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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 mechanism for agranulocytosis induced by antithyroid drugs is not established. The few available studies have proposed an immune-mediated process against mature granulocytes. We investigated the effect of methimazole and propylthiouracil and serum from a patient with methimazole-induced agranulocytosis on marrow myeloid colony growth. In the presence of normal serum or patient's recovery serum, antithyroid drugs had no effect on the growth of CFU-GM colonies from normal or patient's marrow. However, the patient's serum obtained during agranulocytosis inhibited the in vitro myeloid colony growth from both autologous and allogeneic bone marrow. These results are compatible with an immune-mediated mechanism for methimazole-induced agranulocytosis rather than a direct toxic effect of the drug on abnormally sensitive cells.

Some characteristics of both erythroid and granulocyte monocyte progenitors in human cord blood were compared to those in adult blood and bone marrow. The number of progenitors in cord blood was higher than that in adult blood and bone marrow. Most colonies in cord blood culture were monocyte-macrophage, whereas those from adult blood were largely eosinophilic. Cord blood progenitors had a slower sedimentation velocity than that reported for marrow, but sedimented faster than that for adult blood. A significant proportion of progenitors in cord blood as well as adult marrow was found to be in the DNA synthetic phase of the cell cycle whereas progenitors in adult blood were not. Cord blood BFU-E were more resistant than adult blood BFU-E but cord blood CFU-GM were not different from adult blood CFU-GM with regard to radiation sensitivity. Cord blood CFU-GM appeared to be more radio-resistant than adult marrow GFU-GM. From these results is seems clear that progenitors in cord blood differ in some aspects from those in adult blood and bone marrow.

We report a case of dyskeratosis congenita ( DCG ) with neutropenia, lymphocytopenia and thrombocytopenia. Peripheral blood T lymphocytes (T cells) were proved to have a suppressive effect on the colony forming unit granulocyte-macrophage (CFU-GM). Splenectomy caused a transient increase of neutrophil count with the disappearance of the suppressive T cell activity. However, pancytopenia recurred without re-appearance of suppressive T cell activity.

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.

The effects of the composition of PBPC grafts from matched related donors (MRDs) and matched unrelated donors (MUDs) have not been compared. In a single-center study, the compositions of 55 MRD PBPC grafts and 33 MUD grafts were studied for their effect on the rate of engraftment in patients who had evidence of donor cell engraftment on day +28. The MUD grafts came more frequently from young male donors and contained more CD34(+) cells but similar numbers of colony-forming units granulocyte-macrophage (CFU-GM) and burst forming units-erythroid. The recovery of neutrophils to500/mm(3) was equally fast in both groups, but recovery of platelets to20,000/mm(3) was significantly delayed in the MUD group (P0.001). The MUD group also required more transfusions of platelets and red cells. Patients receiving grafts containing low numbers of CFU-GM had markedly delayed platelet recovery. The patients with the slowest engraftment tended to have prolonged transportation times. Storage experiments suggested a major loss of viable CD34(+) cells and CFU-GM when undiluted PBPC products are stored at room temperature. The data suggest that a fraction of the MUD grafts suffer during transportation. In vitro proliferation assays should be part of the validation and auditing of transportation of MUD grafts.

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 the last 20 years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods has approved the colony-forming unit granulocytes–macrophages test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime.

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.

Bone marrow (BM) suppression occurs following trauma/hemorrhagic shock (T/HS) in experimental animals as well as following severe injury in humans. Although the pathophysiology of BM suppression remains poorly understood, mesenteric lymph is thought to play an important role in T/HS-induced BM suppression; however, the direct effect of mesenteric lymph on BM in vitro has never been studied. In addition, recent studies in rats have also shown that female and castrated male rats are protected against T/HS-induced BM failure. We therefore hypothesized that mesenteric lymph is a source of factor(s) causing direct BM suppression and that the effects of mesenteric lymph are gender dependent. To test this hypothesis, we subjected noncastrated (NC) and castrated (C) male and proestrus female rats to T/HS or trauma sham shock (T/SS). Mesenteric lymph collected 3 h postshock was plated (4% v/v) with BM cells collected from unmanipulated male or female rats for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) colony growth. The T/HS lymph collected from NC-male rats but not from female rats caused a 50% inhibition of CFU-GM and BFU-E colony growth compared with cells cultured without lymph (P < 0.05 versus all other groups (ANOVA + Tukey). T/HS lymph collected from C-male rats also caused no significant inhibition of CFU-GM and BFU-E colony growth compared with cells cultured without lymph. Female and male BM progenitor cells had a similar response to mesenteric lymph from all groups tested. These results show that mesenteric lymph from NC-male rats suppresses CFU-GM and BFU-E progenitor growth in vitro, whereas the lymph from C-male and female rats did not. The effects of mesenteric lymph were the same regardless of whether the target BM was from male or female rats. The results therefore indicate that BM failure in male rats is directly mediated by factors present within the mesenteric lymph that appear to be modulated by castration, and protection against BM failure in female rats occurs at a systemic rather than a local level. Further studies are needed to elucidate potential therapeutic effects of lymph manipulation in hematopoiesis after injury.

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.

In vitro clonogenic assays have been developed and widely used since many years to investigate the proliferation and the differentiation both of pluripotent haemopoietic stem cells (PHSC) and of the different progenitors of blood cell lineages: megakaryocytes (Colony Forming Unit-Mk) granulocyte -macrophage (CFU-GM), erythrocytes (BFU-E/CFU-E). As these techniques have been introduced, they appeared to be very useful to investigate the pathogenic mechanisms of drug induced blood disorders and also for screening compound during preclinical safety study. Because the integrity of the hematopoiesis is also essential to guarantee the immunological function, the application to the toxicology of these clonogenic assays, provides an essential tool for better understanding the in vivo observation (experimental and clinical) by also helping in predicting the degree of a possible in vivo hematotoxic in drug treated patients. The review introduces to basic concepts on hematopoiesis and on classical evaluation of a drug hematotoxic phenomenon. It describes the application of each clonogenic test to assess the specific hematotoxicity action. Moreover it report the most recent studies on standardisation, prevalidation and validation of such assays and critically reviews a model for predicting myelotoxicity by discussing the main aspects referred to the evaluation of the in vivo acute neutropenia by using the in vitro CFU-GM assay.

In previous works, we have demonstrated that the myeloprotective properties of several natural and synthetic compounds are partly responsible for their antitumor activity in the Ehrlich ascites tumor (EAT) model. In this work, we present information that may be useful to the study of pharmacological and toxicological properties of compounds that affect the hematological compartment. Clonogenic studies in EAT-inoculated mice demonstrated a rapid decrease in bone marrow CFU-GM, whereas a progressive increase in splenic CFU-GM and cellularity was observed, followed by splenomegaly. Bone marrow cellularity declined on the third day after tumor challenge, returning to normal values thereafter. Serum from EAT-bearing mice produced detectable colony-stimulating activity in vitro. Similar results were observed with the conditioned medium from Ehrlich tumor cell cultures, but not with the cell-free Ehrlich tumor ascitic fluid. Tumor inoculation also resulted in a more striking depletion in the number of non-adherent cells in long-term bone marrow cell cultures (LTBMCs) with no bone marrow stroma formation. We speculate that the physiological alterations induced by the EAT growth can be used to assess the ability of compounds to modulate the hematopoietic response.

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.

Objective: The aim of this study was to clarify the appropriate timing for peripheral blood progenitor cells (PBPC) harvesting after platinum-based mobilization chemotherapy by measurement of the circulating CD34+ cell concentrations. Patients and Methods: PBPC were collected for autotransplantation via a total of 68 leukaphereses in 16 patients with gynecological cancer. Circulating CD34+ cell concentrations were measured by CD34-side scatter parameter analysis. Results: Data could be fitted into a linear regression line described by the equation y=0.33+4.14×x (R 2=0.256), where y=the number of harvested colony-forming unit granulocyte macrophage (CFU-GM (×105/kg) and x=the percentage of circulating CD34+ cells and y=1.747+44.53×x (R2= 0.475), where y=the number of harvested CD34+ cells (×106/kg) and x=the percentage of circulating CD34+ cells. Failure to mobilize sufficient CFU-GM numbers (>1×105/kg) occurred in 29 of 31 leukaphereses when the percentage of circulating CD34+ cells was less than 0.10%. Conclusions: Harvesting procedure may be avoided when circulating CD34+ cells showed less than 0.10% after platinum-based mobilization chemotherapy.

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.

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu. In the last twenty years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods (ECVAM) has approved the Colony forming Unit-Granulocytes-Macrophages (CFU-GM) test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip (LoC) platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime.

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.

BACKGROUND: Ex vivo expansion strategies with different cytokine combinations are currently used by several groups as a means of increasing the number of HPCs for a variety of special clinical applications. Because there is little information on the potential role of IL-10 in such ex vivo expansion models, the effect of this cytokine on the generation of myeloid progenitor cells in suspension cultures was investigated. STUDY DESIGN AND METHODS: On the basis of data from the literature and from new experiments, the combination of SCF and IL-3 at concentrations of 100 ng per mL and 100 U per mL, respectively, was chosen as the standard cocktail. The addition of IL-10 to such cultures resulted in a marked and dose-dependent potentiation of myeloid progenitor cell production. RESULTS: Using unmanipulated leukapheresis components from 13 individuals (including lymphoma and cancer patients and normal donors), the expansion multiple of CFU–GM after 14 days as compared with pre-expansion values was 9.54 ± 2.31 times by SCF/IL-3 and 46.38 ± 7.37 times by the combination of SCF/IL-3 and 100 ng per mL of IL-10 (p

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely spread environmental pollutant. Homopoietic system is one of the targets of TCDD in laboratory animals including monkeys. The present study is the hemopoietic cell kinetics in mice, from the severe depression in cellularity of bone marrow and CFU-GM, to their recovery after the intraperitoneal injection of high dosage of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The bone-marrow cellularity and CFU-GM were severely decreased to 37.8% and 48% of the control, respectively until day 1 after exposure to TCDD. They were, however, soon recovered, even overshot the control value. Subsequently, they tended to show decrease and oscillation again to and under the control value. In conclusion, our cell kinetic study has proven the oscillation in bone-marrow cellularity and CFU-GM during the recovery period, of which the observation seems to be useful to extend our understanding in the hematotoxicity of TCDD.

The pattern of emergence of multipotential (CFU-A) and committed (CFU-GM and BFU-E) progenitor cells in peripheral blood has been examined in patients with Hodgkin's disease and non-Hodgkin's lymphoma. Mobilization protocols used chemotherapy with or without granulocyte colony-stimulating factor (n = 8 and n = 5, respectively). In all patients, the numbers of CFU-A, CFU-GM and BFU-E peaked simultaneously, rather than sequentially, suggesting that marrow regeneration after these mobilization protocols occurred from progenitors at all stages of differentiation. We conclude that peripheral blood stem cell harvest strategies based on peak values for total progenitor numbers will also capture maximum numbers of multipotential progenitors. However, the variable relationship between CFU-A and CFU-GM numbers suggests that overall progenitor cell numbers can give only a broad estimate of the absolute numbers of multipotential progenitors in an individual harvest.

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.

Objective We analyzed the incidence of primitive (LTC-IC) and committed (CFU-mix, BFU-E, CFU-GM) hematopoietic progenitors detected under steady-state conditions and upon progenitor cell mobilization in a cohort of healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF). Materials and methods Healthy donors ( n = 30) of HLA-mismatched or -matched stem cell transplants were mobilized with rhG-CSF (8 μg/Kg body weight subcutaneously twice daily until completion of leukapheresis). PBPC collections were started after 4 days of rhG-CSF therapy. Results Steady-state incidence of bone marrow LTC-IC, but not committed progenitors, significantly correlated with the numbers of mobilized CD34 + cells ( r = 0.6, p = 0.004), CFU-GM ( r = 0.79, p = 0.0005) and CFC ( r = 0.76, p = 0.001) detected after 4 days of rhG-CSF therapy. Statistically significant correlations were also found between steady-state blood CFU-GM and peak numbers of CD341 cells ( r = 0.68, p = 0.001), numbers of day 4 CD341 cells ( r = 0.52, p = 0.005), CFU-GM ( r = 0.63, p = 0.002), and CFC ( r = 0.61, p = 0.003). Conclusion Our data show that in normal volunteers baseline marrow LTC-IC and blood CFU-GM correlate with rhG-CSF-mobilized PBPC. The potential clinical relevance of these findings in the identification of poor mobilizers will be tested in a prospective study.

Peripheral blood CD34/4 5 + cell (CD34/45) enumeration is an expensive and labour-intensive investigation but remains the standard assay for optimizing yield and timing of peripheral blood stem cell harvesting (PBSCH). The present study examined the value of the Sysmex SE9000 parameters (WBC, neutrophil count, and immature myeloid index (IMI)) and Sysmex R2000 reticulocyte parameters (absolute, high and medium fluorescence reticulocytes) in predicting the optimum timing of PBSCH in comparison to peripheral blood CD34/45. Sixty-four PBSCH from 23 patients with haematological malignancies were assessed with a variety of mobilization regimes used. Reticulocyte parameters showed high interpatient variability and did not prove clinically useful. IMI did not consistently predict satisfactory PBSCH yield except when > 1000 × 10 6 /l. Peripheral blood CD34/45 was the most useful predictor of yield. IMI > 20 × 10 6 /l was, however, a useful surrogate for predicting a rise in peripheral blood CD34/45 from nadir and proved to be superior to WBC or neutrophil count. A rising IMI is a marker of early regeneration and has a role in determining when to initiate enumeration of peripheral blood CD34/45.

There are considerable differences in haemopoietic activity between young children and adults on the one hand, and between adults and the elderly on the other. A fundamental unanswered question is whether these differences relate to discrete stages or are part of a continuous process. We have sought to define aspects of the haematological ageing process, and have found that results from children with bone marrow failure syndromes differ from age-matched reference values. Haemopoietic cells were obtained from umbilical cord blood, from blood and bone marrow of healthy individuals and from the blood of young patients with bone marrow failure syndromes. Clonogenic myeloid progenitors (CFU-GM) were grown in semi-solid medium to measure their frequency; the proliferative capacity of myeloid progenitors was measured by replating colonies and observing secondary colony formation. We found that the frequency of CFU-GM in normal marrow increased and their proliferative capacity decreased exponentially with age. The proliferative capacity of CFU-GM in normal blood also decreased exponentially with age. This relationship extrapolated back to the levels of proliferation measured for cord blood CFU-GM (age = 0). The proliferative capacities of CFU-GM from children with bone marrow failure syndromes were severely reduced compared with age-matched reference values. These results indicate that a decline in haemopoietic progenitor cell function begins at birth and continues throughout life. This decline may occur prematurely in childhood marrow failure syndromes with a predisposition to leukaemia.

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.

No treatment has proved its efficiency in CLL. Autologous transplantation is now under consideration for the youngest patients. We assayed progenitor cells (CFU-GM, BFU-E, CD34) in the peripheral blood of 28 untreated CLL patients and found an increase of all these progenitors in CLL compared to controls. There was no statistical difference between stage A versus stages B and C for CFU-GM and BFU-E. In contrast, CD34 cells were higher in stages B and C as compared to stage A. This finding could be explained by a high number of circulating clonal cells in advanced stages of the disease.

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).