Calcium Pyrophosphate Dihydrate Crystals Increase the Granulocyte/Monocyte Progenitor (GMP) and Enhance Granulocyte and Monocyte Differentiation In Vivo.

Calcium pyrophosphate dihydrate (CPPD) crystals are formed locally within the joints, leading to pseudogout. Although the mobilization of local granulocytes can be observed in joints where pseudogout has manifested, the mechanism of this activity remains poorly understood. In this study, CPPD crystals were administered to mice, and the dynamics of splenic and peripheral blood myeloid cells were analyzed. As a result, levels of both granulocytes and monocytes were found to increase following CPPD crystal administration in a concentration-dependent manner, with a concomitant decrease in lymphocytes in the peripheral blood. In contrast, the levels of other cells, such as dendritic cell subsets, T-cells, and B-cells, remained unchanged in the spleen, following CPPD crystal administration. Furthermore, an increase in granulocytes/monocyte progenitors (GMPs) and a decrease in megakaryocyte/erythrocyte progenitors (MEPs) were also observed in the bone marrow. In addition, CPPD administration induced production of IL-1ß, which acts on hematopoietic stem cells and hematopoietic progenitors and promotes myeloid cell differentiation and expansion. These results suggest that CPPD crystals act as a "danger signal" to induce IL-1ß production, resulting in changes in course of hematopoietic progenitor cell differentiation and in increased granulocyte/monocyte levels, and contributing to the development of gout.

Protein arginine methyltransferase 1 is required for maintenance of normal adult hematopoiesis.

Protein arginine methyltransferase 1 (PRMT1) is the predominant asymmetric (type I) methyltransferase in mammalian cells. Mounting evidence suggested that PRMT1 is essential to embryonic development and tumor pathogenesis, but its role in normal adult hematopoiesis is less studied. We used a Prmt1 conditional knockout (KO) mouse model to identify the role of PRMT1 in normal adult hematopoiesis. The results indicated that deletion of PRMT1 results in anemia and leukopenia, reducing terminal erythroid and lymphocyte differentiation. Additionally, we found a significant decrease of megakaryocyte progenitors (MkPs) compared with similarly treated littermate control mice. The frequency of short-term hematopoietic stem cells (ST-HSCs) and granulocyte-macrophage progenitors (GMPs) populations were significantly lower in PRMT1f/f/Mx1-CRE bone marrow (BM) compared with littermate control mice. Importantly, in-vitro replating assays and BM transplantation results revealed that PRMT1 KO results in reduced hematopoietic stem and progenitor cells (HSPCs) self-renewal capacity. Thus, we conclude that PRMT1 is required for hematopoietic differentiation and the competitive fitness of HSPCs, and we believed that PRMT1 serves as a key epigenetic regulator of normal hematopoiesis that occurs throughout life.

Fate Mapping via Ms4a3-Expression History Traces Monocyte-Derived Cells.

Most tissue-resident macrophage (RTM) populations are seeded by waves of embryonic hematopoiesis and are self-maintained independently of a bone marrow contribution during adulthood. A proportion of RTMs, however, is constantly replaced by blood monocytes, and their functions compared to embryonic RTMs remain unclear. The kinetics and extent of the contribution of circulating monocytes to RTM replacement during homeostasis, inflammation, and disease are highly debated. Here, we identified Ms4a3 as a specific gene expressed by granulocyte-monocyte progenitors (GMPs) and subsequently generated Ms4a3TdT reporter, Ms4a3Cre, and Ms4a3CreERT2 fate-mapping models. These models traced efficiently monocytes and granulocytes, but no lymphocytes or tissue dendritic cells. Using these models, we precisely quantified the contribution of monocytes to the RTM pool during homeostasis and inflammation. The unambiguous identification of monocyte-derived cells will permit future studies of their function under any condition.

Trib1 regulates eosinophil lineage commitment and identity by restraining the neutrophil program.

Eosinophils and neutrophils are critical for host defense, yet gaps in understanding how granulocytes differentiate from hematopoietic stem cells (HSCs) into mature effectors remain. The pseudokinase tribbles homolog 1 (Trib1) is an important regulator of granulocytes; knockout mice lack eosinophils and have increased neutrophils. However, how Trib1 regulates cellular identity and function during eosinophilopoiesis is not understood. Trib1 expression markedly increases with eosinophil-lineage commitment in eosinophil progenitors (EoPs), downstream of the granulocyte/macrophage progenitor (GMP). Using hematopoietic- and eosinophil-lineage-specific Trib1 deletion, we found that Trib1 regulates both granulocyte precursor lineage commitment and mature eosinophil identity. Conditional Trib1 deletion in HSCs reduced the size of the EoP pool and increased neutrophils, whereas deletion following eosinophil lineage commitment blunted the decrease in EoPs without increasing neutrophils. In both modes of deletion, Trib1-deficient mice expanded a stable population of Ly6G+ eosinophils with neutrophilic characteristics and functions, and had increased CCAAT/enhancer binding protein α (C/EBPα) p42. Using an ex vivo differentiation assay, we found that interleukin 5 (IL-5) supports the generation of Ly6G+ eosinophils from Trib1-deficient cells, but is not sufficient to restore normal eosinophil differentiation and development. Furthermore, we demonstrated that Trib1 loss blunted eosinophil migration and altered chemokine receptor expression, both in vivo and ex vivo. Finally, we showed that Trib1 controls eosinophil identity by modulating C/EBPα. Together, our findings provide new insights into early events in myelopoiesis, whereby Trib1 functions at 2 distinct stages to guide eosinophil lineage commitment from the GMP and suppress the neutrophil program, promoting eosinophil terminal identity and maintaining lineage fidelity.

Sympathetic Neuronal Activation Triggers Myeloid Progenitor Proliferation and Differentiation.

There is a growing body of research on the neural control of immunity and inflammation. However, it is not known whether the nervous system can regulate the production of inflammatory myeloid cells from hematopoietic progenitor cells in disease conditions. Myeloid cell numbers in diabetic patients were strongly correlated with plasma concentrations of norepinephrine, suggesting the role of sympathetic neuronal activation in myeloid cell production. The spleens of diabetic patients and mice contained higher numbers of tyrosine hydroxylase (TH)-expressing leukocytes that produced catecholamines. Granulocyte macrophage progenitors (GMPs) expressed the ß2 adrenergic receptor, a target of catecholamines. Ablation of splenic sympathetic neuronal signaling using surgical, chemical, and genetic approaches diminished GMP proliferation and myeloid cell development. Finally, mice lacking TH-producing leukocytes had reduced GMP proliferation, resulting in diminished myelopoiesis. Taken together, our study demonstrates that catecholamines produced by leukocytes and sympathetic nerve termini promote GMP proliferation and myeloid cell development.

Identification of a Siglec-F+ granulocyte-macrophage progenitor.

In recent years multi-parameter flow cytometry has enabled identification of cells at major stages in myeloid development; from pluripotent hematopoietic stem cells, through populations with increasingly limited developmental potential (common myeloid progenitors and granulocyte-macrophage progenitors), to terminally differentiated mature cells. Myeloid progenitors are heterogeneous, and the surface markers that define transition states from progenitors to mature cells are poorly characterized. Siglec-F is a surface glycoprotein frequently used in combination with IL-5 receptor alpha (IL5Rα) for the identification of murine eosinophils. Here, we describe a CD11b+ Siglec-F+ IL5Rα- myeloid population in the bone marrow of C57BL/6 mice. The CD11b+ Siglec-F+ IL5Rα- cells are retained in eosinophil deficient PHIL mice, and are not expanded upon overexpression of IL-5, indicating that they are upstream or independent of the eosinophil lineage. We show these cells to have GMP-like developmental potential in vitro and in vivo, and to be transcriptionally distinct from the classically described GMP population. The CD11b+ Siglec-F+ IL5Rα- population expands in the bone marrow of Myb mutant mice, which is potentially due to negative transcriptional regulation of Siglec-F by Myb. Lastly, we show that the role of Siglec-F may be, at least in part, to regulate GMP viability.

Treatment of MCPT8DTR mice with high- or low-dose diphtheria toxin leads to differential depletion of basophils and granulocyte-macrophage progenitors.

Basophils have been recently recognized to play important roles in type 2 immune responses during allergies and parasitic infection, largely due to the development of novel tools for the in vivo study of these cells. As such, the genetically-engineered MCPT8DTR mouse line has been used to specifically deplete basophils following treatment with diphtheria toxin (DT). In this study, we showed that DT-injected MCPT8DTR mice exhibited a striking decrease of eosinophils and neutrophils in skin when subjected to a hapten fluorescein isothiocyanate (FITC)-induced allergic contact dermatitis (ACD) experimental protocol. Unexpectedly, we found that loss of skin eosinophils and neutrophils was not due to a lack of basophil-mediated recruitment, as DT injection caused a systemic reduction of eosinophils and neutrophils in MCPT8DTR mice in a time-dependent manner. Furthermore, we found that hematopoietic stem-cell-derived granulocyte-macrophage progenitors (GMPs) expressed MCPT8 gene, and that these cells were depleted upon DT injection. Finally, we optimized a protocol in which a low-dose DT achieved a better specificity for depleting basophils, but not GMPs, in MCPT8DTR mice, and demonstrate that basophils do not play a major role in recruiting eosinophils and neutrophils to ACD skin. These data provide new and valuable information about functional studies of basophils.

GMP-ing to Spatial Conclusions about Emergency and Leukemic Myelopoiesis.

Hematopoietic stem cells (HSCs) in the bone marrow (BM) form mature blood cells of all lineages through expansion of lineage-biased progenitors. In a recent issue of Nature, Hérault et al. (2017) uncover a unique spatiotemporal mechanism of granulocyte-macrophage progenitors (GMPs) employed in emergency hematopoiesis that is also hijacked in leukemia.

Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis.

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1ß, G-CSF, TGFß and CXCL4) and activation of an inducible Irf8 and ß-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.

C/EBP-Induced Transdifferentiation Reveals Granulocyte-Macrophage Precursor-like Plasticity of B Cells.

The lymphoid-myeloid transdifferentiation potentials of members of the C/EBP family (C/EBPα, ß, δ, and ε) were compared in v-Abl-immortalized primary B cells. Conversion of B cells to macrophages was readily induced by the ectopic expression of any C/EBP, and enhanced by endogenous C/EBPα and ß activation. High transgene expression of C/EBPß or C/EBPε, but not of C/EBPα or C/EBPδ, also induced the formation of granulocytes. Granulocytes and macrophages emerged in a mutually exclusive manner. C/EBPß-expressing B cells produced granulocyte-macrophage progenitor (GMP)-like progenitors when subjected to selective pressure to eliminate lymphoid cells. The GMP-like progenitors remained self-renewing and cytokine-independent, and continuously produced macrophages and granulocytes. In addition to their suitability to study myelomonocytic lineage bifurcation, lineage-switched GMP-like progenitors could reflect the features of the lympho-myeloid lineage switch observed in leukemic progression.

Identification of an atypical monocyte and committed progenitor involved in fibrosis.

Monocytes and macrophages comprise a variety of subsets with diverse functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein ß (C/EBPß), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb-/- haematopoietic cells. Adoptive transfer of SatM into Cebpb-/- mice resulted in fibrosis. Notably, SatM are derived from Ly6C-FcεRI+ granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6C-FcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPß licenses differentiation of SatM from their committed progenitor.

Differential Regulation of Lympho-Myelopoiesis by Stromal Cells in the Early and Late Phases in BALB/c Mice Repeatedly Exposed to Lipopolysaccharide.

Chronic lipopolysaccharide (LPS) exposure to mice reduces the lymphoid compartment and skews the hematopoietic cell compartment toward myeloid-cells, which is considered to be a direct effect of LPS on hematopoietic stem cells. However, the effect of chronic LPS exposure on stromal-cells, which compose the hematopoietic microenvironment, has not been elucidated. Here, we investigated early- and late-phase effects of repeated LPS exposure on stromal-cells. During the early phase, when mice were treated with 5 or 25 µg LPS three times at weekly intervals, the numbers of myeloid-progenitor (colony forming unit-granulocyte macrophage (CFU-GM)) cells and B lymphoid-progenitor (CFU-preB) cells in the bone-marrow (BM) rapidly decreased after each treatment. The number of CFU-GM cells recovered from the initial decrease and then increased to levels higher than pretreatment levels, whereas the number of CFU-preB cells remained lower than pretreatment levels. In the BM, expression of genes for positive-regulators of myelopoiesis including granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6 and negative-regulators of B lymphopoiesis including tumor necrosis factor (TNF)-α was up-regulated, whereas expression of positive-regulators of B lymphopoiesis including stromal cell-derived factor (SDF)-1, IL-7, and stem cell factor (SCF) was down-regulated. During the late phase, the number of CFU-preB cells remained lower than pretreatment levels 70 d after the first treatments with 5 and 25 µg LPS, whereas the number of CFU-GM cells returned to pretreatment levels. IL-7 gene expression in the BM remained down-regulated, whereas gene-expression levels of SDF-1 and SCF were restored. Thus, chronic LPS exposure may impair stromal-cell function, resulting in prolonged suppression of B lymphopoiesis, which may appear to be senescence similar to the hematological phenotype.

Quantifying intrinsic and extrinsic control of single-cell fates in cancer and stem/progenitor cell pedigrees with competing risks analysis.

The molecular control of cell fate and behaviour is a central theme in biology. Inherent heterogeneity within cell populations requires that control of cell fate is studied at the single-cell level. Time-lapse imaging and single-cell tracking are powerful technologies for acquiring cell lifetime data, allowing quantification of how cell-intrinsic and extrinsic factors control single-cell fates over time. However, cell lifetime data contain complex features. Competing cell fates, censoring, and the possible inter-dependence of competing fates, currently present challenges to modelling cell lifetime data. Thus far such features are largely ignored, resulting in loss of data and introducing a source of bias. Here we show that competing risks and concordance statistics, previously applied to clinical data and the study of genetic influences on life events in twins, respectively, can be used to quantify intrinsic and extrinsic control of single-cell fates. Using these statistics we demonstrate that 1) breast cancer cell fate after chemotherapy is dependent on p53 genotype; 2) granulocyte macrophage progenitors and their differentiated progeny have concordant fates; and 3) cytokines promote self-renewal of cardiac mesenchymal stem cells by symmetric divisions. Therefore, competing risks and concordance statistics provide a robust and unbiased approach for evaluating hypotheses at the single-cell level.

Conditional knockout mice demonstrate function of Klf5 as a myeloid transcription factor.

Krüppel-like factor 5 (Klf5) encodes a zinc-finger transcription factor and has been reported to be a direct target of C/EBPα, a master transcription factor critical for formation of granulocyte-macrophage progenitors (GMP) and leukemic GMP. Using an in vivo hematopoietic-specific gene ablation model, we demonstrate that loss of Klf5 function leads to a progressive increase in peripheral white blood cells, associated with increasing splenomegaly. Long-term hematopoietic stem cells (HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) were all significantly reduced in Klf5(Δ/Δ) mice, and knockdown of KLF5 in human CD34(+) cells suppressed colony-forming potential. ST-HSCs, MPPs, and total numbers of committed progenitors were increased in the spleen of Klf5(Δ/Δ) mice, and reduced ß1- and ß2-integrin expression on hematopoietic progenitors suggests that increased splenic hematopoiesis results from increased stem and progenitor mobilization. Klf5(Δ/Δ) mice show a significant reduction in the fraction of Gr1(+)Mac1(+) cells (neutrophils) in peripheral blood and bone marrow and increased frequency of eosinophils in the peripheral blood, bone marrow, and lung. Thus, these studies demonstrate dual functions of Klf5 in regulating hematopoietic stem and progenitor proliferation and localization in the bone marrow, as well as lineage choice after GMP, promoting increased neutrophil output at the expense of eosinophil production.

Trim33/Tif1γ is involved in late stages of granulomonopoiesis in mice.

Trim33/Tif1γ (Trim33) is a member of the tripartite motif family. Using a conditional hematopoietic-specific Trim33 knock-out (Trim33(Δ/Δ)) mouse, we showed previously that Trim33 deficiency in hematopoietic stem cells leads to severe defects in hematopoiesis, resembling the main features of human chronic myelomonocytic leukemia. We also demonstrated that Trim33 is involved in hematopoietic aging through TGFß signaling. Nevertheless, how Trim33 contributes to the terminal stages of myeloid differentiation remains to be clarified. We reveal here the crucial role of Trim33 expression in the control of mature granulomonocytic differentiation. An important component of Trim33-deficient mice is the alteration of myeloid differentiation, as characterized by dysplastic features, abnormal granulocyte and monocyte maturation, and the expansion of CD11b(+)Ly6G(high)Ly6C(low) myeloid cells, which share some features with polymorphonuclear-myeloid-derived suppressor cells. Moreover, in Trim33(Δ/Δ) mice, we observed the alteration of CSF-1-mediated macrophage differentiation in association with the lack of Csf-1 receptor. Altogether, these results indicate that Trim33 deficiency leads to the expansion of a subset of myeloid cells characterizing the myelodysplastic/myeloproliferative neoplasm.

Transcriptional regulation of mast cell and basophil lineage commitment.

Basophils and mast cells have long been known to play critical roles in allergic disease and in immunity against parasitic infection. Accumulated evidence also supports that basophils and mast cells have important roles in immune regulations, host defense against bacteria and viruses, and autoimmune diseases. However, origin and molecular regulation of basophil and mast cell differentiation remain incompletely understood. In this review, we focus on recent advances in the understanding of origin and molecular regulation of mouse and human basophil and mast cell development. A more complete understanding of how basophils and mast cells develop at the molecular level will lead to development of interventions that are more effective in achieving long-term success.

Variability in subjective review of umbilical cord blood colony forming unit assay.

BACKGROUND: The goal of this study was to quantitatively evaluate the reproducibility of current manual counting methods of colony forming units (CFUs) from umbilical cord blood samples METHODS: Fresh and reconstituted frozen cells from 10 cord blood samples were cultured under standard conditions. The number of BFU-Es, CFU-GMs, and CFU-GEMMs were counted by three expert reviewers using the standard microscope method and manually traced CFUs on digital images of cell cultures. RESULTS: The mean colony count based on the traced digital images was 82 (22% CV) and 52 (15% CV) for the fresh and frozen samples, respectively. This was significantly greater than that observed using the microscope, 61 (13% CV) for fresh and 43 (16% CV) for frozen. The difference was mainly due to the reviewers observing more CFU-GMs in the digital images than through the microscope review. All three reviewers agreed on the presence of a colony 72% of the time based on the digital review in both fresh and frozen samples. Reviewer agreement with respect to colony type in the fresh samples was 38% (22%CV), 25% (51%CV), and 6% (115%CV) for BFU-Es, CFU-GMs, and CFU-GEMMs, respectively. Reviewer agreement increased for BFU-Es and CFU-GMs in the frozen samples where fewer colonies were present. CONCLUSIONS: Although this study showed marked variability between reviewers, the analysis of manually traced digital images has the potential to improve inter-observer variation when compared to current methods by identifying features that lead to discrepancies in colony counting and providing cases with consensus results. © 2016 International Clinical Cytometry Society.

[Relationship among the Oxygen Concentration, Reactive Oxygen Species and the Biological Characteristics of Mouse Bone Marrow Hematopoietic Stem Cells].

OBJECTIVE: To investigate the effects of oxygen concentration and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and to analyzed the relationship among the oxygen concentration, ROS and the biological characteristics of mouse HSC through simulation of oxygen environment experienced by PB HSC during transplantation. METHODS: The detection of reactive oxygen species (ROS), in vitro amplification, directional differentiation (BFU-E, CFU-GM, CFU-Mix), homing of adhesion molecules (CXCR4, CD44, VLA4, VLA5, P-selectin), migration rate, CFU-S of NOD/SCID mice irradiated with sublethal dose were performed to study the effect of oxgen concentration and reactive oxygen species on the biological characteristics of mouse BM-HSC and the relationship among them. RESULTS: The oxygen concentrations lower than normal oxygen concentration (especially hypoxic oxygen environment) could reduce ROS level and amplify more Lin(-) c-kit(+) Sca-1(+) BM HSC, which was more helpful to the growth of various colonies (BFU-E, CFU-GM, CFU-Mix) and to maintain the migratory ability of HSC, thus promoting CFU-S growth significantly after the transplantation of HSC in NOD/SCID mice irradiated by a sublethal dose. BM HSC exposed to oxygen environments of normal, inconstant oxygen level and strenuously thanging of oxygen concentration could result in higher level of ROS, at the same time, the above-mentioned features and functional indicators were relatively lower. CONCLUSION: The ROS levels of BM HSC in PB HSCT are closely related to the concentrations and stability of oxygen surrounding the cells. High oxygen concentration results in an high level of ROS, which is not helpful to maintain the biological characteristics of BM HSC. Before transplantation and in vitro amplification, the application of antioxidancs and constant oxygen level environments may be beneficial for transplantation of BMMSC.

Extraction and identification of collagen-derived peptides with hematopoietic activity from Colla Corii Asini.

ETHNOPHARMACOLOGICAL RELEVANCE: Colla Corii Asini is a widely used traditional Chinese medicine to treat anemia with a long history due to its stimulating effect in hematopoiesis, but the components contributing to this effect are still unknown. In this study, we aimed to establish a methodology to isolate the bioactive components and provide pharmacological basis for its usage in treating anemia. METHODS: 5-FU and γ-ray radiation induced anemic mice models were generated by treating with 5-FU at 150mg/kg body weight and γ-rays by a 4MV linear accelerator by total body irradiation using female ICR mice respectively. Oral administration of fraction A was performed by gastric lavage at 1g/kg and 2g/kg body weight for 12 days and 25 days and peripheral blood sample was collected from ocular sinus red blood cell (RBC) and white blood cell (WBC) counts every 3 days and 5 days for 5-FU and radiation induced models, respectively. Next, fraction A was separated to A1 and A2 using cation exchange chromatography (IEC) based on ionic strength. Fraction A1 was further separated using reverse phase chromatography (RPC) based on the hydrophobicity first with 0-10% linear gradient, then 20%, 30%, 50% constant gradient of 60% acetonitrile in neutral Na2HPO4 buffer. Peak fractions were pooled, evaporatively dried, and dissolved in ultrapure water. Finally, fraction A11 was analyzed combining tandem mass spectrometry and proteomic tools and two peptides (peptide 11 and 16) were identified. The hematopoietic effects of multiple fractions and the two peptides were measured using colony-forming units-erythroid (CFU-E), an indication of late erythroid progenitor cells and colony-forming units granulocyte-monocyte (CFU-GM), an indication of granulocyte and monocyte progenitor cells respectively on hematopoietic progenitor cells prepared from bone marrow (Till and Mcculloch 1961). RESULTS: Fraction A at 1g/kg and 2g/kg could increase RBC and WBC counts in 5-FU and radiation induced anemic mice models. Fraction A1 at 0.1mg/ml and 0.5mg/ml, exhibited stronger hematopoietic activity than fraction A2, both of which were subfractions from fraction A using IEX, by elevated CFU-E and CFU-GM of mouse bone marrow cells. Furthermore, fraction A11 at 0.1mg/ml showed stronger CFU-E and CFU-GM than fractions A12 to A14 from RPC separation. Finally, peptide 11 and peptide 16 were identified from tandem mass spectrometry and peptide 11 increased CFU-E and CFU-GM in a dose dependent manner. CONCLUSIONS: We combined multiple approaches including chromatography, mass spectrometry, cell-based assays, as well as animal studies to identify and demonstrate that the hematopoietic effect of Colla Corii Asini is at least in part from the peptidic components identified using our methodology. This is the first time to isolate peptidic components from Colla Corii Asini, and to provide molecular basis for its usage in treating anemia, which may particularly have the potential to benefit cancer patients suffering from myelosuppression due to radiotherapy or chemotherapy.

Clinical outcomes and graft characteristics in pediatric hematopoietic stem cell transplantation: Effect of granulocyte-colony stimulating factor priming.

In this study, we aimed to determine the effect(s) of G-CSF priming on graft and transplantation parameters and compare these findings with those obtained without priming. A total of 64 pediatric patients transplanted from HLA-matched family donors were enrolled in the study. Twenty-nine patients received G-CSF primed marrow (G-BM group) and 35 patients received steady state bone marrow (S-BM group). Number of total nucleated cells (TNC) and CD34(+) cells, CFU-GM colony number, neutrophil and platelet engraftment times, total length of stay in hospital, overall and disease free survival, and occasions of acute and chronic GvHD has been compared between these two groups. Granulocyte colony stimulating factor primed bone marrow (G-BM) yielded higher numbers of CD34(+) cells, TNCs, and CFU-GM colony numbers compared to those obtained in S-BM. The neutrophil engraftment time, platelet engraftment time, length of stay in hospital, overall survival and disease free survival were not different between G-BM and S-BM groups. Also the cumulative incidence of grades II-IV acute and chronic GvHD were similar. It was observed that the use of G-CSF did not increase the risk of acute or chronic GvHD. We concluded that use of G-CSF for stem cell mobilization is an effective and safe method in children.