Granulocyte-monocyte Colony-forming Units Research Articles

Plerixafor Overcomes Lenalidomide-Associated Stem Cell Collection Failure and Affects Collected Myeloid and Erythroid Progenitors

▪High-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-HCT) remains standard of care for transplant-eligible multiple myeloma (MM) patients (pts). MM frontline therapy typically includes lenalidomide (LEN). However, studies have indicated that LEN-containing regimens are associated with an increase in peripheral blood stem cell (PBSC) collection failure. Since most health insurance carriers do not reimburse for PBSC collection if there is no intent to proceed with auto-HCT soon after collection, a common current practice is early auto-HCT after a few cycles of LEN-containing regimen. This may prevent a subset of pts from achieving the deepest possible response prior to transplant. Recently, plerixafor has been used sporadically to ameliorate the suppressive effect of LEN on PBSC collection, however its effect on collected graft composition is unknown. Here, we examine the influence of plerixafor on PBSC mobilization, graft composition, and auto-HCT outcomes.MethodsPts who underwent auto-HCT between 2007 and 2016 at our institution were included. After excluding pts without complete progenitor cell assays, 94 pts formed the final analysis set. The primary end point was PBSC collection failure, defined as the inability to collect at least 5×10^6 CD34+ cells/kg on first apheresis attempt. Secondary endpoints were frequency, in-vitro proliferation, and differentiation capability of hematopoietic progenitors. Granulocyte-Monocyte Colony-Forming Units (CFU-GM) and Burst-Forming Erythroid Units (BFU-E) colonies were scored after 14 days of incubation. Effects of baseline and treatment variables on PBSC collection failure were examined by logistic regression. Significant variables were entered in a multivariate regression model.ResultsPBSC mobilization was achieved with G-CSF and cyclophosphamide (21 pts), G-CSF alone (8 pts), or with G-CSF and plerixafor (65 pts). Pts that received LEN-containing regimens as the immediate line of therapy before collection (LENpos, N=51) were compared to those who did not (LENneg, N=43). Median number of LEN cycles was 4 (range 1-13). 15 pts (16%) had day 1 collection failure with no difference between LENpos and LENneg groups (17% vs. 14%, respectively; P=0.392). Only 1 pts (1.1%) failed final collection. Collection days and median number of collected CD34+ cells were similar in both groups. Older age (OR: 1.85, 95% CI: 1.11-2.54, P=0.001), lower platelets on day of stem cell collection (OR: 1.87, 95% CI: 1.09-2.98, P=0.021), and lack of plerixafor use for PBSC mobilization (OR: 1.96, 95% CI: 1.11-3.52, P<0.001) were risk factors associated with worse stem cell collection in univariate analysis (Table 1). All three parameters detected by univariate analysis retained statistical negative predictive significance. LEN exposure did not affect the collection failure rate (OR: 1.15, 95% CI: 0.81-1.36, P=0.356). There was no difference between LENpog and LENneg groups in median ratio of CFU-GM/total nucleated cells (TNC) (10.5 vs. 11.7 10^-4, P=0.381) neither BFU-E/TNC (7.2 vs. 7.9 10^-4, P= 0.253), indicating similar LEN effect on collected myeloid and erythroid progenitors. The number of collected CFU-GM and BFU-E correlated with collected TNC in both groups (LENpos: R2:0.400, P=0.031, LENneg: R2: 0.405, P=0.001; and LENpos: R2:0.357, P=0.009, LENneg: R2: 0.292, P=0.001; respectively, Figure 1). However there were no difference between LENpos and LENneg group. LENpos pts that did not use plerixafor had significantly lower collected CFU-GM and BGU-E compared to the remainder of pts (P=0.003 and P=0.021, respectively; Figure 3). Mean time to neutrophil and platelet engraftments were similar in both groups (Figure-2). There was one platelet engraftment failure in the LENneg group. There was no detected difference in progression-free survival between the two groups (3-year PFS rate: 71% vs. 74%, HR: 0.91, CI: 0.72-1.16, p=0.27).ConclusionsIn our cohort of heavily pre-treated pts, the length of LEN usage prior to PBSC collection did not affect PBSC collection, graft quality or engraftment when plerixafor was added to the mobilization regimen, suggesting plerixafor may overcome potential deleterious effects of LEN on PBSC collection. The impact of LEN on PBSC collection and transplantation needs further study in prospective clinical trials. [Display omitted] DisclosuresDe Lima:Celgene Corporation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Malek:Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Low content of clonogenic progenitors on day+18 is associated with acute graft-versus-host disease and predicts transplant-related mortality

A marrow reaction associated with acute-graft-versus-host disease (a-GVHD) has been demonstrated in experimental models; its existence in human transplantation is controversial. The aim of the present study was to investigate whether clonogenic marrow precursors are an early marker for a-GVHD and transplant-related mortality (TRM). We prospectively studied 133 patients for colony-forming units-granulocyte-monocyte (CFU-GM) at day +18/+19 posttransplantation. CFU-GM frequency below the 25th percentile was predictive of an acute GVHD score I°-IV° when evaluated in multivariate logistic regression analysis (odds ratio = 13.551, 95% confidence interval [CI]: 1.583-116.031, p = 0.01). In the group with a clonogenic frequency below the 25th percentile, the cumulative incidence of GVHD grades II-IV was significantly more frequent with respect to the group with a frequency greater than the 25th percentile, 86% versus 54% (Gray test: p = 0.02). In multivariate Cox proportional analysis, a CFU-GM frequency below the 25th percentile at day +18 was associated with reduced overall survival (OS) (hazard ratio = 1.778, 95% CI: 1.022-3.093, p = 0.04). Patients with a frequency of CFU-GM greater than the 25th percentile had increased TRM with respect to patients with a clonogenic cell frequency greater than the 25th percentile (33.5% vs. 13.0%, p = 0.01). Patients were divided based on median content of viable CD34+ cells, and measurement of viable CD34+ cells was predictive for OS (p = 0.005) and TRM (p = 0.003). A weak correlation was observed between CFU-GM frequency in marrow at day +18 and levels of IL-2 receptor (IL-2R) in plasma (r = -0.226, p = 0.03). We conclude that marrow progenitor cell counts, on day +18 may be a useful marker for identifying patients at risk for severe a-GVHD, TRM, and inferior survival.

Use of Sysmex Hematology Analyzer based Hematopoietic Progenitor Cell (XN-HPC) Count in Allogeneic Peripheral Blood Stem Cell Transplant Setting; Is It Substantially Reliable?

Allogeneic stem cell transplantation remains the only curative option for various benign and malignant hematological disorders. The procedure of allogeneic stem cell transplantation entails the administration of adequate doses of hematopoietic progenitor cells, aiming for complete and sustained hematopoietic reconstitution. Hematopoietic progenitors are obtained either through a bone marrow harvest or from GCSF-mobilized peripheral blood of the identified donor. Over the years various modalities have been utilized to establish the adequacy of harvested stem cell product e.g. Colony-forming unit– granulocyte monocyte (CFU-GM) assay, total mononuclear cell count (MNC), and CD34+ cell count

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells.

Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34+ cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34+ and CD34+CD90+) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.

Hematopoietic stem/progenitor cell differentiation towards myeloid lineage is modulated by LIGHT/LIGHT receptor signaling.

The cytokine LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a member of the tumor necrosis factor (TNF) superfamily. It is expressed primarily on activated T lymphocytes, and detectable on monocytes, granulocytes, and immune dendritic cells. It mainly plays a role in immune regulation including T cell activation and dendritic cell maturation. We recently reported its role as an inducer in embryonic stem cell differentiation, but its role in regulation of adult stem cell has not been defined. In the present study, we examined the expression of LIGHT receptor in Lin- c-kit+ Sca-1+ hematopoietic stem/progenitor cells (HSC/HPCs). We found that HSC express HVEM, a LIGHT receptor, on its surface. We further identified the role of LIGHT in promoting myeloid differentiation of HSCs driven by granulocyte-monocyte colony stimulating factor (GM-CSF). Further studies showed that LIGHT enhances both GM-CSF and GM-CSF receptor (GM-CSFR) expression in HSCs. LIGHT stimulation increases PU.1 expression in HSC/HPCs. In vivo administration of LIGHT increases the colony-forming unit-granulocyte/monocyte (CFU-GM) colony formation and plasma GM-CSF level. Altogether, the data suggest LIGHT promote myeloid differentiation of HSC/HPCs.

Increased haematopoietic activity in patients with atherosclerosis.

Experimental work posits that acute ischaemic events trigger haematopoietic activity, driving monocytosis, and atherogenesis. Considering the chronic low-grade inflammatory state in atherosclerosis, we hypothesized that haematopoietic hyperactivity is a persistent feature in cardiovascular disease (CVD). Therefore, we aimed to assess the activity of haematopoietic organs and haematopoietic stem and progenitor cells (HSPCs) in humans. First, we performed 18F-fluorodeoxyglucose positron emission tomographic (18F-FDG PET) imaging in 26 patients with stable atherosclerotic CVD (ischaemic event >12 months ago), and 25 matched controls. In splenic tissue, 18F-FDG uptake was 2.68 ± 0.65 in CVD patients vs. 1.75 ± 0.54 in controls (1.6-fold higher; P< 0.001), and in bone marrow 3.20 ± 0.76 vs. 2.72 ± 0.46 (1.2-fold higher; P = 0.003), closely related to LDL cholesterol levels (LDLc, r = 0.72). Subsequently, we determined progenitor potential of HSPCs harvested from 18 patients with known atherosclerotic CVD and 30 matched controls; both groups were selected from a cohort of cancer patients undergoing autologous stem cell transplantation. In CVD patients, the normalized progenitor potential, expressed as the number of colony-forming units-granulocyte/monocyte (CFU-GM) colonies/CD34+ cell, was 1.6-fold higher compared with matched controls (P < 0.001). Finally, we assessed the effects of native and oxidized lipoproteins on HSPCs harvested from healthy donors in vitro. Haematopoietic stem and progenitor cells displayed a 1.5-fold increased CFU-GM capacity in co-culture with oxidized LDL in vitro (P = 0.002), which was inhibited by blocking oxidized phospholipids via E06 (P = 0.001). Collectively, these findings strengthen the case for a chronically affected haematopoietic system, potentially driving the low-grade inflammatory state in patients with atherosclerosis.

Post-thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes.

Standard quality assurance (QA) of cryopreserved peripheral blood stem cells (PBSC) uses post-thaw viable CD34(+) cell counts. In 2013, concerns arose at Great Ormond Street Hospital (GOSH) about 8 patients with delayed engraftment following myeloablative chemotherapy with cryopreserved cell rescue, despite adequate post-thaw viable cell counts in all cases. Root cause analysis was undertaken; investigations suggested the freeze process itself was a contributing factor to suboptimal engraftment. Experiments were undertaken in which a single PBSC product was divided into three and cryopreserved in parallel using a control-rate freezer (CRF) or passive freezing method (-80°C freezer) at GOSH, or the same passive freezing at another laboratory. Viable CD34(+) counts were equivalent and adequate in each. Granulocyte-monocyte colony-forming unit assays demonstrated colonies from the products cryopreserved using passive freezing (both laboratories), but no colonies from products cryopreserved using the CRF. The CRF was shown to be operating within manufacturer's specifications with freeze-profile within acceptable limits. This experience has important implications for quality assurance for all transplant programmes, particularly those using cryopreserved products. The failure of post-thaw viable CD34(+) counts, the most widely used routine QA test available, to ensure PBSC function is of great concern and should prompt reassessment of protocols and QA procedures.

Liquid Storage of Hematopoietic Stem Cells Versus Proliferative Potential Colony-Forming Unit Granulocyte-Monocytes: Validation of Cell Processing

BackgroundThe material for transplantation must be of the highest quality. As far as we know, short-term storage is one of the crucial points of stem cell banking. According to the quality assurance system in a stem cell bank, each step of cell processing must be validated. The aim of this study was to assess the influence of short-term storage conditions into a clonogenic assay. MethodsMaterial was collected from mobilized peripheral blood by means of leukapheresis from 15 patients. Samples were stored at 4°C and 20°C; samples were evaluated on the day of leukapheresis and after 24 hours and after 48 hours of storage. The number of colony-forming unit granulocyte-monocyte (CFU-GM) precursors was analyzed with the use of in vitro culture. The material was evaluated before freezing and after thawing. ResultsThe average number of CFU-GM precursors in the material stored at 4°C before freezing on the day of collection was 84/105 nuclear cells (nc) and after 24 hours and 48 hours of storage was, respectively, 62/105 nc (P = .011719) and 36/105 nc (P = .02088). The average of the CFU-GM precursors in material stored at 20°C after 24 hours and 48 hours of storage amounted to 33/105 nc (P = .004439) and 2/105 nc (P = .00346), respectively. ConclusionsIn our study, the number of colonies of CFU-GM after 24 hours and 48 hours of storage, both at 4°C and 20°C, was significantly reduced compared with the number of colonies on the day of collection. Significantly greater numbers of CFU-GM precursors were observed in the material stored before freezing at 4°C in comparison with the material stored at 20°C.

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

Ethnopharmacological relevanceColla 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. Methods5-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). ResultsFraction 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. ConclusionsWe 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.

Effect of cryopreservation on umbilical blood cells and its mechanism

To evaluate the effect of cryopreservation on clonogenic ability and apoptosis rate of mono-nuclear cells and CD34+ cells in umbilical blood (UB), and to choose the index to present the freezing injury and optimize the cryopreservation of UB. The mono-nuclear cells (MNC) and CD34+ cells were separated from UB and frozen.After 30 days, they were thawed in warm water. Clonogenic capacity and clonogenic recovery before and after the cryopreservation was compared. We also used Annexin V-FITC-PI to investigate the apoptosis rate of the cells before and after the cryopreservation of these 2 types of cells. The number of colony forming unit-granulocyte/monocyte (CFU-GMs) was not changed after freezing and thawing in both MNCs and CD34+ cells, while the number of colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) was obviously reduced after freezing in CD34+ cells. The 2 types of cryopreserved cells had certain degree of apoptosis before the cryopreservation. MNC-type cryopreservation increased the cells apoptosis a little, while CD34+-type cryopreservation increased more. The cells have certain degree of apoptosis before the cryopreservation. The freezing and thawing procedure does affect the early stage progenitor cells-CFU-GEMM in the CD34+- type cryopreserved cells in UB. The damage may be induced by the cell apoptosis.

Rituximab associated neutropenia: Description of three cases and an insight into the underlying pathogenesis

SummaryBackgroundTo describe Rituximab associated neutropenia (RAN), and to explore its underlying mechanism.Case ReportWe describe three patients with RAN. The effect of patient’s plasma on colony forming unit, Granulocyte-Monocyte (CFU-GM) was measured by the addition of plasma to the culture of a healthy bone-marrow. Repeated tests were performed after recovery of white count.In the leukopenic period the patient’s plasma inhibited CFU growth completely. Control plasma did not have such an effect. Addition of patient’s cell supernatant to bone marrow cells did not change the number of CFU. The same effect was demonstrated in normal control. After recovery the patient’s plasma did not inhibit colony formation, similar to control.ConclusionsRAN is a clinically significant side effect. It may take place during treatment or several months afterwards. Circulating antibodies in the plasma may be responsible for this unique BM toxicity.

βig-h3 Regulates Differentiation and Survival of Human Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 2591Adult hematopoietic stem cells (HSC) reside in dedicated niches in the bone marrow (BM). Within this specialized microenvironment, the various interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are critical for the maintenance of the HSC population and the concomitant development of the distinct blood cell lineages. Comparative gene-expression profiling of purified HSC identified ECM proteins that are differentially expressed in homeostatic and regenerative conditions. The ECM protein βig-h3 was one of the proteins upregulated in regenerative conditions. Therefore, we characterized the role of βig-h3 in the regulation of HSC self-renewal and differentiation.A comparison between human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB), revealed the highest βig-h3 expression in BM-HSPC (3.9-fold increased compared to MPB, 1.7-fold increased compared to CB), which may implie a role for βig-h3 in retaining HSC in the BM.To examine the functional relevance of βig-h3 in HSC, we first increased βig-h3 expression by transducing human HSPC with a lentiviral βig-h3-SIN-GFP expression vector or a control SIN-GFP vector. Over-expression of βig-h3 (80-fold) in HSPC decreased colony-forming-unit-granulocyte-monocyte (CFU-GM) formation from 130 (SEM=47) to 73 (SEM=19, n=3) CFU-GM per 500 plated CD34+ cells, while megakaryopoiesis was accelerated and the number of mature megakaryocytic cells increased from 16% (SEM=6%) to 30% (SEM=7%, n=4) at day 14 of culture. Ectopic expression of βig-h3 did not affect differentiation along the erythroid or granulopoietic lineage. The development of megakaryocytes at the cost of pluripotent CFU-GM suggests that βig-h3 drives differentiation.In addition, βig-h3 expression in HSPC was reduced by two different short-hairpin-RNAs (shRNA) expressed from lentiviral vectors, which resulted in decreased proliferation (from 19.6- to 5.8-fold per input cell at day 13) and increased apoptosis (from 13.5% to 25.3% at day 13) in liquid HSPC cultures, as analyzed by Annexin V staining. Similarly, knock-down of βig-h3 in various cell lines also resulted in a decreased proliferation and increased apoptosis. Knock-down of βig-h3 in primary HSPC dramatically reduced CFU-GM from 73 (SEM=8.7) to 31 (SEM=14.4, n=6) CFU-GM per 500 CD34+ cells plated, and reduced colony-forming-unit-erythrocyte (CFU-E) formation from 30 (SEM=6.5) to 9 (SEM=1.6, n=4) CFU-E per 500 CD34+ cells plated. This can be explained by increased apoptosis of βig-h3 knock-down cells. Notably, co-culture of βig-h3 knock-down HSPC with stromal feeder cells, known to express high levels of βig-h3, showed no difference compared to control HSPC in cobblestone area formation within two weeks, indicating that stromal cells can counteract apoptosis in βig-h3 knock-down cells. Remarkably, long-term-culture CFU-GM (LTC-CFU) formation of HSPC that were co-cultured with stromal cells during two weeks, was even significantly increased (1.9-fold, n=2) in βig-h3 knock-down cells, indicating that decreased endogenous levels of βig-h3 stimulates the maintenance or expansion of HSPC on stroma.In conclusion, ectopic expression of βig-h3 decreased CFU-GM in HSPC and accelerated differentiation towards megakaryocytes, suggesting that βig-h3 might drive lineage commitment of HSC. Conversely, knock-down of βig-h3 in HSPC stimulated LTC-CFU formation, indicating that decreased βig-h3 levels in HSPC maintain their undifferentiated state. In absence of stroma, however, knock-down of βig-h3 induces apoptosis, indicating βig-h3 as an essential survival factor, which expression levels regulate differentiation and maintenance of HSC. Disclosures:No relevant conflicts of interest to declare.

Maitake beta-glucan promotes recovery of leukocytes and myeloid cell function in peripheral blood from paclitaxel hematotoxicity

Bone marrow myelotoxicity is a major limitation of chemotherapy. While granulocyte colony stimulating factor (G-CSF) treatment is effective, alternative approaches to support hematopoietic recovery are sought. We previously found that a beta-glucan extract from maitake mushroom Grifola frondosa (MBG) enhanced colony forming unit-granulocyte monocyte (CFU-GM) activity of mouse bone marrow and human hematopoietic progenitor cells (HPC), stimulated G-CSF production and spared HPC from doxorubicin toxicity in vitro. This investigation assessed the effects of MBG on leukocyte recovery and granulocyte/monocyte function in vivo after dose intensive paclitaxel (Ptx) in a normal mouse. After a cumulative dose of Ptx (90-120 mg/kg) given to B6D2F1mice, daily oral MBG (4 or 6 mg/kg), intravenous G-CSF (80 microg/kg) or Ptx alone were compared for effects on the dynamics of leukocyte recovery in blood, CFU-GM activity in bone marrow and spleen, and granulocyte/monocyte production of reactive oxygen species (ROS). Leukocyte counts declined less in Ptx + MBG mice compared to Ptx-alone (p = 0.024) or Ptx + G-CSF treatment (p = 0.031). Lymphocyte levels were higher after Ptx + MBG but not Ptx + G-CSF treatment compared to Ptx alone (p < 0.01). MBG increased CFU-GM activity in bone marrow and spleen (p < 0.001, p = 0.002) 2 days after Ptx. After two additional days (Ptx post-day 4), MBG restored granulocyte/monocyte ROS response to normal levels compared to Ptx-alone and increased ROS response compared to Ptx-alone or Ptx + G-CSF (p < 0.01, both). The studies indicate that oral MBG promoted maturation of HPC to become functionally active myeloid cells and enhanced peripheral blood leukocyte recovery after chemotoxic bone marrow injury.

JAK2 V617F mutation in myelodysplastic syndrome (MDS) with del(5q) arises in genetically discordant clones

The 2008 World Health Organization (WHO) proposed revision of the classification of MDS recognizes a deletion (5q) subtype with mutation of Janus kinase-2 (JAK2 V617F). We investigated the clonal origin of this gene mutation in a patient with del(5q) MDS presenting with thrombocytosis and normal hemoglobin. Analysis of colony forming units-granulocyte-monocyte (CFU-GM) and erythropoietin-independent growth of bone marrow (BM) and peripheral blood (PB) burst forming units-erythroid (BFU-E) showed that del(5q) and JAK2 V617F existed in progenitors derived from independent clones. Fifty percent of endogenous erythroid colonies (EEC) harbored the JAK2 V617F mutation whereas fluorescent in situ hybridization (Fish) with a chromosome 5 (q31.1) probe showed only a diploid allele compliment. Assessment of transcriptional clonality by iduronate-2-sulfatase (IDS) gene polymorphism suggested that JAK2 V617F was acquired in at least two independent multipotent stem cell progeny. Our findings indicate that JAK2 V617F mutant clones may arise in genetically discordant clones independent of del(5q).

Maitake Beta-Glucan Enhances Umbilical Cord Blood Stem Cell Transplantation in the NOD/SCID Mouse

Beta glucans are cell wall constituents of yeast, fungi and bacteria, as well as mushrooms and barley. Glucans are not expressed on mammalian cells and are recognized as pathogen-associated molecular patterns (PAMPS) by pattern recognition receptors (PRR). Beta glucans have potential activity as biological response modifiers for hematopoiesis and enhancement of bone marrow recovery after injury. We have reported that Maitake beta glucan (MBG) enhanced mouse bone marrow (BMC) and human umbilical cord blood (CB) cell granulocyte-monocyte colony forming unit (GM-CFU) activity in vitro and protected GM-CFU forming stem cells from doxorubicin (DOX) toxicity. The objective of this study was to determine the effects of MBG on expansion of phenotypically distinct subpopulations of progenitor and stem cells in CB from full-term infants cultured ex vivo and on homing and engraftment in vivo in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse. MBG promoted a greater expansion of CD34+CD33+CD38- human committed hematopoietic progenitor (HPC) cells compared to the conventional stem cell culture medium (P = 0.002 by ANOVA). CD34+CXCR4+CD38- early, uncommitted human hematopoietic stem cell (HSC) numbers showed a trend towards increase in response to MBG. The fate of CD34+ enriched CB cells after injection into the sublethally irradiated NOS/SCID mouse was evaluated after retrieval of xenografted human CB from marrow and spleen by flow cytometric analysis. Oral administration of MBG to recipient NOS/SCID mice led to enhanced homing at 3 days and engraftment at 6 days in mouse bone marrow (P = 0.002 and P = 0.0005, respectively) compared to control mice. More CD34+ human CB cells were also retrieved from mouse spleen in MBG treated mice at 6 days after transplantation. The studies suggest that MBG promotes hematopoiesis through effects on CD34+ progenitor cell expansion ex vivo and when given to the transplant recipient could enhance CD34+ precursor cell homing and support engraftment.

Hematopoietic Stem - and Progenitor Cells Are Differentially Mobilized in Response to Flt3-Ligand

Flt3 is a tyrosine kinase receptor expressed mainly on primitive hematopoietic cells and its ligand, Flt3-ligand (FL), is a slowly mobilizing agent in mice when administered for 5–10 days. This provides a time-frame to study the HPC and HSC migration kinetics in detail. BALB/c mice were injected for 3, 5, 7 and 10 days with FL (10 ug/day, intraperitoneally). Mobilization of hematopoietic stem- and progenitor cells was studied using colony-forming-unit granulocyte/monocyte (CFU-GM) and cobblestone-area-forming-cell (CAFC) assays. The radioprotective capacity of mobilized peripheral blood mononuclear cells (PBMC) was studied by transplantation of 1.5 × 106 FL-mobilized PBMNC into lethally irradiated (9.5 Gy) recipients. Hematopoietic progenitor cell mobilization was detected from day 3 onwards and prolonged administration of FL showed a steady increase in mobilized progenitor cells (CFU-GM; 23.1±20.1 [PBS], 219.0±213.7 [3 days FL, n=10], 1,007.8±742 [5 days FL, n=21], 3,526.6±1,406 [7 days FL, n=10] and 19,149±2,338 [10 days FL, n=10]). Administration of FL for 10 days lead to a 5.5-fold increase in CAFC-week 4, compared to FL administration for 5 days (0.69 vs 0.13 CAFC per 105 PBMC respectively) and a 5.0-fold increase in CAFC-week 5 (0.27 vs 0.05 CAFC per 105 PBMC respectively). No CAFC week 4/5 were found in PBMC obtained from PBS-injected control mice. Transplantation of 5-day FL-mobilized PBMC did not radioprotect lethally irradiated recipients (mean survival time (MST): 66 days; MST PBS: 13 days). In contrast, transplantation of PBMC obtained from a 10-day FL mobilization regime radioprotected 100% of the recipients (Table 1). These results indicate that HPC and HSC show different mobilization kinetics in response to FL, resulting in preferential mobilization of HPC at day 5, followed by HSC mobilization at day 10. We speculate that this difference in mobilization kinetics might be due to the selective recruitment of HPC and HSC from their respective bone marrow compartments.Table 1. HPC and HSC frequencies in peripheral blood following Flt3-ligand-induced mobilizationCFU-GM per ml PBCAFC-day 28 per 105cellsCAFC-day 35 per 105cells% Radioprotection day 1505 days Flt3-L1,007 (n=21)0.1260.0540 (n=10)10 days Flt3-L19,149 (n=10) (19.0)*0.694 (5.5)*0.272 (5.0)*100 (n=10)* fold increase compared to 5 day FL

The haemotoxicity of azathioprine in repeat dose studies in the female CD-1 mouse.

Azathioprine (AZA) is a cytotoxic immunosuppressive drug used in the prevention of rejection in organ transplants and the treatment of auto-immune diseases. However, AZA is haemotoxic causing significant bone marrow depression. The present studies were to characterize the haemotoxicity of AZA in the female CD-1 mouse. In Experiment 1, a dose-ranging study, with AZA gavaged daily for 10 days, clinical evidence of toxicity was evident at 125 mg/kg and above. Experiment 2 was a dose-response study with AZA gavaged daily for 10 days at 40-120 mg/kg. At day 1 after the final dose, AZA induced a dose-related pancytopaenia, reduced femoral marrow cellularity, increases in serum levels of the cytokine fms-like tyrosine kinase 3 ligand, reduction in granulocyte-monocyte colony-forming units and erythroid colonies, and increased bone marrow apoptosis. Histology demonstrated hepatocyte hypertrophy, thymic atrophy, reduced splenic extramedullary haemopoiesis, and reduced cellularity of sternal bone marrow. In Experiment 3, AZA was dosed for 10 days at 100 mg/kg with autopsies at 1, 3, 9, 22, 29, 43 and 57 days postdosing. At 1, 3 and 9 days, haematological parameters reflected changes in Experiment 2. At 22/29 days, many blood parameters were returning towards normal; at 43/57 days, most parameters compared with controls. However, there was some evidence of a persistent (i.e. residual/late-stage) mild reduction in RBC and erythroid progenitor cell counts at day 43/57. We conclude that the CD-1 mouse provides an acceptable model for the haemotoxicity of AZA in man.

CD34+ CD38− and CD34+ HLA-DR− cells in BM stem cell grafts correlate with short-term engraftment but have no influence on long-term hematopoietic reconstitution after autologous transplantation

Prior studies have demonstrated that relatively immature hematopoietic stem cells, including CD34(+) CD38(-) and CD34(+) HLA-DR(-) subsets, correlate with short-term hematopoietic reconstruction (SHR) after transplantation. The aim of this study was to investigate whether these immature CD34(+) subsets also correlate with long-term hematopoietic reconstitution (LHR) in recipients of ABMT. We examined stem cell grafts from 58 patients with B-cell lymphoma or CLL who underwent ABMT after myeloablative conditioning. We determined whether total mononuclear cell dose (MNC), colony-forming unit-granulocyte-monocyte (CFU-GM), CD34(+) cell dose and CD34(+) cell subsets (CD34(+) CD38(-) and CD34(+) HLA-DR(-) were associated with SHR and/or LHR. Time to neutrophil engraftment (TNE) and time to platelet engraftment (TPE) were used to measure SHR, while platelet counts at day 100 and 1 year post-ABMT were used as indicators for LHR. CD34(+) cell dose and CD34(+) cell subsets were significantly associated with SHR. However, at day 100 and 1 year post-transplant only total CD34(+) cell dose was associated with LHR. The association of total CD34(+) cell dose with LHR persisted after adjusting for age, sex and disease. None of the CD34(+) cell subsets analyzed showed evidence of significant association with LHR.

Hematologic abnormalities in Shwachman Diamond syndrome: lack of genotype-phenotype relationship

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by short stature, exocrine pancreatic insufficiency, and hematologic defects. The causative SBDS gene was sequenced in 20 of 23 unrelated patients with clinical SDS. Mutations in the SBDS gene were found in 75%, being identical in 11 patients. Hematologic parameters for all 3 lineages were determined over time such as absolute neutrophil counts (ANCs), granulocyte functions, and erythroid and myeloid colony formation (erythroid burst-forming unit [BFU-E] and granulocyte-monocyte colony-forming unit [CFU-GM]) from hematopoietic progenitor cells, percentage of fetal hemoglobin (HbF), and platelet counts. Persistent neutropenia was present in 43% in the absence of apoptosis and unrelated to chemotaxis defects (in 65%) or infection rate. Irrespective of the ANC in vivo, abnormal CFU-GM was observed in all patients with SDS tested (14 of 14), whereas BFU-E was less often affected (9 of 14). Cytogenetic aberrations occurred in 5 of 19 patients in the absence of myelodysplasia. One child died during allogeneic bone marrow transplantation. In conclusion, neutropenia and defective chemotaxis did not result in severe clinical infection in SDS. CFU-GMs were impaired in all patients tested. From the SBDS sequence data, we conclude that in patients with genetically proven SDS a genotype-phenotype relationship in SDS does not exist in clinical and hematologic terms.

Effect of Propofol Exposure on Hematopoiesis in Mouse Bone Marrow

Background: It is evident that inhalational anesthetics, such as nitrous oxide, possess a certain degree of myelodepressive effect in humans. However, unlike nitrous oxide, propofol is frequently recommended for the anesthesia of oncologic patients or for harvesting bone marrow from donors. To evaluate the possible toxicity of propofol on hematopoietic stem cells, the in vitro sensitivity of colony growth to propofol was assessed using murine clonogenic hematopoietic progenitor cells. Methods: Femoral and tibial marrow cells were obtained from 4- to 6-week-old male BALB/c mice. Propofol was diluted in culture medium (30, 300 and 1 mM) and added into methylcellulose semi-solid culture media. After 14 days of culturing, the numbers of colony-forming unit granulocyte/monocyte (CFU-GM) colonies were counted. An advance liquid culture (RPMI 1640) of 5 hours duration was also applied prior to culturing in semisolid media to assess the short term exposure toxicity. Results: The colony counts were significantly decreased compared to the control at higher concentrations than 1 mM (P ＜ 0.05). The pre-exposure to propofol did not affect the number CFU-GM colony count at the concentrations of 30 and 300 or under conditions of co-culture. Conclusions: No myelodepressive effect was observed in mouse bone marrow cells with exposure of propofol at concentrations under 300.