Granulocyte-macrophage Colony-forming Units Research Articles

Control of AC133/CD133 and impact on human hematopoietic progenitor cells through nucleolin.

AC133 is a prominent surface marker of CD34+ and CD34- hematopoietic stem/progenitor cell (HSPC) subsets. AC133+ HSPCs contain high progenitor cell activity and are capable of hematopoietic reconstitution. Furthermore, AC133 is used for prospective isolation of tumor-initiating cells in several hematological malignancies. Nucleolin is a multifunctional factor of growing and cancer cells, which is aberrantly active in certain hematological neoplasms, and serves as a candidate molecular target for cancer therapy. Nucleolin is involved in gene transcription and RNA metabolism and is prevalently expressed in HSPCs, as opposed to differentiated hematopoietic tissue. The present study dissects nucleolin-mediated activation of surface AC133 and its cognate gene CD133, via specific interaction of nucleolin with the tissue-dependent CD133 promoter P1, as a mechanism that crucially contributes to AC133 expression in CD34+ HSPCs. In mobilized peripheral blood (MPB)-derived HSPCs, nucleolin elevates colony-forming unit (CFU) frequencies and enriches granulocyte-macrophage CFUs. Furthermore, nucleolin amplifies long-term culture-initiating cells and also promotes long-term, cytokine-dependent maintenance of hematopoietic progenitor cells. Active β-catenin, active Akt and Bcl-2 levels in MPB-derived HSPCs are nucleolin-dependent, and effects of nucleolin on these cells partially rely on β-catenin activity. The study provides new insights into molecular network relevant to stem/progenitor cells in normal and malignant hematopoiesis.

139 CD45−CD34+ Circulating Progenitor Cells Levels Are Associated With the Presence of Coronary Artery Disease (CAD) in Patients With Nonalcoholic Fatty Liver Disease (NAFLD)

Background and Aim: Angiogenic growth factors (AGF) play a crucial role in regulating hematopoietic stem and progenitor cell homing and differentiation. Patients with NAFLD and CAD have lower levels of AGF (Elsheikh et al. DDW 2014). Thus, in this study we investigated the level and functions of circulating progenitor cells (CPC) in patients with NAFLD and CAD. Methods: We prospectively enrolled 82 patients undergoing elective coronary angiography. Each patient had fasting serum, clinical data and abdominal ultrasound (US). All US were read centrally using a standard protocol. NAFLD was defined as radiologic fatty liver in the absence of other causes of liver disease and excessive alcohol use. Patients were divided into NAFLD with CAD (n=24), NAFLD without CAD (n=13), only CAD (n= 31) and Non-NAFLD and Non-CAD (n=14). Circulating progenitor cells (CPC) were quantified by flow cytometry based on the expression of (CD34+, CD133+, CD34+ CD133+) in presence or absence of the hematopoietic marker (CD45). To study the functional capacity of CPC, colony-forming unit (CFU) assay was used. These include erythroid progenitors (burst-forming unit erythroid (BFU-E)); granulocyte/macrophage progenitors (CFU-granulocyte, macrophage (CFU-GM); and multi-potential progenitors (CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM)). Results: The levels of the CD45-CD34+, CD45CD133+ and BFU-E were higher in NAFLD patients with CAD (median, 15%, 2% and 60 colonies, respectively) than NAFLD patients without CAD (median, 9%, 1% and 37 colonies , respectively, all p-values≤0.05). We also found that the levels of the CD45-CD133+ were higher in NAFLD patients with CAD (median, 15%) than patients with only CAD (median, 11%, p 25th quartile of CD45-CD34+, CD45-CD133+ and total CFU levels had the highest risk for CAD [OR: 7.00 (1.34-36.68), 5.00 (1.07-23.46) and 7.00 (1.10-44.61) respectively]. After age adjustment, only CD45-CD34+ circulating progenitor cells remain associated with increased risk of CAD in patients with NAFLD [OR: 8.71 (1.21-62.51)]. Conclusions: Our results indicate that, levels of circulating progenitor cells CD45-CD34+ may be associated with increased risk of CAD in NAFLD patients. Increased circulating progenitor cells levels could be due to the low levels of AGF leading to impaired homing and differentiation capacity.

Acute GVHD after allogeneic hematopoietic transplantation affects early marrow reconstitution and speed of engraftment

Our aim was to study the influence of acute graft-versus-host disease (a-GVHD) on primary engraftment times after allogeneic transplantation. Primary engraftment and frequency of marrow granulocyte-macrophage colony-forming units and erythroid burst-forming units, at day +18, were studied in 126 allogeneic transplants. Patients were grouped according to the time when a-GVHD treatment with corticosteroids was started. The no-a-GVHD group are those who, during the first 3months, had no need for a-GVHD treatment; the early-a-GVHD group are those who needed a-GVHD treatment within 19days; and the postengraftment-a-GVHD group are those who were not on corticosteroid treatment at the time of engraftment but needed it after day +19. The no-a-GVHD group reached a neutrophil count (N)>0.5×10(9)/L in a median of 17.8days. The postengraftment-a-GVHD group reached N>0.5×10(9)/L in a median of21.4days (p=0.0003). The early-a-GVHD group had N>0.5×10(9)/L in a median of +17.0days (p=0.23). When factors important for engraftment were studied in a multivariate analysis, postengraftment a-GVHD was a significant factor in delayed neutrophil and platelet engraftment. Both the early-a-GVHD and postengraftment-a-GVHD groups showed a significant reduction in frequency of granulocyte-macrophage colony-forming units and erythroid burst-forming units found in marrow at day +18. In conclusion, a-GVHD may influence early marrowreconstitution and is a relevant factor for primary myeloid and platelet engraftment.

Interleukin-10 inhibits autonomous myelopoiesis in patients with myelofibrosis.

The spontaneous formation of colony-forming units granulocyte/macrophage (CFU-GM) in semisolid cultures has been shown to be due to the endogenous release of cytokines and/or to the hypersensitivity of cells against growth factors. We have reported that increased autonomous CFU-GM growth is an in vitro characteristic of myelofibrosis (MF) which may reflect aberrant hematopoiesis in vivo. Because of its cytokine synthesis-inhibiting action, we speculated that interleukin-10 (IL-10) may inhibit pathological overproduction of myeloid cells in MF by suppression of autonomous myelopoiesis. In this study, IL-10 significantly inhibited autonomous CFU-GM formation in vitro from peripheral blood mononuclear cells (PB MNC) in 10 of 11 patients with MF tested. In all patients, there was a mean inhibition of 69% ranging from 35% to 100%. Suppression of autonomous CFU-GM formation by IL-10 was dose dependent and reversible by the addition of anti-IL-10 antibodies. Our results indicate that IL-10 is a potentially useful molecule to affect aberrant myelopoiesis in patients with MF.

Biochemical and Functional Analysis of Novel KRAS Insertions in MPN and Other Cancers

The somatic KRAS mutations found in ~25% of human cancers commonly encode amino acid substitutions at codons 12, 13, and 61. Each of these mutations lead to elevated levels of active Ras-GTP by reducing intrinsic Ras GTPase activity and conferring resistance to GTPase activating proteins (GAPs). Amino acids 12, 13, and 61 play key roles in binding the γ phosphate of GTP and facilitating GAP-mediated catalysis. The germline KRAS point mutations found in Noonan syndrome encode different amino acid substitutions that have less severe biochemical consequences. Together, these observations and the structural conservation of the Ras/GAP switch through evolution suggest that a limited spectrum of mutations can constitutively activate Ras and cause disease. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm characterized by driver Ras pathway mutations in 85% of cases, including common somatic KRAS and NRAS substitutions. We unexpectedly identified a novel somatic KRAS insertion, 66GQEEYSA67, in the bone marrow of a patient who meets most, but not all diagnostic criteria, for JMML and lacks any other Ras pathway mutation. This seven amino acid insertion is located in the conserved Switch II domain of Ras, which binds to and activates PI3 kinase and other Ras effectors. We characterized the functional and biochemical consequences of this mutation, and of a similar five amino acid insertion identified in two lung cancers and one colon cancer archived in the COSMIC database (66EEYSA67). We find that unlike WT K-Ras, the expression of both insertion mutants supports cytokine independent growth of IL-3 dependent pro-B Ba/F3 cells similar to oncogenic K-RasG12D. Ba/F3 cells expressing K-Ras66GQEEYSA67 or K-Ras66EEYSA67 mutant proteins have markedly elevated levels of Ras-GTP that are insensitive to IL-3 stimulation, and result in constitutive ERK and Akt phosphorylation. Confirming that these Switch II insertion mutants have oncogenic potential in the hematopoietic system, fetal liver cells transduced with KRas66GQEEYSA67, KRas66EEYSA67, or KRasG12D formed cytokine independent colony forming unit granulocyte macrophage (CFU-GM) colonies and also exhibited GM-CSF hypersensitivity, which is a cellular hallmark of JMML. Biochemical characterization of recombinant K-Ras66GQEEYSA67 and K-Ras66EEYSA67 proteins showed them to have reduced intrinsic GTP hydrolysis, and to accumulate in the GTP-bound conformation when compared to oncogenic K-RasG12D. Furthermore, whereas recombinant p120 GAP markedly accelerated the rate of GTP hydrolysis by WT K-Ras, K-Ras66GQEEYSA67 and K-Ras66EEYSA67 were completely resistant. The most likely structural explanation is that these insertions perturb critical contacts between glutamine 61 in the Switch II domain of Ras and GAPs. We conclude that KRAS66GQEEYSA67 and KRAS66EEYSA67 are oncogenic driver mutations in JMML and other cancers. These data illuminate a new class of oncogenic RAS mutations and are of immediate diagnostic relevance. Furthermore, these data reveal unanticipated structural flexibility in a domain of Ras that plays a critical role in effector binding, which has implications for potential mechanism of resistance to emerging therapeutic approaches. DisclosuresBollag:Plexxikon: Employment. Zhang:Plexxikon: Employment.

Abstract 1167: Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets

Abstract Fatty diets can induce low-grade inflammation that we report is increased by chronic alcohol consumption (CAC). CAC as 16.6% of total calories when administered in combination with the Lieber-DeCarli high-fat diet increases inflammation included hepatic and splenic extramedullary hematopoiesis (EMH) as assessed by flow cytometry, immunohistochemistry and a colony forming unit-granulocyte macrophage (CFU-GM) assay. Further, an increased number of hepatic myeloid derived suppressor cells (MDSCs) CD11b+Gr1+ cells that were predominantly Ly6cbr are observed. The increase in MDSCs is associated with an increased number of hepatic non-parenchymal cells including adipocytes (Oil Red O). Consistent with the increased number of hepatic MDSCs and EMH is a decrease in bone marrow cellularity and progenitor cells measured by flow cytometry (Lin-CD11b-Gr1-Sca-1+) and CFU-GM/femur. Unexpectedly, we observed demineralization and osteolytic lesions by micro computed tomography (micro CT) in all bones examined including femur, tibia, fibula and vertebral column that was associated with osteoclast activity (Trap+). Osteolysis was most notable in the fibula and vertebral spurs associated with osteoclast channels, and the demineralization appeared to be associated with areas of active myelopoiesis. The low grade, chronic inflammation associated with the Lieber-DeCarli fatty diet and CAC accelerated the induction of orthotopic 4T1 mammary tumors, resulting in extensive bone osteolysis, demineralization and increased metastases at aberrant sites including splenic, cardiac, hepatic, and extensive lymph node foci in addition to peritoneal and pleural effusions. The latter were haemorrhagic with a predominant nucleated cell infiltrate composed of bands, segs and myelocytes, supporting EMH. These results support the suggestion that a high-fat diet and CAC together increase tumor induction, metastasis and pathology in association with MDSC mobilization, expansion and EMH resulting in increased numbers of osteoclasts, associated bone demineralization and suppression of T-cell frequency and function. These results support the development of combination therapy strategies incorporating multiple molecular therapeutics that inhibit OCs, MDSCs and their associated mediators. Citation Format: Anand Dusad, Saraswoti Khadge, Geoffrey M. Thiele, Michael J. Duryee, Holly C. Britton, Lynell W. Klassen, Alicia J. Dafferner, Tracy Farrell, Timothy R. McGuire, Carlos D. Hunter, Karen C. Easterling, Karen J. O'Kane, John Graham Sharp, James E. Talmadge. Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2014-1167

The orally bioavailable MDM2 antagonist RG7112 and pegylated interferon α 2a target JAK2V617F-positive progenitor and stem cells

The Philadelphia chromosomal-negative chronic myeloproliferative neoplasms (MPNs) originate at the level of the hematopoietic stem cell (HSC). The protracted clinical course of the MPNs has limited the use of potentially toxic treatment modalities, which may eliminate the responsible malignant clone. Treatment with low doses of RG7112, an orally available small-molecule inhibitor of p53-MDM2, both alone and combined with pegylated interferon α 2a (Peg-IFNα 2a), significantly decreased MPN colony-forming unit-granulocyte macrophage and burst-forming unit-erythroid numbers and preferentially eliminated the total number of JAKV617F(+) MPN hematopoietic progenitor cells. The effects of RG7112 and Peg-IFNα 2a on MPN progenitor cells were dependent on blocking p53-MDM2 interactions and activating the p53 pathway, thereby increasing MPN CD34(+) cell apoptosis. Treatment of polycythemia vera (PV) and primary myelofibrosis (PMF) CD34(+) cells with low doses of RG7112 and Peg-IFNα 2a before their transplantation into immune-deficient mice decreased the degree of donor-derived chimerism as well as the JAK2V617F allele burden, indicating that these drugs can each alone or in combination deplete MPN HSCs. These results provide a rationale for the use of combinations of low doses of RG7112 and Peg-IFNα 2a for the treatment of PV or PMF patients with the intent of altering their natural history.

Do two different stem cell grafts: G-CSF stimulated and unstimulated bone marrow differ according to hematopoietic colony forming capacity?

The study was designed to compare colony forming capacity of granulocyte-colony stimulating factor (G-CSF) stimulated bone marrow (G-BM) with standard unstimulated bone marrow (U-BM) of healthy donors of pediatric patients. CFU-Assay results of 26 healthy donors of pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) were analyzed retrospectively. 13 of donors received 10 μg/kg per day of G-CSF as a single injection for 3 consecutive days and other 13 of donors had unstimulated BM. Colony forming capacity of hematopoietic stem cells evaluated with Colony Forming Unit-Assay (CFU-Assay) with in semi-solid agar culture medium after 14-18 days of culture period. CFU-Assay results of G-BM and U-BM (expressed as means) were; Burst Forming Unit-Erythroid (BFU-E): 15.20 × 10(4)/kg and 8.38 × 10(4)/kg, Colony Forming Unit-Granulocyte Macrophage (CFU-GM): 10.35 × 10(4)/kg and 5.67 × 10(4)/kg, Colony Forming Unit-Erythroid (CFU-E): 0.59 × 10(4)/kg and 0.33 × 10(4)/kg, CFU-Granulocyte Erythroid Macrophage Megakaryocyte (CFU-GEMM): 0.52 × 10(4)/kg and 0.53 × 10(4)/kg respectively. BFU-E and CFU-GM capacity of G-BM was increased and statistically significantly different than standard U-BM (p ⩽ 0.01). In conclusion, increased colony forming capacity of hematopoietic stem cells of G-BM when compared with standard unstimulated BM could be a major advantage for transplantation.

NS-018, a selective JAK2 inhibitor, preferentially inhibits CFU-GM colony formation by bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients

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

FEASIBILITY OF COLLECTING UMBILICAL CORD BLOOD IN JORDAN AND THE EFFECT OF MATERNAL AND NEONATAL FACTORS ON HEMATOPOIETIC STEM CELL CONTENT

BackgroundCord blood transplant is an accepted treatment for many malignant and non-malignant diseases. We sought to determine the feasibility of collecting cord blood in Jordan and the effect of maternal and fetal factors on the quality of the cord blood units.MethodsA total of 124 cord blood units were collected, and 75 (60%) cord blood units were included in this analysis. Cord blood volume, total nucleated cell (TNC) count, cell viability and CD34+ content were measured, and clonogenic assay was performed.ResultsThe mean volume of the collected units was 68.9 ml (range 40–115) with mean nucleated cell count of 6.5 x 108 (range 1–23.0). Our results showed a positive correlation between the volume of cord blood and TNC count (p=0.008), cell viability (p=0.001), CD34+ content (p=0.034) and the length of the umbilical cord (p=0.011). In addition, our results showed an inverse relation between the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) concentration and the gestation duration (p=0.038).ConclusionWe conclude that it is feasible to collect cord blood units in Jordan with excellent TNC and CD34+ cell content. The volume of cord blood collected was associated with higher TNC count and CD34+ count. Efforts toward establishing public cord blood banks in our area are warranted.

Allogeneic compact bone-derived mesenchymal stem cell transplantation increases survival of mice exposed to lethal total body irradiation: a potential immunological mechanism

Background Radiation-induced injury after accidental or therapeutic total body exposure to ionizing radiation has serious pathophysiological consequences, and currently no effective therapy exists. This study was designed to investigate whether transplantation of allogeneic murine compact bone derived-mesenchymal stem cells (CB-MSCs) could improve the survival of mice exposed to lethal dosage total body irradiation (TBI), and to explore the potential immunoprotective role of MSCs. Methods BALB/c mice were treated with 8 Gy TBI, and then some were administered CB-MSCs isolated from C57BL/6 mice. Survival rates and body weight were analyzed for 14 days post-irradiation. At three days post-irradiation, we evaluated IFN-γ and IL-4 concentrations; CD4+CD25+Foxp3+ regulatory T cell (Treg) percentage; CXCR3, CCR5, and CCR7 expressions on CD3+ T cells; and splenocyte T-bet and GATA-3 mRNA levels. CB-MSC effects on bone marrow hemopoiesis were assessed via colony-forming unit granulocyte/macrophage (CFU-GM) assay. Results After lethal TBI, compared to non-transplanted mice, CB-MSC-transplanted mice exhibited significantly increased survival, body weight, and CFU-GM counts of bone marrow cells (P<0.05), as well as higher Treg percentages, reduced IFN-γ, CXCR3 and CCR5 down-regulation, and CCR7 up-regulation. CB-MSC transplantation suppressed Th1 immunity. Irradiated splenocytes directly suppressed CFU-GM formation from bone marrow cells, and CB-MSC co-culture reversed this inhibition. Conclusion Allogeneic CB-MSC transplantation attenuated radiation-induced hematopoietic toxicity, and provided immunoprotection by alleviating lymphocyte-mediated CFU-GM inhibition, expanding Tregs, regulating T cell chemokine receptor expressions, and skewing the Th1/Th2 balance toward anti-inflammatory Th2 polarization.

Dominant Negative MCP‐1 Blocks Human Osteoclast Differentiation

Human osteoclasts were differentiated using receptor activator of NFκB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) from colony forming unit-granulocyte macrophage (CFU-GM) precursors of the myeloid lineage grown from umbilical cord blood. Gene expression profiling using quantitative polymerase chain reaction (Q-PCR) showed more than 1,000-fold induction of chemokine MCP-1 within 24 h of RANKL treatment. MCP-1 mRNA content exceeds that of other assayed chemokines (CCL1, 3, 4, and 5) at all time points up to day 14 of treatment. MCP-1 induction preceded peak induction of calcium signaling activator calmodulin 1 (CALM1) and transcription factors JUN and FOS, which were at 3 days. Key osteoclast related transcription factors NFATc1 and NFATc2 showed peak induction at 7 days, while marker genes for osteoclast function cathepsin K and tartrate resistance acid phosphatase (TRAP) were maximally induced at 14 days, corresponding with mature osteoclast function. To test whether the early and substantial peak in MCP-1 expression is part of human osteoclast differentiation events, a dominant negative inhibitor of MCP-1 (7ND) was added simultaneously with RANKL and M-CSF, resulting in blockade of CALM1, JUN and NFATc2 induction and strong inhibition of human osteoclast differentiation. These data show that a cascade of gene expression leading to osteoclast differentiation depends on intact early MCP-1 induction and signaling in human osteoclasts.

Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis

The tumor suppressor p53 is thought to play a role in megakaryocyte (MK) development. To assess the influence of the p53 regulatory pathway further, we studied the effect of RG7112, a small molecule MDM2 antagonist that activates p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and platelet production. This drug has been previously been evaluated in clinical trials of cancer patients where thrombocytopenia was one of the major dose-limiting toxicities. In this study, we demonstrated that administration of RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we identified two distinct mechanisms by which RG7112-mediated activation of p53 affected human megakaryocytopoiesis and platelet production in vitro. RG7112 promoted apoptosis of MK progenitor cells, resulting in a reduction of their numbers and RG7112 affected mature MK by blocking DNA synthesis during endomitosis and impairing platelet production. Together, the disruption of these events provides an explanation for RG7112-induced thrombocytopenia and insight into the role of the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis.

Transfusion begets anemia

Following trauma, transfusion of aged stored blood is often necessary yet associated with increased morbidity and mortality. Despite blood replacement, many patients have a prolonged anemia requiring further transfusions. The effects of aged blood on bone marrow (BM) hematopoiesis have not been studied, and we hypothesized that stored blood suppresses BM function. Blood from Sprague-Dawley rats was stored for 1, 14, or 28 days with the industry preservative citrate-phosphate-dextrose-adenine-1 (CPDA-1). For in vitro studies, 5% supernatant was incubated with normal rat BM and cultured for erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units. Data were compared with cultures of BM alone, 5% control plasma (negative control), and 12% CPDA-1. For in vivo studies, rats were transfused with stored supernatants (5% estimated blood volume (EBV) over 30 minutes). BM from each recipient was cultured for CFU-E and CFU-GM at 3 hours after transfusion. Data were compared with cultures of BM alone. Difference between groups determined by analysis of variance and Tukey's multiple comparison test. In vitro exposure to CPDA-1, control plasma, or 1-day supernatant (D1) had no effect on BM growth compared with BM alone. In vitro exposure to 14-day (D14) and 28-day (D28) supernatant significantly suppressed CFU-E by 60% and CFU-GM growth by 71% (both p < 0.05) compared with D1 or medial alone. There were no differences between D14 and D28. In vivo exposure to D14 reduced BM CFU-E and CFU-GM growth by 55% (both p < 0.05) compared with D1 supernatant. Plasma from aged blood adversely affects CFU-E and CFU-GM growth in rats. The effect is not mediated by CPDA-1. Transfusion of aged stored blood may contribute to BM dysfunction in critically ill patients, resulting in persistent anemia and the need for further transfusion. This BM dysfunction may also partly explain the observed increased susceptibility to infection.

Study for the improvement of umbilical cord blood sampling using a new trial apparatus

The aim of this study was to evaluate the usefulness of the trial umbilical cord blood sampling bag for unrelated cord blood transplantation. Data were obtained from 100 vaginal deliveries. In 50 cases, umbilical cord blood (UCB) was taken with the traditional Kawasumi type UCB sampling bag. In another 50 cases, UCB were taken with trial UCB sampling bag offered by NIPRO Co. We compared the sampling volume between the two groups. Furthermore, 10 cases in each group were matched by sampling volume; we examined the quality of UCB on the number and concentration of nucleated cells, mononuclear cells, CD34+ cells and colony-forming unit granulocyte macrophage and the numbers tested positive for bacteria. Whereas there were no significant differences in gestational weeks at sampling, the ratio of primipara women to multipara women, maternal age, and neonatal weight between the two groups, the sampling UCB volumes with the trial sampling bag were significantly higher than those with traditional sampling bags (P < 0.05). In addition, this phenomenon was more significant in the latter part of the study period (P < 0.05). On the other hand, there were no significant differences in the quality of UCB between the two groups. Once clinicians have become accustomed to the trial UBC sampling bag, this method might be a useful method for collecting UCB for unrelated cord blood transplantation.

The JAK2-Selective Inhibitor NS-018 Preferentially Suppresses CFU-GM Colony Formation By Bone-Marrow Cells From High-Risk Myelodysplastic Syndrome Patients

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

Identification Of CD34+ Microparticles In Cord Blood Units

Introduction Cell-derived microparticles (MPs), small membrane vesicles, are considered as markers of cell activation, as well as apoptosis and recently was shown to participate in important normal biological functions and pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Recently platelet MPs have been detected in patients undergoing allogeneic stem cell transplantation. Umbilical cord blood is an alternative stem cell source for transplantation where the presence of MPs has not been extensively studied. The aim of this study is to quantify the MPs of CD34+ cells in cord blood units and to investigate their relationship with different parameters. Methods Cord blood units (CBUs) (n=41) were processed using the Sepax automated method (Biosafe). After processing, cryopreservation was performed using 10% DMSO in a controled rate freezer (IceCube, Sy-lab). Units were thawed after two weeks. Adjusted variables of the CBUs included their net volume, the absolute number of CD34+ per graft and their viability as determined by flow cytometry, the number of leukocytes as recorded with an hematology analyzer and the absolute number of erythroblasts as estimated by optical microscope after Giemsa staining. Mononuclear cells were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony forming unit granulocyte/macrophage (CFU-GM), burst forming unit erythroid (BFU-E) and Colony-Forming Unit-Granulocyte, Erythrocyte, Monocyte/macrophage, Megakaryocyte (CFU-GEMM) colony growth. The MPs were isolated after centrifugation of the plasma and their number was determined after incubation with Annexin V and CD34 by flow cytometry. Electron microscope photographs were obtained, after the proceedings for TEM. Statistical analysis was done using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables. Results The mean number of AnnV+/CD34+MPs increased during post-processing (76,7±15,9) compared to the number of pre-processing AnnV+/CD34+MPs (73±22 ) and decreased during post-thawing (52,4 ±16,7). On univariate analysis found that the number of AnnV+/CD34+MPs (pre-processing vs. post-processing) has statistically significant mean positive correlation (p&lt;0,012, Pearson's rho =0,823) whereas the number of MPs post-thawing was not statistically significant. Pre-processing AnnV+/CD34+MPs has statistically significant correlation with the absolute number of pre-processing CD34+ cell per unit (p&lt;0,0001, Spearman's rho =0,835), post-processing (p&lt;0,0001, Spearman's rho =0,814) and post-thawing CD34+ cells per unit (p&lt;0,0001, Spearman's rho =0,993). Correlation of post-processing CFUs and AnnV+/CD34+MPs showed that the number of AnnV+/CD34+MPs has statistically significant mean negative correlation to the number of CFU-GEMM (p&lt;0,028, Spearman's rho = -0,348). Conclusions These data confirm the identification of stem cell-derived MPs from cord blood samples. Moreover, the number of pre- and post- processing AnnV+/CD34+MPs was statistically correlated. In addition, the absolute number of CD34 (pre-, post- processing and post-thawing) was associated with the AnnV+/CD34+MPs pre- processing. Thus it remains to be shown if the stem cell derived MPs could be a useful parameter to predict the quality of the cord blood unit. Disclosures: No relevant conflicts of interest to declare.

Activities Of Commonly Used Tyrosine Kinase Inhibitors Against Wild-Type C-KIT and Potential Impact On Normal Hematopoiesis

The receptor tyrosine kinase (RTK) c-KIT is important in early hematopoiesis, and impaired c-KIT activity is associated with abnormal erythroid and megakaryotic function in animal models. Several approved and investigational small molecule tyrosine kinase inhibitors have off-target activity against c-KIT, with resultant potential for hematopoietic toxicity. To investigate the association between c-KIT inhibition by TKIs and myelosuppression, we determined the inhibitory activity of a series of TKIs against c-KIT using immunoblot estimates of IC50 concentrations, and compared those results with the compounds' effects on human hematopoiesis in progenitor cell assays. For this study, we used quizartinib, crenolanib, sorafenib, imatinib, pazopanib, and dasatinib. IC50s against FLT3 and c-Kit were established for each kinase with assays performed both in culture medium (RPMI/10%fetal bovine serum) and 100% human plasma. Colony formation assays with normal human bone marrow were performed in quadruplicate with clinically achievable levels of each drug, adjusting for plasma protein binding (and based on published pharmacokinetic data from plasma samples of human subjects). Out of the six TKIs studied, most showed limited to no inhibition of the formation of colony-forming unit-granulocyte macrophage (CFU-GM). Three TKIs, quizartinib, pazopanib, and dasatinib, demonstrated inhibition of burst-forming unit-erythroid (BFU-E) colonies. The highest doses of these drugs inhibited growth of BFU-E colonies by 30% versus untreated control. These same three drugs are also the most potent c-Kit inhibitors of this group, with IC50s of 35, 7.5, and 1.4 nM, respectively, in culture medium. The remaining TKIs, crenolanib, sorafenib, and imatinib, showed far less BFU-E inhibition, none inhibiting colony formation by more than 25%. These TKIs also had higher c-Kit IC50 values of 65, 395, and 122 nM, respectively. We conclude that c-KIT inhibition correlates with eythroid progenitor cell suppression in vitro, and that TKIs that achieve c-KIT inhibitory levels in vivo would be predicted to cause anemia. During clinical development of TKIs that have c-KIT inhibitory activity, attention should be paid to the therapeutic index between the targeted kinase (e.g., FLT3) and c-KIT, so as to avoid unnecessary myelosuppresive effects. Dose range determinations to spare c-Kit inhibition would lead to a decreased risk of disrupting normal hematopoiesis, making treatment far more manageable for these patients. Disclosures:Levis:Ambit Biosciences: Consultancy.

Definition of the Variables Affecting Efficacy of Immunodepletion Ex Vivo of Peripheral Blood Progenitor Cell Grafts by Alemtuzumab (Campath in the Bag)

The immunodepleting effects of alemtuzumab on peripheral blood progenitor cell (PBPC) grafts for stem cell transplantation need to be better defined. The optimal graft cell concentration, antibody dose, need for complement, and whether alemtuzumab is infused with the graft during transplantation remain unclear. PBPC from 6 normal allogeneic stem cell donors harvested by apheresis were first quantitated and the cellular content defined by flow cytometry. Mononuclear cells were then incubated with incremental concentrations of alemtuzumab (.00001, .0001, .001, and .01 mg/mL) for 30 minutes at 20°C or in cell dose responses with 1, 5, and 10 × 106 mononuclear cells/mL added to a fixed dose of .001 mg/mL of alemtuzumab with or without a source of complement. Cells were enumerated and analyzed by flow cytometry before and after exposure to alemtuzumab. To determine the presence of unbound anti-CD52, the supernatant of the cell dose responses were tested using the ELISA assay. Selected CD34+ lineage-negative cells were incubated with antibody at the same working concentrations and conditions and cultured in granulocyte-macrophage colony-forming unit assay. The colony numbers were compared with control cultures devoid of the antibody. Incremental concentrations of alemtuzumab led to a significant (2 log) reduction in CD3, CD4, and CD8 populations, which plateaued at .001 mg/mL. Addition of complement led to a further significant reduction in the CD4 and CD8 cells. The maximum CD4 (3 log) and CD8 (2 log) cell death was obtained at 10 × 106 cells/mL. Analysis of supernatants for soluble alemtuzumab by ELISA showed a significant reduction in the free antibody concentration when the cell number was increased from 1 to 10 × 106 cells/mL implying utilization/binding of the antibody by target cells. Incremental concentrations of alemtuzumab did not affect the number of granulocyte-macrophage colony-forming units. Alemtuzumab depletes all cells expressing the CD52 antigen and has higher activity on CD3, CD8, and particularly on CD4 cells, which are depleted in excess of 2 logs. From this study, we were able to derive that the optimal cell kill in the graft without detectable free alemtuzumab in the supernatant can be achieved with 1 mg of antibody per 100 mL containing 10 × 109 cells and active complement (AB serum).

Comparison of the Ex Vivo Expansion of UCB-Derived CD34+ in 3D DBM/MBA Scaffolds with USSC as a Feeder Layer.

Ex vivo expansion of hematopoitic stem cells is an alternative way to increase umbilical cord blood (UCB)-CD34+ cells for bone marrow transplantation. For this purpose demineralized bone matrix (DBM) and mineralized bone allograft (MBA) as two scaffolds based on bone matrix and stem cell niche, were simultaneously used to enhance the effect of human mesenchymal progenitor cells (MPCs) - unrestricted somatic stem cells (USSCs) - as a feeder layer. USSCs were isolated and characterized by morphological and immunological analysis then seeded on both scaffolds as a feeder layer. UCB-CD34(+) were isolated by MACS method and were co-culture expanded by USSC in 3D and 2D environments. After 3 weeks expansion, cells were counted and were assessed by karyotype, flow cytometry, clonogenic activity, and long-term culture-initiating cells (LTC-IC). Co-culture expansion in DBM and MBA was 29.22-fold and 27.77-fold, no significant differences in colony and LTC-IC were obtained. Maximum number of colonies belonged to the day 14 with the 73% CFU-GM (Colony Forming Unit- Granulocyte/Macrophage) in contrast to the day 0 which was BFU-E/CFU-E (Burst/Colony Forming Unit-Erythroid). Flow cytometry indicated that the percentage of CD34+ marker was decreased in USSC co-culture and the highest percentage was observed in simple 2D culture. Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion.