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We have originally reported that colony-forming units granulocyte/macrophage (CFU-GM) formation is an in vitro feature of chronic myelomonocytic leukemia (CMML) and a strong predictor for short survival. Elucidation of the molecular basis underlying this in vitro phenomenon could be helpful to define molecular features that predict inferior outcome in patients. We studied the correlation between the mutational landscape and spontaneous colony formation in 164 samples from 125 CMML patients. As compared to wildtype samples, spontaneous in vitro CFU-GM formation was significantly increased in samples containing mutations in NRAS, CBL and EZH2 that were confirmed as independent stimulatory factors by multiple regression analysis. Inducible expression of mutated RAS but not JAK2 was able to induce growth factor independence of Ba/F3 cells. Whereas high colony CFU-GM growth was a strong unfavorable parameter for survival (p &lt, 0.00001) and time to transformation (p = 0.01390), no single mutated gene had the power to significantly predict for both outcome parameters. A composite molecular parameter including NRAS/CBL/EZH2, however, was predictive for inferior survival (p = 0.00059) as well as for increased risk of transformation (p = 0.01429). In conclusion, we show that the composite molecular profile NRAS/CBL/EZH2 derived from its impact on spontaneous in vitro myeloid colony formation improves the predictive power over single molecular parameters in patients with CMML.

Gamma interferon inducible lysosomal thiol reductase (GILT), is known to be involved in immunity, but its role in hematopoiesis has not been previously reported. Herein, we demonstrate using gilt knockout (-/-) mice that loss of gilt associates with decreased numbers and cycling status of femoral hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) with more modest effects on splenic progenitor cells. Thus, GILT is associated with positive regulation of hematopoietic progenitor cells in mice, mainly in bone marrow.

The investigational anticancer agent batracylin (BAT; 8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) causes γ-H2AX foci development in exposed tumor cells and has demonstrated activity against solid tumors and adriamycin-resistant leukemia. Reports indicate BAT has wide interspecies variation of adverse effects, including myelosuppression, kidney, bladder, and liver damage, including biliary hyperplasia. The effects of BAT and its metabolite N-acetyl batracylin (NAB) were evaluated in the CFU-GM bone marrow toxicity assay, rat kidney (NRK) cells, bladder epithelial (RBLAK) cells, and rat precision cut liver slices (PCLS). Exposure effects were evaluated biochemically and histologically. Human, dog, and rat exhibited similar CFU-GM IC90 values for BAT (21–29 μM). The ATP assay and γ-H2AX staining showed time- and concentration-dependent toxicity in RBLAK (more severe than NRK at IC50<20 μM after 96 hr BAT exposure). BAT (5 μM and 25 μM) caused biochemical and histology changes to PCLS by day 3 and 25 μM produced centrilobular hepatotoxicity. NAB (≤5 μM) produced no toxicity in CFU-GM, NRK, or RBLAK cells. However, both BAT and NAB caused biliary epithelial cell proliferation in PCLS. Our studies demonstrated species similarities in sensitivity to BAT-induced myelosuppression, and implicate the metabolite NAB in biliary hyperplasia.

The reports regarding the mutual influence between the central nervous system and the immune system constitute a vast and somewhat controversial body of literature. Stress is known to disturb homeostasis, impairing immunological functions. In this study, we investigated the hematopoietic response of Chlorella vulgaris (CV)-treated mice exposed to single (SST) and repeated stress (RST). We observed a reduction in the numbers of hematopoietic progenitors (HP) in the bone marrow and long-term bone marrow cultures (LTBMC) using flow cytometry and a coinciding decrease in the number of granulocyte–macrophage colonies (CFU-GM) after treatment with both stressors, but SST caused a more profound suppression. We observed a proportional increase in the colony-stimulating activity (CSA) of the serum of animals subjected to SST or RST. In the bone marrow, SST and RST induced a decrease in both mature myeloid and lymphoid populations but did not affect pluripotent hematopoietic progenitors (Lin−Sca-1+c-kit+, LSK), and again, a more profound suppression was observed after SST. We further quantified the levels of interleukin-1α (IL-1α) and interleukin-6 (IL-6) and the number of myeloid cells in LTBMC. Both SST and RST reduced the levels of these cytokines to similar degrees. The myeloid population was also reduced in LTBMC, and SST induced a more intense suppression. Importantly, CV treatment prevented the changes produced by SST and RST in all of the parameters evaluated. Together, our results suggest that CV treatment is an effective tool for the prophylaxis of myelosuppression caused by single or repeated stressors.

Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3mg/m3 FA for 2weeks, mimicking occupational exposure, and were monitored for another 7days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk.

The regulation of blood cell production (hematopoiesis) by CCN proteins is an area of increasing interest to hematologists. There is some discordance in the literature in this area due to the use of mixed or ill-defined cell populations for experiments. Expression of, and response to, CCN proteins is specific to both cell type and differentiation status. Here, we describe methods to prepare defined hematopoietic cell populations and associated functional assays.

Chlorella vulgaris (CV) was examined for its modulating effects on the reduction induced by lead (Pb) on the numbers of marrow hematopoietic stem cells (HSCs) (c-Kit(+)Lin(-)), granulocyte-macrophage progenitors (Gr1(+)Mac1(+)) and total bone marrow cellularity. In mice gavage-treated daily with 50mg/kg dose of CV for 10 days, concomitant to a continuous offering of 1300 ppm lead acetate in drinking water, the treatment with the algae recovered the significantly reduced numbers of these cell populations to control values. As CV may have a myelostimulating effect through the induction of cytokines, we evaluated its modulating effects on the production of IL-1α, TNF-α, IFN-γ, IL-10 and IL-6. Our results demonstrated that lead significantly impairs the production of IFN-γ, IL-1α and TNF-α and increases the production of IL-10 and IL-6 and that these effects are successfully modulated by the CV treatment. The activity of NK cells, reduced in Pb-exposed animals, was raised to levels higher than those of controls in the exposed group treated with CV. Treatment with the algae also stimulated the production of IFN-γ, IL-1α, TNF-α and NK cells activity in normal mice. In addition, zinc bone concentrations, reduced in lead-exposed mice, were partially, but significantly, reversed by the treatment with CV.

Proton pump inhibitors (PPI) are being proposed as potent antitumor agents, owing to their ability to specifically induce tumor cell death by reversing H+ ion homeostasis. As tumor growth induces myelosuppression in tumor-bearing hosts, it remains unclear if PPI can also modulate tumor-induced myelosuppression. Thus, we studied the effect of in vivo administration of pantoprazole (PPZ), a PPI, on myelopoiesis in a murine model of a transplantable T cell lymphoma, designated as Dalton’s lymphoma (DL). Intraperitoneal administration of PPZ to tumor-bearing mice resulted in an enhanced bone marrow cellularity, inhibited induction of apoptosis and augmented bone marrow cell (BMC) survival. BMC of PPZ-administered tumor-bearing mice showed elevated number of F4/80 positive cells, augmented colony forming ability and differentiation in bone marrow-derived macrophages (BMDM) with higher expression of F4/80 and CD11c markers. This study also presents evidences to indicate that PPZ-dependent augmentation of myelopoiesis in the tumor-bearing host is dependent on an enhanced expression of M-CSF and receptors for M-CSF & GM-CSF in BMC, along with a modulation in the expression of cell survival regulatory molecules PUMA, Bcl2, p53 and caspase-activated DNase (CAD). BMDM obtained from PPZ-administered tumor-bearing mice also showed an augmented expression of TLR-2, tumoricidal activity, production of NO and monokines: IL-1, IL-6 & TNF-α. The study discusses the possible mechanisms underlying PPZ-dependent augmentation of myelopoiesis. Taken together, the present study proposes that a PPZ-dependent alleviation of tumor-induced myelosuppression could contribute to an augmented myelopoiesis.

Ethnopharmacological relevance The Ashaninkas, indigenous people of Peru, use cat's claw (Uncaria tomentosa) to restore health. Uncaria tomentosa has antioxidant activity and works as an agent to repair DNA damage. It causes different effects on cell proliferation depending on the cell type involved; specifically, it can stimulate the proliferation of myeloid progenitors and cause apoptosis of neoplastic cells. Neutropenia is the most common collateral effect of chemotherapy. For patients undergoing cancer treatment, the administration of a drug that stimulates the proliferation of healthy hematopoietic tissue cells is very desirable. It is important to assess the acute effects of Uncaria tomentosa on granulocyte-macrophage colony-forming cells (CFU-GM) and in the recovery of neutrophils after chemotherapy-induced neutropenia, by establishing the correlation with filgrastim (rhG-CSF) treatment to evaluate its possible use in clinical oncology. Materials and methods The in vivo assay was performed in ifosfamide-treated mice receiving oral doses of 5 and 15 mg of Uncaria tomentosa and intraperitoneal doses of 3 and 9 μg of filgrastim, respectively, for four days. Colony-forming cell (CFC) assays were performed with human hematopoietic stem/precursor cells (hHSPCs) obtained from umbilical cord blood (UCB). Results Bioassays showed that treatment with Uncaria tomentosa significantly increased the neutrophil count, and a potency of 85.2% was calculated in relation to filgrastim at the corresponding doses tested. An in vitro CFC assay showed an increase in CFU-GM size and mixed colonies (CFU-GEMM) size at the final concentrations of 100 and 200 μg extract/mL. Conclusions At the tested doses, Uncaria tomentosa had a positive effect on myeloid progenitor number and is promising for use with chemotherapy to minimize the adverse effects of this treatment. These results support the belief of the Ashaninkas, who have classified Uncaria tomentosa as a ‘powerful plant’.

Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001-50microg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3-3mg/kgb.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data. We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC(50) values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC(50): 2.45+/-1.08 and 1.82+/-0.8microM for humans, and 11.08+/-2.92 and 1.81+/-0.20microM for mice, respectively), than the mature CFU-E (AFB1 IC(50): 12.58+/-5.4 and 40.27+/-6.05microM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.

The effect of aging on the myelopoietic progenitor cell (CFU-GM) numbers in mice was studied with special emphasis on biological rhythms in hemopoiesis. Female C3H mice, 16-, 21- and 26-month-old, were investigated versus 3-month-old controls every 3 hours during the 24-h period at three different times of the year. Strong circadian rhythms were observed in both CFU-GM concentration and content in the femur of all age-groups. The amplitudes and the 24-h mean values were declining in mice aged 21 and 26 months. From 16 months of age, a significant advance of circadian peak phases was observed. The overlapping was variable during the 24-h period. The present results may explain previous inconsistencies regarding myelopoietic progenitor cell numbers in aging mice. Some seasonal differences in rhythmicity patterns were also observed. Fitting of original data to single sinus functions was highly significant, although important details sometimes were obscured.

The density distribution in Percoll gradients of clonogenic cells forming colonies in diffusion chambers (CFU-D) or in agar culture (CFU-GM) was studied in chronic myeloid leukaemia (CML). The density distribution of CFU-GM in CML was homogeneous with peaks within 1.058-1.061 g/ml, which is slightly lower than normal. CFU-D, on the other hand, showed heterogeneous distributions both in CML and in normal controls. Two separable populations of CFU-D were recognized, one with the same or lower density than CFU-GM that formed almost exclusively neutrophilic colonies in diffusion chambers, and a second population concentrating in the density range 1.068-1.075 g/ml which primarily formed macrophage colonies. The second population contained colony-forming cells derived from both Fc-receptor positive and Fc-receptor negative precursor cells, suggesting that at least some colonies in diffusion chambers arise from Fc-receptor positive granulopoietic cells of intermediate maturity and/or monocytes. The concentration of CFU-D in peripheral blood was increased 40- to 100-fold in 2 patients currently off treatment who had increased WBC counts.

This paper reports on the production of tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor (GM-CSF) by cultured mononuclear adherent cells derived from bone marrow of 25 patients affected by myelodysplastic syndrome (MDS) of different FAB subtypes. Mean production of GM-CSF was much lower than in controls, without significant differences among different subtypes. Mean production of TNF was similar in MDS patients and in controls, but noteworthy differences were observed between patients with RA, RAEB and RAEB-t and patients with RARS and CMML. Growth of bone marrow granulocyte macrophage and erythroid progenitors did not correlate with TNF and GM-CSF production, although in MDS subtypes with higher GM-CSF levels, colony growth was slightly higher than in subtypes with lower GM-CSF production.

The radiosensitivity of human cord blood haematopoietic stem cells was measured by assessing colony formation in methylcellulose cultures and diffusion chambers. The results show that fetal colony forming units, granulocytic, monocytic (CFU-GM), are radiosensitive: colonies formed by electron-irradiated CFU-GM, both in spontaneous and in colony stimulating factor (CSF) supplemented cultures decrease in number in an exponential fashion with increasing doses of irradiation. Do values up to 800 rads were, for highly purified fetal lymphoid cells, 160 +/- 88 rads; for whole buffy coat cells, 155 +/- 75 rads; and for buffy coat cells plus exogenous CSF: 155 +/- 77 rads. Extrapolation numbers (N) approached the value of 1. Among cord blood diffusion chambers progenitor cells (DCPC), radiosensitive and radioresistant subpopulations of myelopoietic stem cells exist, as tested over a range up to 1500 rads. Monocytopoietic CFU-D are radioresistant.

A synthetic analog of a hemoregulatory peptide associated with mature human granulocyte (HP5B) has been investigated for inhibitory effects on human myelopoietic stem cells in vitro. In addition, it has been tested for effects on phagocytosis by human granulocytes and monocytes by use of an automatic flow cytometric method. A dose-dependent inhibition of colony formation was found after preincubation of bone marrow cells for 1 h at 37 degrees C in the range 10(7) -10(-11) mol/l. Above or below these concentrations, no inhibitory effects were seen. The degree of inhibition varied from experiment to experiment, indicating variable responsiveness of the donor cells. Maximal effect was of magnitude 90% inhibition, and the optimal dose was 10(7) mol/l. The peptide had no effect on the kinetics of phagocytosis by measurements of the uptake of fluorescent Zymosan particles or Staphylococcus aureus. This may indicate a selective effect on the precursor cells, with no effect on the functional state of their progeny, the granulocytes and monocytes.

The expression of CD33 and CD34 antigens on human bone marrow cells was examined by fluorescence-activated cell sorting and colony assays. A marked difference of antigen expression was observed on sorted progenitors of the granulocyte/macrophage lineage (CFU-GM) with respect to enrichment and maturation status. Single-color cell sorting revealed no difference of enrichment by anti-CD33 antibody between day-7 and d-14 progenitors, while anti-CD34 antibody preferentially enriched d-14 colony-forming units (CFU). By two-color cell sorting it could be shown that enrichment of d-14 CFU-GM occurred mostly in the CD34+CD33- and to a lesser extent in the double-positive fraction. In contrast, there was no difference in degree of enrichment between d-7 and d-14 CD34-CD33+ CFU-GM. From these data we conclude that, during early myelopoiesis, CD34 antigens are gradually lost while CD33 antigens are acquired.

Changes occurring in human haematopoiesis with advancing age were studied using an in vitro haematopoietic colony assay in 22 elderly subjects with unexplained anaemia, and in 15 elderly and 15 young subjects without anaemia. Both elderly groups were found to have significantly lower numbers of bone marrow early erythroid-committed progenitors (BFU-E) than the young controls. The elderly anaemic group also showed significantly lower numbers of granulocyte/macrophage progenitors (CFU-GM) than the young controls. However, the responses of the erythroid-committed progenitors in the elderly groups to erythropoietin and burst-promoting activity were similar to those observed in the young controls. Therefore, it is probable that anaemia tends to occur easily in elderly individuals as a result of reduction of reserves of haematopoietic progenitors with advancing age.

A contiguous segment attached to the cord blood unit (CBU) is required for verifying HLA types, cell viability, and, possibly, potency before transplantation since such a segment is considered to be representative of the CBU. However, little is known regarding the characteristics of contiguous segments in comparison to main bag units due to the difficulty experienced in accessing a large number of cryopreserved CBUs. In this study, we used 245 nonconforming CBUs for allogeneic transplantation. After thawing the cryopreserved CBU, the number of total nucleated cells (TNCs), CD34+ cells, and CFUs in CB from main bags and segments, as well as cell viability and apoptosis, were examined. The comparative analysis showed that the number of TNCs was significantly higher in CB from main bags, whereas the numbers of CD34+ cells and CFU-GM were significantly higher in CB from segments. While the cell viability of TNCs in segments was higher, the proportion of apoptotic TNCs was also higher. In contrast, no difference was observed between the proportion of apoptotic CD34+ cells in main bags and segments. In the correlation analysis, the numbers of TNCs, CD34+ cells, and CFU-GM in main bags were highly correlated with those in segments, indicating that CB from segments is indeed representative of CB in main bags. Taken together, we conclude that segments have higher CD34+ cells and CFU-GM and lower TNCs than the main cryopreserved bag, although the two compartments are highly correlated with each other.

In this work, a first attempt to set-up a new in vitro experimental protocol with culture in liquid medium and flow cytometry analysis of bone marrow progenitors is described. This protocol is proposed as an alternative to the colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic in vitro assay currently used to assess the toxic potential of new drugs in the bone marrow. This new experimental approach should enable to speed up the procedure of the in vitro haematotoxic potential assessment, to reduce inter-experimental variability and to enhance result accuracy. Preliminary results obtained demonstrated that the progenitor cell count by flow cytometry replacing the light microscopy granulocyte/macrophage colony count represents a tremendous improvement in terms of accuracy and standardisation. Moreover, differential counts of cell sub-populations can be performed by using specific monoclonal antibodies. Furthermore, this method demonstrated to be time-saving, since 4 day cell incubation period is required instead of 7-14 day incubation in the CFU-GM clonogenic assay. On the basis of results obtained so far, the new experimental protocol proposed looks a promising alternative to the CFU-GM clonogenic assay currently used.

In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34(+) cells, and granulocyte-macrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34(+) cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P=0.136 and 0.417, respectively) or platelets (P=0.88 and 0.126, respectively). However, the reinfused viable CD34(+) cells/kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P=0.0001 and 0.001, respectively) and platelets (P=0.023 and 0.010, respectively), whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P=0.011 and 0.007, respectively) but not to platelets (P=0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34(+) cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34(+) cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.

Human osteoclasts can be efficiently generated in vitro from cord blood mononuclear cells and derived CFU-GM colonies. However, CFU-M colonies are poorly osteoclastogenic. Short-term (2–48 h) treatment with GM-CSF stimulates osteoclast formation by proliferating precursors, whereas longer exposure favors dendritic cell formation. Introduction: Osteoclasts (OC) differentiate from cells of the myelomonocytic lineage under the influence of macrophage-colony stimulating factor (M-CSF) and RANKL. However, cells of this lineage can also differentiate to macrophages and dendritic cells (DC) depending on the cytokine environment. The aims of this study were to develop an efficient human osteoclastogenesis model and to investigate the roles of granulocyte macrophage-colony stimulating factor (GM-CSF) and M-CSF in human OC differentiation. Materials and Methods: A human osteoclastogenesis model, using as precursors colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from umbilical cord mononuclear cells cultured in methylcellulose with GM-CSF, interleukin (IL)–3 and stem cell factor (SCF), has been developed. CFU-GM, colony forming unit-macrophage (CFU-M), or mixed colonies were cultured on dentine with soluble RANKL (sRANKL) and human M-CSF with and without GM-CSF. Major endpoints were OC number, dentine resorption, and CD1a+ DC clusters. Results: Osteoclast generation from CFU-GM and mixed colonies treated with M-CSF and sRANKL for 7–14 days was highly efficient, but CFU-M colonies were poorly osteoclastogenic under these conditions. Pretreatment of precursors with M-CSF for 7 or 14 days maintained the precursor pool, but OCs were smaller and resorption was reduced. The effect of GM-CSF treatment was biphasic, depending on the timing and duration of exposure. Short-term treatment (2–48 h) at the beginning of the culture stimulated cell proliferation and enhanced OC formation up to 100%, independent of sRANKL. Longer-term GM-CSF treatment in the presence of sRANKL, however, inhibited OC generation with the formation of extensive CD1a+ DC clusters, accompanied by downregulation of c-Fos mRNA. Delaying the addition of GM-CSF resulted in progressively less inhibition of osteoclastogenesis. Conclusions: Human CFU-GM, but not CFU-M, progenitors have high osteoclastogenic potential. GM-CSF plays an important role in osteoclastogenesis and has a biphasic effect: Short-term treatment potentiates OC differentiation by proliferating precursors, but persistent exposure favors DC formation.

Amifostine is a phosphorylated aminothiol that has besides anti-oxidative and cytoprotective properties, also survival- and growth-promoting effects on hematopoietic progenitor cells. Clinical studies have demonstrated that infusions with amifostine are able to increase erythro-, myelo-, and thrombopoiesis in some patients with myelodysplastic syndromes (MDS). Since clonal and non-clonal progenitors can coexist in early phase MDS, we have studied if amifostine exerts a selective growth-promoting effect on clonal or non-clonal cells. For this purpose, purified CD34(+) marrow progenitors from nine female MDS patients were grown in short- and long-term cultures. Clonality was studied on individual colonies using polymorphisms in the human androgen receptor assay (HUMARA) locus. Three patients had growth of residual non-clonal progenitors at baseline. Continuous exposure to 100nM amifostine exerted a growth-promoting effect on progenitors in 50% of the patients. HUMARA patterns of individual colony-forming unit granulocyte macrophage (CFU-GM; 5/9) and 5 week long-term culture-initiating cells (LTC-IC; 2/9) were compared without and with amifostine exposure. We did not observe preferential stimulation of clonal or non-clonal progenitors. Based on these results, the stimulation of committed and immature progenitor growth in MDS by amifostine, is non-selective and does not favor nor suppress the growth of residual non-clonal cells.

A haematotoxicity model was proposed by Parchment in 1998 to predict the maximum-tolerated dose (MTD) in humans of myelosuppressive antitumour agents by combining data from in vitro clonogenic assays on haematopoietic progenitors and in vivo systemic exposure data in animals. A prospective validation of this model in humans was performed with PNU-159548, a novel agent showing selective dose-limiting myelosuppression in animals. PNU-159548 and its main metabolite, PNU-169884, were tested in vitro on murine, canine and human colony forming units-granulocyte macrophages (CFU-GM) and in vivo on mice and dogs. The IC(90x) ratios (IC(x)=concentration inhibiting x% of colony growth) for CFU-GM and drug plasma protein binding were used to adjust the target plasma concentrations versus time curve (AUC) and predict the human MTD. The predicted MTD was compared with values achieved in phase I studies. Canine CFU-GM were 6-fold more sensitive (P0.01) and murine CFU-GM 1.7-fold less sensitive (P0.05) to PNU-159548 treatment than the human progenitors. PNU-169884 behaved similarly to PNU-159548. The predicted MTDs in humans calculated from data in mice and dogs were 15 and 38 mg/m(2), respectively. Overall, 61 patients were treated in two phase I studies, at doses ranging from 1.0 to 16 mg/m(2). Thrombocytopenia was dose-limiting with a MTD of 14 and 16 mg/m(2) in heavily and minimally pretreated/non-pretreated patients, respectively. Adjusting animal MTD data by means of the CFU-GM ratio between species can predict the human MTD with a good quantitative accuracy. Inhibition of common haemopoietic progenitors by PNU-159548 induced neutropenia/thrombocytopenia in animals and thrombocytopenia in patients, probably due to the higher sensitivity to the compound observed in human colony forming units-megakaryocyte (CFU-MK).

Objective: To evaluate the effects of cryopreservation of APBSCs in −80°C un-controlled freezer and liquid nitrogen, and to search for the efficient combined cryoprotectant and the highest cell concentration for cryopreservating peripheral blood stem cell (PBSC). Methods: To compare the effect of three combined cryoprotectants, evaluation of in vitro proliferative capacity by colony-forming unit-Granulocyte-macrophage (CFU-GM) and burst-forming Unit-erythroid (BFU-E) assay, immunophenotyping for CD34+/CD38− cells by FCM, and viability assessment by trypan blue exclusion were performed. Results: The cryoprotectant 10%DMSO+HES+auto-Plasma resulted in the highest recovery rates. CFU-GM and BFU-E were (78.7±9.8)%, (69.8±14.1)%, respectively. The recovery rates of CFU-GM and BFU-E in A/C groups were (68.3±6.2)%/ (65.8±7.2)% and (63.4±9.7)%/ (60.4±10.5)%, respectively. The recovery rates of CD34+/CD38− cells and cell viability with the three combined reagents were 90% and 80%, respectively. Conclusion: 5%DMSO+ HES+auto-PLASMA is an ideal combined cryoprotectant for cryopreserving PBSC. The cell concentrations may be cryopreserved at 4×108 ml−1.

Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10(6) cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10(5) cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10(6) /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10(5) cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.

Aplidine is a cyclic depsipeptide that was isolated from a Mediterranean marine tunicate, Aplidium albicans. In experimental animals, Aplidine mediated an in vivo inhibitory effect in a number of tumor cell types. In humans, Aplidine is currently used in phase I clinical trials. Aiming to predict the hematotoxicity of Aplidine in humans, samples from human bone marrow (BM) and cord blood (CB) were exposed in vitro to increasing concentrations of the drug and then assayed for the clonogenic ability of myeloid (CFU-GM), erythroid (BFU-E), megakaryocitic (CFU-Meg) and pluripotent (CFU-Mix) hematopoietic progenitors. We investigated whether predictions of the hematotoxicity of Aplidine based on bone marrow (BM) cultures were reproduced when a more readily available source of human hematopoietic cells, cord blood cells, was used in experiments involving 24-h exposures. Although hematopoietic progenitors derived from bone marrow were generally more sensitive than those derived from cord blood, differences on the IC50, IC70 and IC90 varied within a relatively small range of 1.6-6.2-fold. Moreover, data obtained from cord blood cultures confirmed the observation made in bone marrow assays indicating that the myeloid (CFU-GM) and the erythroid (BFU-E) progenitors were the least sensitive to Aplidine. Regardless of the origin of the hematopoietic progenitors (bone marrow or cord blood) the toxicity of Aplidine in human hematopoietic progenitors (IC50: 150-2250 nM) was lower than that observed in previous studies with tumoral cell lines.

In pharmaceutical research,in vitro toxicity tests, for assessing the potential toxicity of new chemical entities are necessary in the early stages of the developmental process, when no information is available about the metabolism or even the target organ toxicity of the compounds to be tested.In vitro specific organ toxicity tests, such as the granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay, are useful tools for predicting the adverse effects of new compounds on the blood-forming system, provided that some reference points are available, e.g., toxicological information about compounds belonging to the same chemical class and structure–activity relationship data. Furthermore, when no information is available about metabolism, thein vitro system should cover as many possibilities as possible, to avoid false positive or false negative results. In fact, while many compounds are metabolized to a variety of inactive chemical species, some undergo bioactivation to form more active metabolites. The addition of a metabolic activation system to the CFU-GM assay enables assessment of direct and metabolism-mediated toxicity. The regulatory agencies and industry value the concept of assays performed with and without metabolic activation, since they often have to take decisions about compounds with unknown mechanisms of action. CFU-GM assay, designed in this way, is an example of such a mechanism-naive assay.

The aim of the study was to investigate the influence of gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) used alone and in combination with 2-chlorodeoxyadenosine (2-CdA) on the colony growth of normal granulocyte-macrophage progenitor cells (CFU-GM) from 15 hematologically healthy donors as well as on CFU-GM from 15 chronic myelogenous leukemia (CML) patients in semisolid cultures in vitro. dFdC and 2-CdA were used either separately or in the following combinations of concentrations: (1) 0.25 nM of dFdC and 2.5 nM of 2-CdA; (2) 0.5 nM of dFdC and 5 nM of 2-CdA; (3) 1 nM of dFdC and 10 nM of 2-CdA; (4) 2 nM of dFdC and 20 nM of 2-CdA. We observed that both dFdC and 2-CdA used separately inhibited the growth of colonies formed by normal and CML CFU-GM cells in a dose-dependent manner. Moreover, the combined therapy with dFdC and 2-CdA caused statistically significant inhibition of the colony growth in both normal and CML CFU-GM cultures. In addition, the inhibition of CML CFU-GM colony formation was greater and statistically significant in the case of the combined therapy using higher concentrations of these drugs as compared with inhibition of the growth of normal CFU-GM colonies. These observations were the basis for the evaluation of the interaction type between dFdC and 2-CdA. We have shown that dFdC used in a combination with 2-CdA acts in an additive way on normal and CML CFU-GM cells.

Estrogen is thought to contribute to the onset of systemic lupus erythematosus (SLE) in women through mechanisms that are not completely understood. Although estrogen serves as a negative regulator in normal hematopoietic development, little research has been conducted examining alteration in hematopoietic development triggered by estrogen in lupus-susceptible individuals. We examined whether estrogen and other factors could influence colony formation of bone marrow cells obtained from normal and lupus-susceptible mice. Bone marrow cells isolated from New Zealand Black (NZB) and lupus-prone New Zealand Black and New Zealand White cross (NZB/W) mice were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) alone or in combination with estrogen, thrombopoietin (TPO), tamoxifen, estrogen and TPO, or estrogen and tamoxifen, and plated in methylcellulose culture medium. Plates were scored for the number of CFU-GM (colony forming unit granulocyte-macrophage) colonies after 6 d in culture. For females of both mouse strains, estrogen significantly decreased (P < 0.05) the number of GM colonies. Co-treatment of NZB/W cells, but not NZB cells, with TPO or tamoxifen reversed the suppressive action of estrogen (P < 0.05). In contrast, while estrogen did suppress colony formation from cells of NZB/W males (P < 0.05), neither TPO nor tamoxifen reversed this effect. Our results indicate that the sensitivity of bone marrow cells isolated from both female and male NZB/W lupus-prone mice to hormones/growth factors is qualitatively different from cells of NZB mice, and suggest that hematopoietic alterations at the level of the bone marrow may be related to the pathogenesis of SLE.

Bone marrow stromal microenvironment is essential for the maintenance of the hematopoietic stem cell renewal both by cell-cell interaction and cytokine productioN. However, stromal cells also exhibit drug metabolizing activities and they may accumulate the drug and successively affect hematopoietic progenitors by a retarded release. Our study investigated the role of both primary culture of murine bone marrow stroma and established stromal cells (SR-4987) in modulating the “in vitro” toxic activity of Doxorubicin (DXR) against murine granulocyte-macrophage progenitors (CFU-GM). The main part of the study has been performed by a “in vitro” agar bilayer technique based on the CFU-GM assay performed over a feederlayer of stromal cells. The results suggest that bone marrow stromal cells play also an important role in decreasing the toxicity of Doxorubicin. Further SR-4987 stromal cells produce a Doxorubicin metabolite (not belonging to the series of metabolites described in literature) which is completely ineffective in inhibiting the growth of CFU-GM and the activity of topoisomerase I. Our data suggest that bone marrow stromal cells must be considered as a cell population having opposite pharmacological roles in modulating the drug toxicity on hematopoietic progenitors. In our model a mechanism of detoxification concerns the capacity of SR-4987 stromal cells to inactivate the drug. For a better prediction of drug hematotoxicity, it is very important to develop “in vitro” cell models able to discriminate between positive and negative modulation of drug toxicity that stromal cells can exert in the bone marrow microenvironment.

Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.

Semisolid agar cultures of committed granulocyte monocyte precursors (CFU-GM) make it possible to study the regulation of normal and abnormal stem cells. In vitro growth of CFU-GM is dependent on the presence of specific stimulators, called CSA (colony stimulating activity) (Metcalf, 1977; Broxmeyer & Moore, 1978). The influence of CSA on CFU-GM may be inhibited by prostaglandins of the E series (Broxmeyer & Moore, 1978; Kurland et al, 1978). Recently it has been demonstrated that chronic granulocytic leukaemia (CGL) CFU-GM are insensitive to prostaglandin E1 (PGE1) over a wide range of concentrations that are inhibitory to normal CFU-GM development (Aglietta et al, 1980; Pelus et al, 1980; Taetle & Koessler, 1980). We report the effect of PGE1 on CFU-GM from patients with idiopathic myelofibrosis (MF).

The colony-forming units granulocyte and macrophage (CFU-GM) assay, using either rat or dog haematopoietic progenitor cells, assesses the toxicity of new compounds. To identify the characteristics of colony formation in this system, a time-course study of CFU-GM assays using rat and dog bone marrow cells was tested. Neutrophil colonies, macrophage colonies and mixed colonies of neutrophils and macrophages were formed in soft agar medium. Neutrophil colonies reached their maximum number on days 3–4 and decreased markedly thereafter. Macrophage colonies reached their maximum number on days 7–8 and remained steady thereafter. Only a small number of mixed colonies of neutrophils and macrophages were formed beginning around day 4. There were no significant differences between rat and dog bone marrow cells in the occurrence of these maxima, or in any other growth phenomenon. This result suggests that to evaluate the influence of compounds on neutrophil colonies and macrophage colonies, observations should be made on days 4 and 8, respectively.

A class of primitive progenitor cells with high proliferative potential in vitro (HPP-CFC), has been identified in fetal liver and adult bone marrow both in murine and human systems. The kinetic properties of HPP-CFC2 and the more mature granulocyte-macrophage colony forming units (CFU-GM) derived from murine fetal liver on d13, d15 and d19 of gestation, newborn liver and neonatal liver on d3 and d8 postpartum have been evaluated and compared with the kinetic properties of these progenitor cell populations derived from adult bone marrow. The frequency of HPP-CFC2 in fetal liver was found to be greatest on d15 of gestation then subsequently declined in newborn and neonatal liver. Similarly, the highest proportion of HPP-CFC2 engaged in DNA synthesis (53+/-3%) was detected in d15 fetal liver. This proportion decreased to 13+/-2% in the liver 1 wk after birth, which is comparable to the number of HPP-CFC2 derived from adult BM which were in S-phase (10+/-1%). Production of CFU-GM was found to be greater in adult bone marrow than in either fetal or newborn liver. While the proportion of CFU-GM in S-phase was high all 3 tissue samples, the greatest proportion of cycling CFU-GM (50+/-2%) was detected in d15 fetal liver. These results suggest that HPP-CFC2 derived from fetal liver are actively cycling while HPP-CFC2 derived from adult bone marrow are relatively quiescent. In contrast, a high proportion of CFU-GM derived from fetal, newborn liver and adult bone marrow are engaged in DNA synthesis.

The biological target for interferon (IFN)-alpha in chronic myeloid leukemia (CML) is unknown, but one possibility is that amplification of granulocyte-macrophage colony-forming cells (CFU-GM) is reduced. Replating CFU-GM colonies and observing secondary colony formation provides a measure of CFU-GM amplification. Amplification of CML, but not normal, CFU-GM in vitro was significantly inhibited by IFN-alpha (P = 0.02). In 5 out of 15 CML cases studied by fluorescence in situ hybridization, in vitro treatment with IFN-alpha increased the proportion of CFU-GM, which lacked BCR-ABL. The ability of patients' CFU-GM to amplify, and suppression of this ability by IFN-alpha, predicted responsiveness to IFN-alpha therapy in 86% of cases. Investigation of patients on treatment with IFN-alpha showed a threefold reduction in CFU-GM amplification in responders (P = 0.03) but no significant change in nonresponders (P = 0.8). We conclude that IFN-alpha preferentially suppresses amplification of CML CFU-GM to varying degrees. The differing in vitro sensitivities to IFN-alpha and growth kinetics of individual patients' cells could help differentiate those who will or will not benefit from treatment with IFN-alpha.

We evaluated in vitro how growth factors influenced the effect of cytostatic drugs on normal hematopoietic progenitor cells. Bone marrow was obtained from 15 donors for bone marrow transplantation. After separation the mononuclear fraction was incubated with granulocyte colony-stimulating factor (G-CSF) at 5 and 50 ng/ml, with granulocyte/macrophage colony-stimulating factor (GM-CSF) at 1 and 10 ng/ml, and with interleukin 3 (IL-3) at 0.5 and 5 ng/ml for 24 h prior to incubation with cytostatic drugs. These incubations were performed with 0.05 microM mitoxantrone and 0.2 microM daunorubicin for 1 h, and cells were thereafter cultured for colony-forming units--granulocyte/macrophage (CFU-GM) in soft agar for 10-12 days. Incubation with 0.05 microM cytosine arabinoside was performed continuously throughout the culture period. The proliferation of normal hematopoietic progenitor cells stimulated with GM-CSF at 10 ng/ml and with IL-3 at 5 ng/ml was significantly increased to 218% and 215% colonies, respectively, as compared with the control stimulated with conditioned medium only. Stimulation with G-CSF, on the other hand, did not induce any significantly enhanced proliferation relative to the control. Daunorubicin applied in combination with G-CSF at 5 ng/ml or with IL-3 at 0.5 ng/ml exerted a significantly higher degree of cytotoxicity on normal hematopoietic progenitor cells, resulting in 21% and 30% surviving colonies as compared with the 38% recorded for daunorubicin alone (P0.05). Neither G-CSF nor IL-3 at a higher concentration nor GM-CSF exerted a significantly altered degree of toxicity relative to cells incubated with daunorubicin alone. The cytotoxic effect exerted on normal hematopoietic cells by mitoxantrone or ara-C was unchanged or significantly decreased after stimulation with growth factors as compared with the effect on cells incubated with cytostatic drugs alone. We conclude that G-CSF and IL-3 augment the effect of daunorubicin on normal hematopoietic progenitor cells.

We have investigated the effect of PGE 2 on the growth of human blood CD34 + hematopoietic progenitor cells. Cultivation of cells in liquid culture for 3 days with PGE 2 (100–10 nM) stimulates their [ 3 H]thymidine incorporation into DNA. Semisolid cultures of these cells reveal the growth of an elevated number of BFU-E and CFU-GM colonies as compared with controls. Addition of PGE 2 (10 nM) in methycellulose cultures immediately after purification results in an elevated cloning efficiency of both BFU-E and CFU-GM. Finally, by using limiting dilution assays we demonstrated that PGE 2 acts directly at a single cell level. These results indicate, for the first time, that PGE 2 may enhance human erythropoiesis and granulopoiesis by a direct effect on human CD34 + progenitor cells.

Summary The pteridine neopterin (NP) was shown to be produced by monocytes and has been known as a useful maker of immunological activation, although, the biological activity of this agent is still unclear. To elucidate the biological function of NP, the changes in the number of blood leukocyte, bone marrow and spleen hemopoietic progenitor cells after intraperitoneal administration of NP into mice were investigated. Administration of NP increased the number of blood leukocytes about 2 fold higher than that of the control at days 7 and 14. Blood films, made by smearing samples of peripheral blood, showed the increment of granulocytes in blood of NP treated animals. The number of granulocyte-macrophage progenitor cells (CFU-GM) was also increased in both bone marrow and spleen in mice with NP administration. Extensive study showed that NP stimulated the hematological recovery in bone marrow transplanted animals. All these findings suggest that NP has a stimulating activity on hemopoiesis by affecting the proliferation and differentiation of hemopoietic progenitor cells in vivo.

Recurrence of leukemia is a major problem after autologous stem cell transplantation. One potential means of reducing this risk is to purge the autologous transplant in vitro by hyperthermia. We have demonstrated that after a hyperthermic treatment of 120 min at 43 degrees C, the leukemic progenitor cells (CFU-AML) are decreased by 5-log but the normal hematopoietic committed progenitor cells (CFU-GM, BFU-E and CFU-E) are reduced by only 1-log. Moreover, the hyperthermic sensitivity coincides with the stem cell hierarchy, ie CFU-GM are less heat sensitive than BFU-E, while CFU-E are the most sensitive. The impact of pretreatment with the tetrapeptide AcSDKP (Goralatide) on the proliferative activity and heat sensitivity of the normal and leukemic progenitor cells was determined. An incubation of 21 h at 37 degrees C with 10(-9) M Goralatide reduces the number of normal hematopoietic progenitor cells in S-phase and concomitantly decreases their hyperthermic sensitivity. This effect implies that the proliferative activity is the major determinant for the detected differences in hyperthermic sensitivity of the subsets in the normal hematopoietic stem cell compartment. In contrast, the cell cycle progression of leukemic progenitor cells is not affected and hence these cells are not protected from hyperthermia-induced cell killing after preincubation with Goralatide. Thus, the treatment with Goralatide increases the therapeutic window of hyperthermia and increases the potential value of this physical purging technique.

All-trans retinoic acid (tRA) has been shown to promote terminal differentiation of promyelocytic leukemia cells, but frequently induce hyperleukocytosis and pulmonary leakage syndrome. Employing pentoxifylline (PTX), a phosphodiesterase inhibitor which could raise intracellular cAMP and modulate leukocyte activation, we sought to investigate if PTX could enhance tRA-induced promyelocytic leukemic cell differentiation but suppress tRA-augmented growth and activation of human granulocytes. tRA could significantly suppress clonal growth of U937 and HL-60 leukemic cells but enhanced the CFU-GM formation of normal bone marrow cells (22 ± 6 vs. 90 ± 16 CFU/well). PTX significantly augmented tRA suppression of clonal growth of U937 and HL-60 leukemic cells but suppressed tRA-augmented CFU-GM formation of normal bone marrow cells (90 ± 16 vs. 25 ± 9 CFU/well). In addition, PTX enhanced tRA-induced growth inhibition and differentiation of promyelocytic HL-60 leukemic cells, but suppressed respiratory burst activation by the immature granulocytic HL-60 cells and suppressed CD11b adhesion molecule expression by mature granulocytes. PTX similar to dibutyric cAMP promoted HL-60 myelocytic leukemic cell differentiation and growth inhibition, whereas PTX, in contrast to dibutyric cAMP which could augment phorbol myristate acetate (PMA)-elicited respiratory burst activity by immature granulocytes, suppressed the PMA-elicited respiratory burst activity by immature and mature granulocytes. PTX did not raise the intracellular cAMP level of HL-60 cells, but partly suppressed the dibutyric cAMP-elicited elevation of intracellular cAMP level. Results from these studies suggest that PTX might act through different signaling pathways to enhance tRA-induced myelocytic leukemic cell differentiation but prevent from hyperreactive normal granulopoiesis and granulocyte activation.

Following chemotherapy in chronic myeloid leukaemia (CML), some peripheral blood (PB) cells may be Philadelphia (Ph) chromosome negative. The BCR-ABL mRNA status of residual Ph+ progenitors is not known. We examined the BCR-ABL mRNA status of individual colony-forming-unit granulocyte-macrophage (CFU-GM) colonies derived from PB harvested following chemotherapy. Seven patients were treated with 200 mg/m2/day cytarabine and 20 mg/m2/day Idarubicin and followed by Lenograstim. PB collections commenced daily when the white blood cell count reached 0.6 x 10(9)/l and continued until at least 5 x 10(8)/kg nucleated cells were obtained. CD34+ cells, Ph status, and CFU-GM were estimated at each harvest. For each patient, up to 24 individual CFU-GM colonies were analysed for BCR-ABL status. Two cases were BCR-ABL negative on all colonies and completely Ph-, and another case was BCR-ABL positive in all colonies and completely Ph+. In contrast, in two patients all colonies were BCR-ABL negative, despite virtually complete Ph+ metaphases. The final assessible case had five of nine BCR-ABL negative colonies, despite 94% Ph+ metaphases. After chemotherapy priming, the PB may contain Ph+ CFU-GM that do not express BCR-ABL.

Purpose: 20(S)-Camptothecin (CAM), topotecan (TPT, active ingredient in Hycamtin) and 9-amino-20(S)-camptothecin (9AC) are topoisomerase I inhibitors that cause similar dose-limiting toxicities to rapidly renewing tissues, such as hematopoietic tissues, in humans, mice, and dogs. However, dose-limiting toxicity occurs at tenfold lower doses in humans than in mice. The purpose of the current study was to determine whether hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to these compounds that is evident in vivo. Methods: Drug-induced inhibition of in vitro colony formation by a myeloid progenitor in human, murine, and canine marrow colony-forming unit-granulocyte/macrophage (CFU-GM) provided the basis for interspecies comparisons at concentrations which inhibited colony formation by 50% (IC50) and 90% (IC90). Results: Murine IC90 values were 2.6-, 2.3-, 10-, 21-, 5.9-, and 11-fold higher than human values for CAM lactone (NSC-94600) and sodium salt (NSC-100880), TPT (NSC-609699), and racemic (NSC-629971), semisynthetic and synthetic preparations (NSC-603071) of 9AC, respectively. In contrast, canine IC90 values were the same as, or lower than, the human IC90 values for all six compounds. Conclusions: The greater susceptibility of humans and dogs to the myelotoxicity of camptothecins, compared to mice, was evident in vitro at the cellular level. Differential sensitivity between murine and human myeloid progenitors explains why the curative doses of TPT and 9AC in mice with human tumor xenografts are not achievable in patients. Realizing the curative potential of these compounds in humans will require the development of therapies to increase drug tolerance of human CFU-GM at least to a level equal to that of murine CFU-GM. Because these interspecies differences are complicated by species-specific effects of plasma proteins on drug stability, not all in vitro assay conditions will yield results which can contribute to the development of such therapies.

The effects of native thrombospondin (TSP), an 18 kd recombinant protein comprising residues 1-174 of TSP (TSP1-174) with heparin-binding domain and a fusion protein comprising residues 559-669 of TSP (TSP559-669) on murine hematopoiesis, were studied by using different in vitro culture systems. TSP by itself did not show an inhibitory effect on colony-forming unit-megakaryocyte (CFU-MK) growth in a serum-free agar system and on the growth of colony-forming unit-granulocyte and macrophage (CFU-GM) in a plasma clot system. It was, however, found that in the plasma clot culture system when using aplastic anemia serum as the source of thrombopoietin or C-Mpl ligand (TPO), TSP and TSP1-174, but not TSP559-669, were able to inhibit the growth of CFU-MK from unfractionated and lineage negative (Lin-) bone marrow cells in a dose-dependent manner. A statistically significant suppression was seen at 1 microg/ml of TSP and 5 microg/ml of TSP1-174. This inhibitory effect of TSP was further found in both the serum-free agar system and the plasma clot system without aplastic anemia serum, where megakaryocyte colony growth was stimulated by recombinant TPO, basic fibroblast growth factor (bFGF), or interleukin-3 (IL-3). In a methylcellulose system, where a combination of stem cell factor (SCF), IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were used, TSP inhibited the growth of colony-forming unit-granulocyte-erythroblast, megakaryocyte, and macrophage (CFU-GEMM) but not CFU-GM and burst-forming unit-erythroblast (BFU-E). Interestingly, this inhibitory effect of TSP on megakaryocyte colony growth could be counteracted by Fraxiparin, a low-molecular-weight heparin. These results demonstrate that TSP is a negative modulator of megakaryocytopoiesis and suggest that its inhibitory effect is at least partially mediated by N-terminal heparin-binding domain.

JAK2/STAT signaling promotes survival and expansion of myelodysplastic syndrome (MDS) clones, but little is known about the potential of JAK2/STAT as a therapeutic target in MDS. We investigated the effect of NS-018, a novel antagonist for JAK2, on the colony-forming ability of bone marrow mononuclear cells (BMMNCs) from high-risk MDS patients. NS-018 decreased colony-forming unit-granulocyte/macrophage (CFU-GM) colony numbers from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent than against normal BMMNCs. In addition, NS-018 suppressed the phosphorylation of STAT3 in colony-forming cells from MDS patients. Collectively, NS-018 could be a new therapeutic option for high-risk MDS.

Unstimulated methylcellulose cultures in 25 myelofibrosis (MF) patients were performed to better understand the role of cytokines in the proliferation of MF cells. Compared to controls MF patients show a variable but highly increased spontaneous CFU-GM formation (66 vs 4.8/10(5) PBMNC). There was a marked reduction of autonomous CFU-GM growth by the cytokine-synthesis-inhibiting molecule IL-10 as well as by antibodies against GM-CSF whereas antibodies against IL-3, G-CSF, M-CSF and IL-1β showed heterogeneous effects. Spontaneous CFU-GM growth >100/10(5) PBMNC predicted shorter survival. Constitutive release of GM-CSF seems to contribute to proliferation of MF cells in vitro and possibly in vivo.

Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, multiple dysmorphisms and congenital heart defects. A myeloproliferative disorder (NS/MPD), resembling juvenile myelomonocytic leukemia (JMML), is occasionally diagnosed in infants with NS. In the present study, we performed a functional evaluation of the circulating hematopoietic progenitors in a series of NS, NS/MPD and JMML patients. The different functional patterns were compared with the aim to identify a possible NS subgroup worthy of stringent hematological follow-up for an increased risk of MPD development. We studied 27 NS and 5 JMML patients fulfilling EWOG-MDS criteria. The more frequent molecular defects observed in NS were mutations in the PTPN11 and SOS genes. The absolute count of monocytes, circulating CD34+ hematopoietic progenitors, their apoptotic rate and the number of circulating CFU-GMs cultured in the presence of decreasing concentrations or in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated. All JMML patients showed monocytosis >1,000/μl. Ten out of the 27 NS patients showed monocytosis >1,000/μl, which included the 3 NS/MPD patients. In JMML patients, circulating CD34+ cells were significantly increased (median, 109.8/μl; range, 44–232) with a low rate of apoptosis (median, 2.1%; range, 0.4–12.1%), and circulating CFU-GMs were hyper-responsive to GM-CSF. NS/MPD patients showed the same flow cytometric pattern as the JMML patients (median, CD34+ cells/μl, 205.7; range, 58–1374; median apoptotic rate, 1.4%; range, 0.2–2.4%) and their circulating CFU-GMs were hyper-responsive to GM-CSF. These functional alterations appeared 10 months before the typical clinical manifestations in 1 NS/MPD patient. In NS, the CD34+ absolute cell count and circulating CFU-GMs showed a normal pattern (median CD34+ cells/μl, 4.9; range, 1.3–17.5), whereas the CD34+ cell apoptotic rate was significantly decreased in comparison with the controls (median, 8.6%; range, 0–27.7% vs. median, 17.6%; range, 2.8–49.6%), suggesting an increased CD34+ cell survival. The functional evaluation of circulating hematopoietic progenitors showed specific patterns in NS and NS/MPD. These tests are a reliable integrative tool that, together with clinical data and other hematological parameters, could help detect NS patients with a high risk for a myeloproliferative evolution.

The 'discordant maturation hypothesis' proposes that the most mature proliferating cells in chronic-phase chronic myeloid leukaemia (CML) are responsible for the expansion of the Ph-positive population. To evaluate this hypothesis we used a delta assay for primitive haemopoietic cells (P delta assay for P delta cells) which allows investigation of the kinetics of granulocyte-macrophage progenitor (CFU-GM) production. The frequencies of these primitive (P delta) cells were similar in CML blood (14.5/10(5) mononuclear cells), CML marrow (17.3/10(5)) and normal marrow (11.6/10(5)) The average frequency in normal blood is only 0.58/10(6). The absolute numbers of P delta cells in CML patients are therefore greatly increased. The average numbers of CFU-GM produced by individual P delta cells were reduced in CML blood (8.1) and marrow (11.6) compared with normal marrow (28.5). This is consistent with a reduced probability of differentiation at the single cell level in CML. Although the absolute number of CFU-GM produced by individual CML P delta cells was subnormal there was a relative increase in the number of day 7 CFU-GM compared with the number of day 14 and 21 CFU-GM, which agrees with the 'discordant maturation hypothesis'. This bias towards day 7 colony formation could reflect accelerated maturation by the CFU-GM produced by P delta cells or, alternatively, the production of CFU-GM with shorter than normal maturation pathways. Overall, these results suggest that discordant maturation does not by itself account for myeloid expansion in CML. It is more likely that myeloid expansion in CML is due mainly to an increase in the number of primitive haemopoietic progenitor cells.

The potent anti-neoplastic actions displayed in vitro by bryostatins have led to the introduction of short-term bryostatin-1 infusions in phase I clinical trials. Because bryostatin (bryo) undergoes a rapid clearance in vivo, we were interested in its scheduling effects on acute myeloid leukemia (AML) clonogenicity. Therefore, we assessed the primary plating efficiency (PE1) of AML samples in response to several bryo concentrations after various preincubation periods in a semi-solid colony forming assay. Whereas continuous exposure to 10 nM bryo during the assay period reduced the PE1 in nearly all samples tested, preincubation of eight AML patients' specimens for 1, 2, 3 or 4 days with bryo in a dose range of 0.1-10 nM showed a heterogenous PE1 response. Stimulatory as well as inhibitory effects on leukemic clonogenic growth were seen within individual specimens depending on dose and preincubation time with no single incubation time or concentration that caused unequivocal common overall inhibition of clonogenic growth in most or all of the samples. Higher doses of bryo also failed to result in specific inhibition of leukemic cells: 4/8 samples showed an increased clonogenic response to 250 nM bryo whereas normal bone marrow progenitor cells were consistently inhibited in their clonogenicity at this dose. A marked similarity, i.e. undulatory effects with increasing bryo concentrations, was found for HL60 leukemic cells. In conclusion, the effects of bryo on clonogenic leukemia cell growth are strongly dependent on scheduling and dose varying between and within individual AML samples. These results caution against in vivo bryo pulse therapy, as currently applied, for treatment of AML.

We investigated the effect of all-trans retinoic acid (ATRA) alone and in combination with interferon-alpha (IFN-alpha) on the granulocyte-macrophage (GM) colony formation of peripheral blood progenitors isolated from patients with chronic myeloid leukemia (CML) (n = 12) or other myeloproliferative disorders (n = 10) as well as from healthy controls (n = 7). The ATRA or IFN-alpha alone inhibited slightly, but not significantly, the GM colony growth in CML. Granulocyte-macrophage colony formation decreased significantly (P

In the present study, the ability of peripheral blood (PB) progenitor cells to form granulocyte-macrophage (GM) colonies spontaneously in methylcellulose was investigated in healthy controls and patients with myeloproliferative disorders (MPDs). Spontaneous colony formation was observed in only one of the 18 control cases (6%), but in 22 of the 29 MPD patients (76%). The incidence of spontaneous GM colonies correlated both with the number of blast cells and the amount of c-kit positive cells present in the initial sample. Spontaneous GM colony growth in PB mononuclear cells isolated from patients with MPDs seems to be a frequent phenomenon in contrast to the healthy controls and may present a marker of malignancy.