU-GM confirmed that the organization of the pig CFU-GM progenitor cell compartment is similar and comparable to that in miniature swine, other animal species and humans. Svinja je životinja koja se koristi kao model u različitim biomedicinskim istraživanjima, a mogia bi biti i interesantan model u ispitivanju fiziologije i patolofizioloških promena hematopoetskog sistema. U cilju razvoja eksperimentalnog modela svinje u istraživanju hematopoeze, stanadardizovan je esej za odredivanje i karakterizaciju opredeljenih matičnih ćelija granulocitno-monocitne loze iz kostne srži i krvi odrasle svinje. Stimulacija proliferacije i diferencijacije ovih matičnih ćelija postignuta je dodavanjem medijuma kondicioniranog leukocitima (LCM - Leukocyte conditioned medium) bogatog faktorima rasta. Broj CFU-GM (Colony forming unit- granulocyte-macrophage) je direktno zavisio od koncentracije LCM-a. Procenat CFU-GM ćelija u S fazi ćelijskog cikiusa odredjivan je tehnikom 'suicida' korišćenjem citozin arabinozida (Ara-C) i iznosio je 34.7% za CFU-GM iz kostne srži i 22.2% za CFU-GM iz periferne krvi. Podaci dobijeni za broj i karakteristike CFU-GM iz kostne srži i periferne krvi potvrđuju da je ovaj odeljak matičnih ćelija kod odraslih svinja organizovan na isti način kao i kod minijaturnih svinja, drugih vrsta životinja i ljudi.

Sixty patients with malignancy were enrolled in a study of high dose chemotherapy and peripheral blood stem cell transplantation (PBSCT). The International Journal of Cell Cloning were harvested prior to PBSCT using 75 cycles of high-dose Cyclophosphamide (Cy) mobilization (4 or 7 G/m2) with collection of a median of 4.6 × 108/kg mononuclear cells (MNC) (range 0.2 - 9.5) and 21.6 × 104/kg colony forming unit - granulocyte /macrophage (CFU-GM) (range 0.1 -220). Forty seven patients were mobilized once, 11 required 2 cycles and 2 required 3 cycles. Eight patients (13%) failed to reach the optimim CFU-GM target (total of > 15 × 104 CFU-GM/kg) following Cy mobilization. In a multivariate regression analysis, a number of factors identified those patients who were likely to achieve optimum stem cell collections. These included the use of the higher Cy mobilization dose (P

The effects of rhIL-3 alone and rhIL-3 followed by GM-CSF on circulating granulocyte-macrophage progenitor cells (CFU-GM) were compared in seven patients with advanced malignancies who were treated as part of a two-stage phase I study. In the first treatment cycle, patients received rhIL-3 for 15 days at dosages of 60 μg/m2 or 250 μg/m2. The second treatment cycle consisted of a 5-day course of rhIL-3 at the same dose level followed by rhGM-CSF at a fixed dose of 250 μg/m2 for an additional 10 days. During treatment with 60 μg/m2 rhIL-3, the number of circulating CFU-GM per volume blood increased to 286±283% of day 0 values at day 8, and then declined to baseline or less in 3 of 4 patients after 15 days. RhIL-3 at 250 μg/m2 was more effective, resulting in an increase of peripheral blood CFU-GM to 491 ± 194% of day 0 levels after 15 days. A considerably more pronounced effect on circulating CFU-GM was observed during sequential treatment with rhIL-3 and rhGM-CSF at both the high and low dosage of rhIL-3. The enhancement was greatest at the end of the second treatment cycle (day 15), with CFU-GM frequencies increased to 734±399% of initial values. This relative increase in the number of circulating CFU-GM correlated with the increase of absolute neutrophil counts. These results indicate that sequential treatment with rhIL-3 and rhGM-CSF is superior to treatment with single factors with regard to neutrophil counts and elevations of circulating progenitor cells.

Recombinant human stem cell factor (SCF), the ligand for human c-kit, by itself stimulates little in vitro proliferation by marrow hematopoietic colony-forming cells (CFC) from baboons, but stimulates increases in the number and size of colonies formed in response to IL-3, G-CSF, GM-CSF, and erythropoietin alone or in combination. SCF given to baboons stimulates increases in peripheral blood cells of multiple myeloid and lymphoid lineages and increases marrow cellularity. The present studies demonstrate that SCF given to baboons also stimulates significant increases in CFU-GM, BFU-E, CFU-MIX, HPP-CFC, and detectable CD34+ cells in blood. These changes are dose dependent. SCF stimulated a doubling of marrow cellularity and modest increases in the frequency of CFC, consistent with an absolute increase in the total number of CFC in marrow. No adverse events were observed. The findings suggest a role for SCF (c-kit ligand) in stimulating expansion of hematopoietic CFC of multiple types in blood and marrow for potential therapeutic purposes.

The munbers of MNC and CFU-GM in peripheral blood began to rise 1 hour after intravenous injection of DS and achieved peak value within 3–5 hours. The toxicity test suggested that the critical drawback of DS for clinical application was hypersensitive reaction rather than LD toxicity. The threshold molecular weight for causing hypersensitive reaction was slightly higher than 10000D. Compared to other kind of mobilizers, such as Cy, Ara—C, DNR, etc., DS may not only be used in patients, but also be used in normal individuals. The safe and effective dose of DS for human being is about 15–20 mg/kg.

Although interferon (IFN)-alpha has no specific inhibitory effect on the plating efficiency of granulocyte-macrophage colony-forming cells (CFU-GM) from patients with chronic myeloid leukaemia (CML), it does selectively inhibit the replating ability (secondary colony formation) of CML CFU-GM. Thus, amplification of CFU-GM may be a target for IFN-alpha and other agents used in the treatment of CML. Here we examined whether cytarabine (Ara-C) or all-trans retinoic acid (ATRA) exert similar effects and whether they might in combination with IFN-alpha enhance its efficacy. We found that Ara-C preferentially inhibits the formation of CML CFU-GM compared to normal CFU-GM, but this inhibition was not increased by addition of IFN-alpha. When Ara-C was added to cultures containing IFN-alpha, the inhibition of replating by CML progenitors was abrogated. ATRA increased significantly the plating efficiency of normal CFU-GM. The addition of IFN-alpha to ATRA had no effect on CML or normal colony numbers. However, addition of ATRA to cultures containing IFN-alpha reversed the selective inhibition of CML CFU-GM replating seen in cultures containing IFN-alpha alone. In four IFN-alpha/Ara-C experiments, secondary CML patient-derived colonies were examined by fluorescence in situ hybridisation (FISH). All of them were Ph chromosome positive. No significant effects on CFU-GM production were observed when CML primitive haemopoietic progenitor cells were investigated in a delta (delta) assay. Thus we conclude that combining IFN-alpha with Ara-C or ATRA neutralises the effect of IFN-alpha on CML CFU-GM. This observation provides a rationale for treating patients with alternating courses of IFN-alpha and Ara-C or ATRA, rather than giving either of these two agents in combination with IFN-alpha.

Primary long-term bone marrow cultures grown in 40 mM HEPES-buffered McCoy's 5A medium produced granulocyte-macrophage colony-forming units (CFU-GM) for a median of 9 weeks compared with 7 weeks with CO2/bicarbonate-buffered cultures. Reducing the medium glucose concentration (from 12.5 to 2.75 mM) extended the culture longevity to 17 weeks. The median period of erythroid colony detection increased from 6 to 8 weeks. Secondary cultures (5 x 106 cord blood mononuclear cells seeded on irradiated stroma) showed statistically similar myeloid and erythroid longevity to primary cultures. Improved control of medium pH significantly improved the capacity of long-term stromal layers to maintain stem cells in vitro.

The granulocyte-macrophage progenitor (CFU-GM) is a multipotent cell that can differentiate to osteoclasts (OCLs), macrophages, or granulocytes. However, the relative potential of CFU-GM to efficiently form OCLs is unknown. In this report we demonstrate that granulocyte-macrophage colony-forming unit (CFU-GM)-derived cells represent an easily obtainable highly purified source of human OCL precursors that form OCLs at very high efficiency (greater than 90%) when cultured with RANK ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and dexamethasone. The OCLs that formed have high bone-resorbing activity and form multiple resorption lacunae per OCL on dentin slices. Similarly, murine marrow-derived CFU-GM also formed OCLs at a high efficiency (>80%) when treated with RANKL, M-CSF, and dexamethasone. In contrast, more committed macrophage colony-forming unit (CFU-M)-derived cells form few OCLs under these conditions.

Many factors affect the hematopoietic reconstitution after allogeneic bone marrow transplantation (alloBMT). We evaluated the impact of the time of marrow cell infusion after BuCy2 therapy on the speed of engraftment in 30 patients (pts.) with chronic myeloid leukemia (22) and acute myeloid leukemia (8). The pts. were divided into two groups. In the first comprising 12 pts., the alloBMT were performed 48 h after conditioning with BuCy2 and the grafts contained a median of 24±6 × 104/kg CFU-GM (colony forming unit-granulocyte/macrophage). The median time to reach granulocytes >0.5 × 109/l and >1.0 × 109/l was 29 (15–49) and 31 (16–86) days, respectively. The recovery of platelets > 20 × 109/l and >50 × 109/l was noted after 22 (7–73) and 39 (11–99) days, respectively. The second group comprised 18 pts. who received transplants 72 h after BuCy2 conditioning. The median number of grafted CFU-GM was 26±7 × 104/kg. Granulocyte reconstitution >0.5 × 109/l and >1.0 × 109/l was observed after 17 (15–24) and 19 (16–31) days, respectively. The engraftment of platelets >20 × 109/l and >50 × 109/l occurred in a median of 18 (8–32) and 35 (16–90) days, respectively. Further studies were undertaken to clear up the observed differences in the time of engraftment between the two groups of pts. The growth abilities of less (7) and more (14) mature CFU-GM were evaluated under the effect of sera taken from the pts. before BuCy2 administration, at the last day of therapy and after conditioning: days 1,2,3,4, and 5. The results of these studies indicate that sera samples from days 1 and 2 after completion of BuCy2 therapy suppress the growth of less and more mature CFU-GM. On the contrary, sera samples from days 3,4 and 5 after conditioning do not affect the CFU-GM-derived colony formation. We conclude that alloBMT carried out 72 h in contrast with 48 h after BuCy2 therapy completion is associated with clearly faster granulocyte reconstitution and normal growth of grafted less and more mature CFU-GM.

Hematopoietic progenitors express HLA-DR molecules. However the significance of HLA-class II molecules on CD34+ cells remains unknown. The primary role of HLA-class-II molecules is antigen presentation although a second role, that of signal transduction, has been established in B cells. The role of HLA-DR in hematopoiesis was examined by determining the ability of CD34+ progenitor cells to differentiate to “Colony Forming Unit Granulocyte-Macrophage” (CFU-GM) and “Burst Forming Unit Erythrocyte” (BFU-E) in the presence of anti-HLA-DR monoclonal antibody. We observed a reduction in the number of CFU-GM which was due in part to down regulation of granulocyte rather than monocyte differentiation. These observations suggest that HLA-DR signals can regulate myelopoiesis. We point out especially the role of the HLA-DR molecule in the switch of CFU-GM between granulocyte or monocyte lineages. Although HLA-DR mediated apoptosis has been described in mature B lymphocytes apoptosis of CD34+ cells was excluded as a mechanism.

In this study we investigated the effects of the treatment with Chlorella vulgaris extract (CVE) on the hematopoietic response of granulocyte-macrophage colony-forming unit (CFU-GM) of mice infected with a sublethal dose of Listeria monocytogenes (1×10 4 organisms/animal). CVE was given orally as 50 mg/kg/day for 5 days. In the CVE treated/infected groups L. monocytogenes was administered at the end of CVE treatment. The colony stimulating activity of the serum (CSA) was also studied in all groups. Although no effects on CFU-GM, as compared to controls, were observed in the groups receiving CVE alone, the extract produced an increase in CSA levels as compared to controls. On the other hand, the presence of the infection led to a significant reduction in the numbers of CFU-GM as observed at 48 and 72 h after the infection, in spite of the significant increase in serum CSA activity. CVE treatment of infected animals restored the numbers of CFU-GM to control levels. In the treated/infected group the increased serum CSA was significantly higher than that observed in the only infected group. The CVE treatment (50 and 500 mg/kg) of mice infected with a dose of 3×10 5 bacteria/animal, which was lethal for all the non-treated controls, produced a dose-response protection which led to a 20 and 52% survival, respectively. These results demonstrated that CVE produces a significant increase in the resistance of the animals infected with L. monocytogenes , and that this protection is due, at least in part, to increased CFU-GM in the bone marrow of infected animals.

Murine bone marrow (BM) cells were cultured in semisolid medium containing interleukin 3 (IL-3) and high doses of G-CSF. Colonies were counted twice, at day 7 and day 14, and the number of granulocyte/macrophage colony-forming units (CFU-GM) accurately estimated by the subtraction of day-14 from day-7 colonies, based on the principle that colonies detectable at day 7 and persisting beyond day 14 are generated by significantly more immature progenitors. The frequency of colonies relative to their size was determined and used to define subsets of high proliferative potential colony-forming cells (HPP-CFC). Two main groups of HPP-CFC were considered: those generating colonies of 0.6-1.8 mm of diameter or larger than 1.8 mm. The characterization of these groups showed that they correspond to different functional subsets of HPP-CFC. The replating ability of colonies was estimated. The percentage of clonogenic progenitors in the S phase of cell cycle was measured by cytosine arabinoside suicide assay. The sensitivity of colonies to 5-fluorouracil (5-FU) in vitro was determined and their survival after an in vivo treatment with 5-FU compared with that of colony-forming units in spleen (CFU-S). This technique allowed identification of: A) CFU-GM; B) relatively mature HPP-CFC, probably corresponding to CFU-S day12; C) more primitive HPP-CFC, relatively resistant to 5-FU in vivo and closely corresponding to CFU-S day 14, and D) very primitive HPP-CFC, resistant to 5-FU in vitro. This simple, rapid, and versatile method allows the detection of a broad range of hematopoietic progenitors in murine BM, from committed progenitors to largely quiescent, primitive stem cells, as well as the evaluation of the progenitors' self-renewal and proliferative potential.

We have designed and used an in vitro bone marrow cell culturing system for investigating pharmacodynamic interactions between platinum anti-cancer drugs and taxanes. With this system, in which the bone marrow progenitor cell CFU-GM is proliferating and differentiating into granulocytes and monocytes, we could show a strong antagonistic cytotoxicity of the combination carboplatin and Taxotere, in three different schedules, and of the combination cisplatin and Taxol, in two out of the three schedules tested. Modulation of intracellular platinum drug accumulation in granulocytes and monocytes does not seem to be a plausible explanation for the observed antagonism. In vitro co-incubation of granulocytes/monocytes with the combination carboplatin and Taxotere did not reveal an effect of Taxotere on intracellular platinum accumulation. Although Taxol reduced intracellular cisplatin levels by 12%, this effect was not significantly different from the co-incubation of cisplatin with Cremophor EL, the solvent for paclitaxel in Taxol. The toxicity data obtained in this study seem to be in accordance with recent clinical trials where combination therapies with platinum drugs and taxanes resulted in marked reductions in myelosuppression in patients. Therefore, these types of assays could be useful as to the assessment of bone marrow toxicities of clinically important drug combinations.

Bryostatin-1 belongs to the family of macrocyclic lactones isolated from the marine bryozoan Bugula neritina and is a potent activator of protein kinase C (PKC). Bryostatin has been demonstrated to possess both in vivo and in vitro anti-leukaemic potential. In samples derived from chronic myeloid leukaemia (CML) patients, it has been demonstrated that bryostatin-1 induces a macrophage differentiation, suppresses colony growth in vitro and promotes cytokine secretion from accessory cells. We investigated the effect of bryostatin-1 treatment on colony-forming unit–granulocyte macrophage (CFU–GM) capacity in the presence of accessory cells, using mononuclear cells, as well as in the absence of accessory cells using purified CD34-positive cells. Cells were obtained from 14 CML patients as well as from nine controls. Moreover, CD34-positive cells derived from CML samples and controls were analysed for stem cell frequency and ability using the long-term culture initiating cell (LTCIC) assay at limiting dilution. Individual colonies derived from both the CFU–GM and LTCIC assays were analysed for the presence of the bcr–abl gene with fluorescence in situ hybridization (FISH) to evaluate inhibition of malignant colony growth. The results show that at the CFU–GM level bryostatin-1 treatment resulted in only a 1.4-fold higher reduction of CML colony growth as compared to the control samples, both in the presence and in the absence of accessory cells. However, at the LTCIC level a sixfold higher reduction of CML growth was observed as compared to the control samples. Analysis of the LTCICs at limiting dilution indicates that this purging effect is caused by a decrease in output per malignant LTCIC combined with an increase in the normal stem cell frequency. It is concluded that bryostatin-1 selectively inhibits CML growth at the LTCIC level and should be explored as a purging modality in CML. © 1999 Cancer Research Campaign

To investigate the hypothesis that commercial kits for CFU-GM (colony forming unit granulocyte-macrophage) assay will reduce the interlaboratory variation noted by many workers, we carried out a quality assurance exercise in 2 parts. There were 8 participants in the first study and each performed CFU-GM assays using their in-house method and a commercial kit (Stem Cell CFU Kit, Gibco) in parallel. In the second exercise there were 10 participants and each performed CFU-GM with in-house methods and with a different commercial medium (Methocult GF H4534, Stem Cell Technologies). Twelve samples of cryopreserved peripheral blood progenitor cells (PBPC) were analysed by each participant in each part of the study. A very wide range of results was found for the different in-house methods, but standardizing the clonogenic assay with the commercial kits did not reduce the variation seen. To improve the reproducibility of CFU-GM assays between laboratories, scrupulous attention should be paid to all the steps involved in the assays, as little progress will be made by using commercial medium in isolation from efforts to reduce other sources of variation.

Cilj istraživanja. Ispitati prinos CD34+ stanica i granulocitno-makrofagnih kolonija (CFU-GM) u leukocitnim pripravcima prije i nakon imunoafinitetnog izdvajanja CD34+ stanica p. krvi za autolognu transplantaciju. Bolesnici i metode. Ispitivanje je obuhvatilo 37 bolesnika dobi 19-66 godina (M:Ž=20:17), oboljelih od ne-Hodgkinova limfoma (n=12), karcinoma dojke (n=11), multiplog mijeloma (n=10), Hodgkinove bolesti (n=3) i karcinoma jajnika (n=1). Protokoli za mobilizaciju CD34+ stanica iz koštane srži u p. krv: ciklofosfamid+G-CSF (n=22), BEAM+G-CSF (n=10) i VIP + G-CSF (n=5). Broj leukafereza za izdvajanje CD34+ stanica: 1 (4/37), 2 (29/37) i 3 (4/37). Izdvajanje CD34+ stanica iz pripravaka leukafereze: SC-kit (anti-CD34-biotin, klon 12.8) i aparat CEPRATEâ SC (Cell-Pro). Rezultati. Medijan broja CD34+ stanica u pripravku leukafereze iznosio je 6.95 (2.7-40.6) x106/kg tjelesne težine, a CFU-GM 20.1 (1.1-117.8) x104/kg. Čistoća izdvojenog pripravka iznosila je 83% (28-96), broj CD34+ stanica 2.7 (0.3-34.7) x106/kg, a CFU-GM 3.1 (0.02-20.8) x104/kg. Prinos CD34+ stanica u izdvojenom pripravku bio je 36.2% (2.7-82.2), a CFU-GM kolonija 15.1% (3.4-49.6). Broj CD34+ stanica korelirao je s brojem CFU-GM u svim pripravcima (r=0.444, p0.1). Broj CFU-GM u produktu leukafereze i izdvojenom produktu korelirao je s parametrima hematološkog oporavka nakon transplantacije. Zaključak. Stopa gubitka CD34+ stanica i broja CFU-GM tijekom izdvajanja CD34+ stanica periferne krvi je različita, a veća je za broj CFU-GM (cca. 1 log). Međutim, podatak da je broj CFU-GM (a ne CD34+ stanica) značajno korelirao s hematološkim oporavkom nakon reinfuzije izdvojenih CD34+ stanica dodatno ističe potrebu za rutinskim izvođenjem testa kolonija in vitro za potrebe autologne transplantacije CD34+ perifernih matičnih stanica.

To delineate the influence of mechanical unloading on the osteogenic potential, the effects of mechanical unloading on the trabecular bone formation and the development of osteoblastic cells from bone marrow cells were examined using neurectomized mice. The trabecular osteoclasts and the osteoclastogenic potential of the bone marrow cells were also assessed. In 14 days after sciatic neurectomy, trabecular bone volume in the proximal tibial metaphysis was significantly reduced. The double-labeled surface and bone formation rate (BFR/BS) were significantly reduced. While the numbers of the total bone marrow cells and the nonadherent cells in the neurectomized limbs were not signficantly smaller than that of the sham-operated loaded limbs, the number of the adherent cells in the unloaded limbs was significantly reduced. The number of CFU-f colonies did not differ, but the number of bone nodules developed from bone marrow cells was significantly reduced in the unloaded limbs. The osteoclast-like cell development was increased when the bone marrow cells were incubated with parathyroid hormone. But, the number of CFU-GM colonies did not increase in the unloaded limbs. These data confirmed that the reduction in trabecular bone formation was associated with the reduction in the adherent stromal cells and bone nodule formation of bone marrow cells in the mice tibia after neurectomy. However, in vitro assays including CFU-f and CFU-GM did not identify the changes by unloading. The mechanical unloading affects the development of the bone cells, but may not greatly enhance the capacity for osteoclastogenesis in the bone marrow.

Mogućnost postizanja klonske remisije agresivnijim i potencijalno eradicirajućim metodama liječenja mlađih bolesnika uz autolognu transplantaciju matičnih stanica čini proučavanje odjeljka ostatnih matičnih stanica u KLL okosnicom istraživanja. Ispitivali smo broj CFU-GM i BFU-E pri koncentraciji zasijavanja od 10E5 MNC/ml, te broj CD34+ stanica u krvi (PK) i koštanoj srži (KS), kao i odnos broja matičnih stanica pri raspodjeli bolesnika po modificiranoj Rai klasifikaciji. Obrađeno je 46 neliječenih bolesnika s KLL, 23 muškarca i 23 žene starosti 37-78 godina (medijan 66). Kod 42 bolesnika medijan BFU-E iznosio je 19 (0-80), a u PK 5 (1-49). Broj usmjerenih matičnih stanica u Ks i PK bolesnika s KLL snižen je u odnosu na referentne vrijednosti. Broj CFU-GM u KS veći je od broja CFU-GM u PK (p

Određivan je učinak tiorfana na hematopoezu u dugotrajnoj kulturi koštane srži (LTBMC). Tiorfan je inhibitor membranske metaloendopeptidaze (CD10). Krioprezervirani buffy coat autologne koštane srži bolesnice s karcinomom ovarija stavljen je u dugotrajnu kulturu (LTC). Neatherirajuće stanice izvađene kod tjednog dohranjivanja izbrojane su, citomorfološki pregledane i testirane u GM-CFU testu klonogenosti. Tiorfan je dodan u kulturu kod prvog i ostalih dohranjivanja u koncentraciji 10-8, 10-10 i 10-12 M po bočici. Kontrolna dugotrajna kultura (LTC) je postavljena s KS-i normalnog davatelja. Ukupna celularnost i klonogenost natherirajućih stanica očekivano se smanjivala za vrijeme kultiviranja, ali više u kulturi bolesnice. U kulturi bolesnice bilo je malo klonogenih GM-CFU stanica, stromalni sloj se slabije raazvio i makrofazi su dominirali u neatherentnoj populaciji stanica čineći 87%, 92%, 87% i 100% redom u 2, 3, 4 i 5. tjednu kultivacije. Dodani tiorfan povisio je postotak makrofaga na 90-92% u drugom tjednu, 96-97% u trećem tjednu i 97-100% u četvrtom tjednu. Normalna dugotrajna kultura (LTC) sadržavala je 37-53% makrofaga tokom 5 tjedana. Visok postotak makrofaga u dugotrajnoj kulturi bolesnice s karcinomom ovarija vjerojatno odražava rezidualno oštećenje hematopoeze antineoplastičkim lijekovima. Učinak tiorfana na stanice u kulturi pripisuje se smanjenom prerađivanju citokina i neuropeptida u kulturi zbog inhibicije membranske metaloendopeptidaze CD10.

The aim of the study was to analyze the relationship between the CD34+ and CFU-GM content in leucocyte products during CD34+ selection (Ceprate, CellPro) in patients undergoing autologous transplantation. A total of 37 patients were analyzed, including 12 pts. With non-Hodgkin lymphoma, 11 with breast carcinoma, 10 with multiple myeloma, 3 with Mb. Hodgkin and 1 pt. with ovarian cancer. Mobilization protocol consisted of cyclophosphamide, BEAM or VIP-protocol, all in combination with G-CSF. Leukaphereses contained a median of 6.95x10E6 CD34+/kg b.w. (2-7-40.6) and 20.1x10E4 CFU-GM/kg b.w. (1.1-117.8) (r=0.412, p

We determined the in vitro cytotoxic activity of the sesquiterpene lactone endoperoxide artemisinin (1) and some chemically prepared derivatives, which have been found to display cytotoxicity to cloned murine Ehrlich ascites tumour (EAT) cells and human HeLa cells and against murine bone marrow using a clonogenic assay for committed progenitor cells of the granulocyte-monocyte lineage (CFU-GM assay). Comparing artemisinin (1) to deoxyartemisinin (2), the endoperoxide group appeared to play a role in cytotoxicity to CFU-GM cells. Dimers of dihydroartemisinin and dihydrodeoxyartemisinin revealed that the stereochemistry of the ether linkage of the dimers was a more important determinant for this cytotoxic activity. The nonsymmetrical dimer of dihydroartemisinin (3) and the corresponding endoperoxide-lacking dimer of dihydrodeoxyartemisinin (5) were equally cytotoxic to CFU-GM cells. Despite the differences between both systems, it may be stated that most compounds displayed higher cytotoxicity to CFU-GM cells than to EAT cells. Dimers of dihydroartemisinin (3, 4) were selected as potential antitumour compounds and subjected to the National Cancer Institute drug-screening programme consisting of about sixty human cancer cell lines derived from nine different tissues. Both compounds displayed the same specific cytotoxicity pattern. Throughout the screen dimer 3 was more active than 4.

Some studies in mice suggest that hematopoietic stem cells can be maintained and possibly expanded ex vivo. As there is a paucity of data from larger animals, we have studied hematologic reconstitution following autologous marrow transplantation in cats. Transplantation of very low density marrow cells (1.050 g/ml), termed "1050 cells," at 2 x 10(5) cells/kg leads to rapid hematopoietic recovery (granulocytes200/microl by day 20+/-2 and platelets50 x 10(3)/microl by day 21+/-3). Recovery rates are comparable when 1-2 x 10(7) nucleated marrow cells/kg are infused, suggesting that reconstituting cells are enriched 50- to 100-fold in the 1050 cell preparation. To explore if the numbers of reconstituting cells could be expanded ex vivo, 1050 cells were cultured in the presence of 5 ng/ml recombinant human interleukin 1beta, 10 ng/ml recombinant canine (rc)G-CSF, 2 U/ml rHu erythropoietin, and 5 ng/ml rc stem cell factor. Maximum numbers of BFU-E and colony-forming units-granulocyte/macrophage (CFU-GM) were generated at day 6. However, when 10(6) 1050 cells/kg (5x that needed for hematologic recovery) were cultured for six days and all resulting cells infused into irradiated donor animals, two of nine (22%) engrafted. Even when flt3 ligand (100 ng/ml) was added to cultures, only two of five animals (40%) engrafted (p = NS versus studies without flt3 ligand). These data confirm that BFU-E and CFU-GM provide inaccurate estimates of reconstituting cells and demonstrate that the number or function of feline reconstituting cells is impaired by in vitro culture with cytokines.

Optimal timing of peripheral blood progenitor cell (PBPC) harvest to collect maximal stem cell numbers is unknown. We assessed the progenitor cell content in 128 PBPC harvests from 21 patients primed with chemotherapy and granulocyte macrophage-colony stimulating factor (GM-CSF) in relation to absolute neutrophil count (ANC) at collection time. Samples were obtained by leukapheresis during rebound from chemotherapy-induced neutropenia while receiving GM-CSF, and assayed by flow cytometry for CD34+ and by colony assays for CFU-GM and BFU-E. The CD34+ cell concentrations per sample tended to be greater at an ANC 3 and decreased with rising ANC (p = 0.001). The CFU-GM and BFU-E concentrations per sample remained relatively constant with rising ANC (p = 0.72, p = 0.90, respectively). Total CD34+ cell number per harvest per kg slightly increased with ANC levels (p = 0.044) whereas the total CFU-GM and the total BFU-E per kilogram increased more modestly with rising ANC (p < 0.001, p < 0.001, respectively). We conclude that after priming with chemotherapy and GM-CSF, PBPC could be collected at different absolute neutrophil counts without greatly affecting total CD34+ cell numbers. The greater concentration of CD34+ progenitor cells at a lower ANC together with the CFU-GM and BFU-E peaking with higher ANC suggest a less mature progenitor cell population at lower ANC levels.

We have studied the regulatory capacity of clonal T-lymphocytes from patients undergoing autologous bone marrow transplantation (ABMT) on the generation of CFU-GM from their harvest marrow preparations To this end, T-lymphocytes from peripheral blood from 5 patients undergoing ABMT isolated 10 d before and 7, 14 and 28 d post-ABMT were placed in limiting dilution conditions (384 wells for each patient at each time point) and polyclonally stimulated. From more than 1600 wells with growth from the 5 patients, preparations from more than 900 wells could be expanded (range between patients 33-452) and identified by immunophenotyping (IP) and flow cytometry (FCM) by their exclusive expression of CD4 or CD8. This was significantly fewer than seen in normal donors, especially so at d 7 and 14 post-ABMT. The ratio between CD4 + and CDB + clones varied between 0.6 and 2.8 (median 1.3) and was significantly lower in the patients compared to normal donors (median 3.1; range 3.0-6.5). When the clonal T-cell preparations were co-cultured with autologous bone marrow cells obtained at the time of harvest and depleted for T-lymphocytes, the vast majority of both CD4 + and CD8 + clones exerted a clear enhancement on the CFU-GM growth with no relation to time of blood sampling in its the magnitude. Moreover, a trend seen in the normal donors towards CD4 + clones being more effective in this enhancement was not observed in ABMT patients. We conclude that clonal T-cells from ABMT patients, irrespective of their phenotype and time of isolation, exert an enhancement on the growth of autologous CFU-GM, which is equal to that seen in normal donors.

It is well established that 1,25(OH)2D3 induces monocyte/macrophage (Mo/Mphi) colonies when added to culture of granulocyte/macrophage progenitors. Recently, we demonstrated that one of the target cells of 1,25(OH)2D3 in Mo/Mphi differentiation is the neutrophilic promyelocyte that is believed to belong to the neutrophilic lineage. This fact overthrows the established theory that normal hematopoietic precursors are committed to respective cell lineages and do not deviate from their own lineage. The lineage switching from the promyelocyte to Mo/Mphi was suggested to be operating in vivo because 1,25(OH)2D3 is a physiological substance produced by Mphi. More recently, we have shown that transient exposure (24 h) of promyelocytes to 1,25(OH)2D3 causes Mo/Mphi differentiation. This strategy could be useful for examining the effects of 1,25(OH)2D3 on the growth and differentiation of normal myeloblasts and myeloid progenitor cells. Recent advances in molecular biology have enabled investigators to identify a number of genes involved in Mo/Mphi differentiation induced by 1,25(OH)2D3. Some of these may be the determinant genes for Mo/Mphi differentiation; however, further studies are required to determine the underlying mechanisms of Mo/Mphi differentiation.

We investigated the influence of TNF alpha and its muteins III, V and VI on the colony growth of normal and CML CFU-GM cells as well as on CFU-L clonogenic blasts from AML patients in cultures in vitro. The muteins were constructed using a synthetic cDNA fragment substituting nucleotides coding for the N-terminal amino acids in the chain of native TNF alpha. We observed that TNF alpha and mutein VI inhibited the growth of colonies formed by both types of CFU-GM and CFU-L cells in a greater degree than muteins III and V. In addition, the percentage of colony growth inhibition after the use of mutein VI was the greatest. These observations were the basis for the evaluation of interaction between 2-CdA and TNF alpha and between 2-CdA and mutein VI. We have confirmed that 2-CdA used together with mutein VI acts synergistically on CFU-GM and CFU-L cells, whereas 2-CdA and TNF alpha show the additive effect.

Determinations of committed haemopoietic progenitor cells, namely CFU-GM (colony-forming unit-granulocyte/macrophage) and of CD34-expression haemopoietic cells as assessed by multiparameter flow cytometry are routine diagnostic tools in haemopoietic cell therapy. Generally, the tests are used to optimise the timing and management of cytapheresis and to assess the engraftment potential of the harvested cells. Both measurements, however, are at best surrogate markers, as an adequate routine test which effectively assesses the short- and long-term repopulating haemopoietic cell is not available. Nonetheless, cell threshold doses have been established. Above these thresholds rapid engraftment is almost invariable but below these thresholds the outcome is variable. In this study we have focussed on the imprecision in counting haemopoietic cells, as assessed as CFU-GM and as CD34-expressing cells. The data on both tests have been analysed from six European institutions. The coefficient of variation in CFU-GM colony counting was about 30%, whereas the coefficient of variation in flow cytometric counting of CD34-expressing cells was about 10%. These data suggest that the technical imprecision in enumerating progenitor cells, particularly CFU-GM, at low levels, might make a major contribution to the clinical variability observed after transplantation of sub-threshold progenitor cell dose.

The results for colony forming unit granulocyte-macrophage (CFU-GM) assays vary substantially between centres. It is possible that colony counting is largely responsible for this discrepancy. In order to examine this exclusively from the many factors that make up the CFU-GM assay, we performed a colony counting exercise involving 11 laboratories. Two-way analysis of variance showed a highly significant difference (P = 0.0001) in the counts obtained from the centres. One centre was found to score consistently high and two others scored consistently low numbers of colonies. This suggests that identification of colonies is a major source of variation between centres.

Background Cell signaling mediated by the JAK2-STAT (Janus kinase 2-signal transducer and activator of transcription) pathway plays a critical role in hematopoiesis, and its aberrant activation is associated with the progression of hematological malignancies. For instance, somatic mutations in the JAK2 gene that lead to constitutive activation of STATs are involved in the pathogenesis of myeloproliferative neoplasms (MPN) and refractory anemia with ringed sideroblasts with thrombocytosis, one of the myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). In addition, insufficient inactivation of the JAK-STAT pathway due to repression of SOCS-1 (suppressor of cytokine signaling 1) through hypermethylation of the SOCS-1 gene is involved in the disease progression of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (Brakensiek et al.; Br. J. Haematol. 2005;130:209). These observations prompted us to investigate the effect of the JAK2-selective inhibitor NS-018, which is in an early-phase clinical trial for MPN, on the colony formation by bone marrow hematopoietic progenitor cells from high-risk de novo MDS patients and the phosphorylation status of STAT3 in these cells. Methods Bone marrow mononuclear cells (BMMNCs) from six MDS patients and three healthy volunteers were collected with informed consent in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. The MDS subtypes of the six patients included two RCMD, three RAEB-1 and one RAEB-2 according to the WHO classification. All MDS patients had complex cytogenetic abnormalities and their prognostic risks were defined to be high (N=1) or very high (N=5) in the IPSS-R. Commercially available normal human CD34+ BM cells were also examined for comparison. Cells were incubated in methylcellulose medium containing cytokines with or without NS-018. Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) were scored on day 14 of culture. Colony-forming cells were then collected and subjected to western blotting analysis. Results We first examined the colony-formation capacity of MDS-derived BMMNCs. CFU-GM colony formation was observed in four of six MDS samples, although the absolute CFU-GM numbers from MDS BMMNCs were less than those from normal BMMNCs and normal CD34+BM cells. MDS-derived BMMNCs produced larger numbers of CFU-GM colonies (78–99% of total colonies) than BFU-E, similar to the findings usually observed in AML, whereas normal BMMNCs and normal CD34+BM cells formed virtually the same numbers of CFU-GM colonies and BFU-E colonies. These results suggest that BMMNCs from four of the MDS patients enrolled in this study had characteristics similar to AML BMMNCs, although they showed less proliferation than AML BMMNCs. We next examined the effect of NS-018 on CFU-GM colony formation. NS-018 treatment decreased the numbers of CFU-GM colonies from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent against MDS-derived than against normal cells (57.2% inhibition in MDS cells vs. 12.8% inhibition in normal cells at 0.5 mM NS-018; see Figure). These results indicate that NS-018 is preferentially efficacious in inhibiting CFU-GM formation from BMMNCs from high risk MDS. In addition, the level of phospho-STAT3 in MDS-derived colony-forming cells was twice as high as in colony-forming cells from normal BMMNCs, and 1.0 mM NS-018 completely suppressed the phosphorylation of STAT3 in CFU-GM colony-forming cells from MDS. Conclusion Our results show for the first time that a JAK2 inhibitor, NS-018, potently suppresses the formation of MDS colonies. NS-018 could be a new therapeutic option for high-risk MDS patients. Disclosures: Kodama: Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Nakaya:Nippon Shinyaku: Employment. Taniwaki:Novartis: Honoraria.

We describe an 'in vitro' model which permits assessment of acute myeloblastic leukaemia (AML) progenitor cells' response to Ara-C alone and in conjunction with recombinant human (rh) cytokines by evaluating cell cycle characteristics of purified AML blast progenitors and their chemosensitivity in clonogenic culture before and after cytokine priming. Parallel investigation of Ara-C/cytokine treatment on normal CFU-GM progenitors was included as these cells are important for post-therapeutic reconstitution of haemopoiesis. Kinetic and clonogenic findings for AML marrows were extremely variable with G+GM-CSF or IL-3 priming. However, inclusion of rhSCF and resultant synergism with the other cytokines, introduced a pronounced element of consistency in AML results. Normal CFU-GM responded to rhG+GM-CSF or IL-3 with increased kinetic activity and sensitivity to Ara-C. rhSCF synergised strongly with the other factors, causing increased cell cycling and attainment of maximal chemosensitivity 'in vitro'. No correlation was evident between 'in vitro' findings for AML samples, patient FAB types or clinical outcome. This study highlights the fact that both normal and AML cells can be targeted by rh cytokines, particularly when rhSCF is included in priming cocktails.

Platelet numbers and circulating haemopoietic progenitor cells were examined in 12 patients with advanced malignancies who were receiving recombinant human interleukin-6 (rhIL-6) as part of an investigation of its thrombopoietic effects. Patients received recombinant glycosylated IL-6 by daily subcutaneous injection for 7 consecutive days in doses of 1, 3 or 10 micrograms/kg/day. Platelet numbers increased reaching a peak on days 12-15 with a mean on day 15 of 198.1% of pre-treatment values. This was accompanied by a significant fall in the mean platelet volume (mean decrease of 10.6%, P = 0.0044). No significant correlation was seen between the IL-6 dose and the change in platelet number. No significant differences were observed between pre- and post-treatment levels of circulating erythroid burst-forming units (E-BFU) and granulocyte macrophage colony-forming units (GM-CFU) but a small significant increase was seen in circulating primitive progenitor cells measured in a plastic-adherent (P delta) assay (P = 0.025). As positive controls, a group of patients treated with cyclophosphamide/G-CSF showed significant increases in GM-CFU (P = 0.018), E-BFU (P = 0.018) and P delta progenitors (P = 0.028). These data suggest that the thrombopoietic effects of IL-6 are mediated at a relatively late stage via effects on megakaryocyte differentiation, with a relatively small effect on circulating haemopoietic progenitors.

During the past few years, hematopoietic cell culture technologies for transplantation therapies have progressed significantly on several fronts. Advances include the discoveries of the growth factors thrombopoietin and Flt-3 ligand, the development of a variety of bioreactor systems, and results from preliminary clinical trials that demonstrate the efficacy of ex vivo expanded hematopoietic cells for transplantation therapy.

The effects of bone marrow (BM) and peripheral blood progenitor (PBPC) concentration during cryopreservation on subsequent hematopoietic engraftment following high-dose chemotherapy were studied in 24 patients. Seventeen BM harvests and 71 PBPC collections were performed between July 1991 and June 1994. The PBPC were frozen at significantly higher cellular concentrations than the BM (medians of 243 x 10(6)/ml versus 73 x 10(6)/ml respectively, p = 0.0003). The recovery of committed progenitor cell colonies (CFU-GM) was significantly lower from PBPC frozen at concentrations above the median, compared with 116% from those frozen at concentrations below the median (p = 0.0467). This phenomenon was not seen in BM, which was generally frozen at a threefold lower concentrations. Despite the lower recovery of CFU-GM when PBPC were frozen at a higher concentration, the patients receiving these grafts achieved good hematopoietic recovery. The higher number of PBPC probably compensated for the loss, and the patients still received a substantial number of clonogenic hematopoietic precursors.

We have compared the CFU-GM growth observed in 29 PBSC samples (8 HL, 21 NHL) to that obtained with 30 bone marrow (BM) “buffy coats” harvested for an autologous BMT program in pts without BM involvement (25 AML, 3 HL, 2 NHL). The CFU-GM tests were performed before cryopreservation, made without noncontrolled heat fusion, and after thawing. The collections of PBSC were made with a CS3000 Plus (Baxter). The apheresis were performed after mobilisation with HD CTX 4 g/m2 and administration of G-CSF (6–15 days). The cultures of CFU-GM were made in methylcellulose for PBSC and in agar monolayer for BM cells, using a conditioned medium from 5637 cell line as source of CSF. Before freezing a mean the CFU-GM growth of from BM cells were higher than in PBSC (35.5 vs 26: P

The effect of granulocyte colony-stimulating factor (G-CSF) on accelerating the rate of neutrophil recovery after peripheral blood stem cell transplantation (PBSCT) remains controversial.We retrospectively analyzed 37 patients who received high-dose chemotherapy followed by PBSCT. Patients were divided into three groups: those who received no G-CSF (control), a low dose (50 mg/m2), or a high dose (150 mg/m2), subcutaneously. Seven patients who received increased numbers of colony forming units-granulocyte macrophage (CFU-GM) (>100x104/kg) and either low-or high-dose G-CSF were separately analyzed. Neutrophil recovery (0.5 x109/1) was accelerated in the G-CSF-treated patients. It occurred at a median of 10 and 9 days in the low-and high-dose G-CSF groups compared with 14 days in the control group (p< .0001). In patients receiving increased numbers of progenitor cells, the time until recovery was 9 days. No statistically significant differences were observed for platelet and reticulocyte recovery, transfusions, days febrile and parenteral antibiotic requirement. Thus, the administration of low-dose G-CSF is recommended in PBSCT.

Bizelesin is a potent synthetic derivative of the anticancer agent CC-1065 that preferentially alkylates and binds the minor grove of DNA. Preclinical animal studies have found bizelesin to be more toxic to beagle dogs than to rodents and that myelosuppression was the dose-limiting toxicity. This toxicity was dose- and time-dependent in all species. Due to the significant difference in the in vivo myelotoxicity between species, it was important to determine which one most closely resembles humans on a pharmacodynamic basis. Therefore, hematopoietic clonal assays were utilized to evaluate the effects of bizelesin on granulocyte-macrophage (CFU-gm) colony formation. Marrow cells were exposed in vitro to bizelesin (0.001-1000 nM) for 1 or 8 h and then assayed for colony formation. There was a 3-log difference in drug concentration at which 100% colony inhibition occurred (1 or 8 h) for murine CFU-gm versus human or canine CFU-gm. The IC70 value after an 8-h bizelesin exposure for human CFU-gm (0.006 +/- 0.002 nM) was 2220-times lower than for murine CFU-gm (13.32 +/- 8.31 nM). At any given concentration, an 8 h drug exposure resulted in greater colony inhibition than a 1 h exposure for all species (P0.05). Increasing exposure time from 1 to 8 h increased toxicity to human and canine CFU-gm much more than to murine CFU-gm. The clinically formulated drug solution was a more potent inhibitor of human colony formation than drug dissolved in DMSO. The IC70 value after a 1-h exposure was 1.7 times lower for human CFU-gm with formulated bizelesin (0.106 +/- 0.105 nM) than bulk drug in DMSO (0.184 +/- 0.044 nM). The results of these in vitro clonal assays were qualitatively consistent with those seen in whole animal studies, suggesting that bizelesin will be a potent myelosuppressive agent in the clinic. Since the dose-limiting toxicity in preclinical models is myelosuppression and the in vitro sensitivity of human and canine CFU-gm is similar, the canine maximum tolerated dose (MTD) is better than the murine MTD to determine a safe starting dose for phase I clinical trials.

Human colony forming units (CFUs) from both malignant and hematopoietic tissues can be assayed in vitro in microcapillary cultures, an alternative cloning system to the Petri dish methodology. For technical reasons, microcapillary culture may be ideally suited for new drug screening by therapeutic index. To achieve the high output required by screening programs, automated quantitation of CFUs is required. Toward this end, this paper reports the development of a prototype CapScan, an image analysis system that uses a novel axial laser illumination system to detect tumor cell colonies and, with technical modifications, CFU-granulocyte-macrophage (CFU-GM) colonies in microcapillary cultures. As currently configured, the CapScan can quantify colonies grown in a rack of 18 microcapillary cultures in 30 minutes or less. The sensitivity and detection specificity of tumor cell colonies is >90% with a coefficient of variance of 5-40%, dependent upon colony number. Over a range of colony numbers, CapScan and manual colony counts showed a linear correlation > -0.9, and yielded identical results in assays of doxorubicin inhibition of clonogenic P388 cells. As an additional advantage, the growth kinetics of individual colonies can also be monitored with the CapScan, making distinctions between cytotoxic and cytostatic drugs possible; colonies of freshly isolated human tumor cells can also be quantified. Thus, a microcapillary-based human tumor cloning assay that tests for resistance and/or sensitivity to chemotherapeutic agents may be useful in drug development programs and may also facilitate the development of chemotherapy for individual patient tumors, especially when tumor availability is limited.

Inhibition of normal hemopoiesis is a regular finding in acute (AML) and chronic (CML) myelogenous leukemias and functional abnormalities of the hemopoietic microenvironment may be involved in this regard. In order to evaluate this possibility we studied the formation of adherent stromal cell layers (ASCL) in long term bone marrow cultures (LTBMC) of 7 patients with CML and 7 patients with AML and examined the ability of these ASCLs to support hemopoiesis after irradiation and a second inoculation of bone marrow cells. The formation of ASCLs was significantly impaired in CML and AML. These CML patients and 3 AML patients did not form typical ASCLs and the cellularity of these layers was greatly reduced. Colony forming unit granulocyte-macrophage (CFU-GM) production from bone marrow cells seeded on normal irradiated ASCLs peaked at week 3 and then gradually decreased by week 8. In CML and AML cocultures CFU-GM numbers decreased rapidly to zero by weeks 4-6 and did not differ significantly from the control cultures which did not contain preestablished ASCLs beginning from week 3. It is suggested that there may be a functional microenvironmental defect in CML and AML that may play a role in the pathogenesis of inhibition of normal hemopoiesis in these diseases.

We have previously reported that serum stem cell factor (SCF) levels are significantly lower in patients with myelodysplastic syndrome (MDS) than normal controls. We have now studied the effects of adding SCF to cultures of blood mononuclear cells from patients with MDS and normal subjects. Three of 17 patients with MDS showed marked increases in erythroid colony numbers with SCF + erythropoietin compared to interleukin-3 + erythropoietin. In two cases (1RA and 1ISA) the erythroid colony numbers became normal. The same RA patient also showed a marked increase in myeloid colony numbers, which were undetectable with granulocyte-macrophage colony-stimulating factor alone, but within the normal range when SCF was added. A fourth patient (ISA) showed a 4.7-fold increase in myeloid colonies with SCF, but no erythroid response. The normal subjects showed a trend towards increased numbers of myeloid colonies with SCF; erythroid colonies did not increase. A correlation was found between the MDS patients' haemoglobin levels and erythroid colony numbers with and without SCF, but there was no correlation between erythroid or myeloid colonies in the presence of SCF and their serum SCF level.

Previous studies have indicated a decline in bone marrow progenitor cell function in subjects aged 75–82 years, possibly causing lower Hb concentrations. We studied the bone marrow with in vitro colony assays and cytogenetic analysis in 24 apparently healthy 88-year-olds with Hb concentrations ranging from moderate anaemia to normal levels. Twenty-two healthy younger subjects, aged 21–57 years, were used as a control group. The 88-year-olds showed significantly lower numbers of myeloid bone marrow progenitors than the controls, and the elderly men had lower numbers of both erythroid and myeloid progenitors than the elderly women. There were no in vitro growth differences between elderly subjects with “low” or “normal” Hb concentrations. Ten out of 14 men had bone marrow cells with a missing Y-chromosome, which did not seem to have any relationship to the erythroid function. No morphological or other cytogenetic indications of a clonal progenitor cell disorder were found. A more rapid decline in Hb concentrations in healthy elderly men as compared to elderly women might be explained by differences in bone marrow progenitor cell function. However, progenitor cell abnormalities do not seem to explain differences in Hb concentrations within groups of apparently healthy men and women of advanced age.