Colony-forming Unit Granulocyte-macrophage Colonies Research Articles

CLL Cell-Derived Exosomes Promote Bone Marrow Fibrosis and Inhibit Normal Hematopoietic Colony Proliferation

Whereas low grade bone marrow (BM) reticulin fibrosis is detected in most patients with CLL, high grade BM fibrosis is associated with anemia, thrombocytopenia and poor prognosis. Although this observation was reported several years ago, the mechanism of BM fibrosis in CLL is still unknown. We have recently found that CD14+ monocyte-derived fibrocytes, known to participate in the initiation of tissue fibrosis in the skin, lung, kidney, liver, and heart, induce BM fibrosis in patients with primary myelofibrosis. Therefore, we wondered whether fibrocytes induce BM fibrosis in CLL. Consistent with our hypothesis, we found increased fibrocytes numbers in CLL patients' BM biopsies as assessed by immunohistochemistry analysis. Because fibrocytes are derived from CD14+ monocytes, thought to derive from the normal hematopoietic clone, we wondered what induces overproduction of fibrocytes in the BM of patients with CLL.Exosomes are 60 -100 nm vesicles released by mammalian cells. Exosomes harbor various intracellular components including transcriptionally active mRNA, non-coding RNAs and proteins. Numerous studies demonstrated that tumor-derived exosomes are stable inter-cellular transport vehicles that deliver tumor cell cargo into other cells. Therefore we wondered whether exosomes affect the generation of BM fibrosis in CLL. In light of recent studies showing that bystander cells engulf CLL-derived exosomes which often alter their function, we hypothesized that CD14+ monocytes engulf CLL-derived exosomes that increase the capacity of these monocytes to differentiate into reticulin producing fibrocytes. To test this hypothesis, we incubated peripheral blood CLL cells in culture medium containing exosome-depleted fetal bovine serum for 2 days and harvested extracellular nanoparticles with CD63-coated beads. By using flow cytometry we found that the CD63+ nanoparticles co-express CD63, CD5, and CD19, and by electron microscopy we confirmed that they were typical 60 to 100 nm cup-shaped exosomes co-expressing CD5 and CD19 as assessed by immuno-gold. To test whether normal monocytes engulf CLL-derived exosomes we incubated CLL-derived exosomes stained with a green FM 1-43 dye, with healthy donor-derived CD14+ monocytes. After 2 hours, excess exosomes were washed out and the monocytes were counterstained with Evans blue. Confocal microscopy analysis showed that these monocytes harbor a significant number of CLL-derived exosomes. We also cultured these monocytes under conditions that favor fibrocyte differentiation and found that normal CD14+ monocytes incubated with CLL-derived exosomes, but not with CLL patients' filtered plasma, express significantly higher levels of CD14 surface antigen than untreated monocytes and form fibrocytes at a 3.6 higher rate than normal monocytes, similar to the level of CLL-derived monocytes. Because we previously found that normal fibrocytes stimulate hematopoietic colony proliferation, we wondered what would be the function of CLL-derived fibrocytes or of fibrocytes generated from normal CD14+ monocytes incubated with CLL exosomes. Compared to untreated controls, the number of colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies was markedly increased when normal BM cells were cultured on top of normal fibrocytes. In contrast, the number of CFU-GM and BFU-E colonies was significantly lower when normal BM cells were cultured on top of CLL-derived fibrocytes or fibrocytes derived from normal CD14+ cells that were incubated with CLL-derived fibrocytes. Furthermore, CLL-derived exosomes, but not normal low-density cell-derived exosomes, inhibited CFU-GM and BFU-E colony growth in a dose-dependent manner, suggesting that CLL-derived exosomes directly inhibit normal myeloid progenitor proliferation.In conclusion: CLL-derived exosomes reprogram monocytes to generate an increased number of reticulin-producing fibrocytes and directly and indirectly inhibit myeloid progenitor proliferation. Taken together our data show how exosomes contribute to hematopoietic dysfunction in CLL and provide a novel perspective on how CLL cells actively modify their microenvironment. DisclosuresVerstovsek:Roche: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Bristol Myers Squibb: Research Funding; Roche: Research Funding; Astrazeneca: Research Funding; Blueprint Medicines Corp: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Blueprint Medicines Corp: Research Funding; Galena BioPharma: Research Funding; Pfizer: Research Funding; CTI BioPharma Corp: Research Funding; Galena BioPharma: Research Funding; CTI BioPharma Corp: Research Funding; Gilead: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Promedior: Research Funding; Celgene: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Research Funding; NS Pharma: Research Funding; NS Pharma: Research Funding; Lilly Oncology: Research Funding; Lilly Oncology: Research Funding; Astrazeneca: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Novartis: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding. Bose:Incyte Corporation: Honoraria. Thompson:Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jain:Incyte: Research Funding; Abbvie: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Verastem: Research Funding; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wierda:Juno: Research Funding; Sanofi: Consultancy, Honoraria; Emergent: Consultancy, Honoraria, Research Funding; Karyopharm: Research Funding; The University of Texas MD Anderson Cancer Center: Employment; Merck: Consultancy, Honoraria; Janssen: Research Funding; GSK/Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Acerta: Research Funding; Genzyme: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Genentech/Roche: Consultancy, Honoraria, Research Funding; Kite: Research Funding.

Impact of Daratumumab on Stem Cell Collection, Graft Composition and Engraftment Among Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplant

Background High dose chemotherapy followed by autologous stem cell transplantation (ASCT) plays an important role in the treatment of transplant-eligible multiple myeloma (MM) patients and yields deep responses, prolongs progression-free survival (PFS) and overall survival (OS). Daratumumab (Dara), a humanized monoclonal antibody that binds to CD38+ on the surface of myeloma cells and is increasingly used upfront to treat MM. Up to 75% of mobilized CD34+ hematopoietic progenitors also express CD38 and, hence exposure to Dara may potentially impact stem cell mobilization, and, given the extended half-life of Dara, also impair engraftment. The Cassiopeia trial (Moreau, P. 2019) showed the utility of Dara in combination with bortezomib and thalidomide as upfront treatment for MM without a negative impact on stem cell collection yield. Similarly, the Griffin trial (Voorhees, PM, 2020) demonstrated the safety of upfront Dara when combined with bortezomib and lenalidomide also with no impact on stem cell collection or engraftment. However, others(Al Saleh, A. 2019) reported delayed neutrophil engraftment in patients treated with Dara. Given the conflicting findings of these studies and scarcity of data on the direct impact of Dara on erythroid and myeloid progenitors, we investigated the effect of Dara on stem cell collection yield, graft composition and time to engraftment among patients who underwent ASCT. Methods We evaluated all patients with MM who underwent ASCT at our institute between 2017-2020 and excluded patients undergoing 2nd transplant or tandem ASCT. Disease risk stratification was assessed as per International Staging System (ISS) for MM. Treatment response prior to transplant was assessed as per response criteria definitions by International Myeloma Working Group (IMWG). Stem cells were collected according to institutional protocol and prior to cryopreservation, 1x105 peripheral blood cells are plated in duplicate in semi-solid media- MethoCult™ H4434 (Stem Cell Technologies, Vancouver, BC) and cultured in 37°C, 5% CO2 following which CFU-GM (colony forming unit- granulocyte macrophage) and BFU-E (burst forming units- erythroid) colonies are scored on day 14 based on morphological recognition, and reported as 104 colonies per kg of recipient weight. CFU-C (colony forming unit combined) is calculated by combining BFU-E and CFU-GM. Results Patients (N=108) that underwent ASCT between 2017-2020 were identified and demographic data are summarized (Table 1). Sixteen patients received Dara as part of upfront treatment prior to stem cell collection. Median age was 62 years old for the entire group with no significant age difference between patients who received Dara vs. those who did not receive. Similarly, there was no difference in race, ISS stage, pre-transplant response, plerixafor or lenalidomide use between the groups. Pts who received Dara were more likely to have received more than one chemotherapy regimen prior to transplant (62.5% vs. 30%, p-value=0.014). Although, total and day 1 collection of CD34+ stem cells was lower in the Dara treated group (7.18 x 106/kg) compared to Dara untreated (8.78 x 106/kg), the difference was not statistically significant. (p-value=0.151). Stem cell product composition based on measurement of BFU-E, CFU-GM and CFU-C colonies were similar independent of Dara use. Prior exposure to Dara was also not predictive of day 1 stem cell collection failure (defined as < 5x106 CD34+ cells/kg) as determined by multivariate analysis. Median time to absolute neutrophil count (ANC) recovery (defined as ANC >1500) was 12 days in both groups (p-value=0.09). Median time to platelet recovery (defined as platelet count >20k) was 12 days in the Darauntreated group vs. 13 days Dara treated group (p-value=0.06). Discussion In this single institute experience, there was a trend towards lower CD34+ collection as well as lower progenitor scoring with Dara use but was not statistically significant and also there was no difference in time to ANC or platelet recovery. The majority of patients in this study received plerixafor for mobilization, which might abrogate the effect of Dara and lenalidomide on the graft and stem cell collection. Further studies to investigate the impact of Dara on CD38+ mobilized hematopoietic cells is warranted. Disclosures Manjappa: Pfizer: Research Funding. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Celgene: Speakers Bureau; Kite pharmaceuticals: Other: Advisory Board; ADC therapeutics: Other: Advisory Board, Research Funding. de Lima:BMS: Other: Personal Fees, advisory board; Incyte: Other: Personal Fees, advisory board; Kadmon: Other: Personal Fees, Advisory board; Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding. Malek:Clegene: Other: Advisory board , Speakers Bureau; Janssen: Other: Advisory board, Speakers Bureau; Sanofi: Other: Advisory board; Amgen: Honoraria; Medpacto: Research Funding; Cumberland: Research Funding; Bluespark: Research Funding; Takeda: Other: Advisory board , Speakers Bureau.

Atrial natriuretic peptide protects against cisplatin-induced granulocytopenia.

Granulocytopenia is the major toxicity associated with cisplatin treatment. Atrial natriuretic peptide (ANP) is a cardiac hormone used clinically for the treatment of acute heart failure in Japan. ANP exerts a wide range of protective effects on various organs, including the heart, blood vessels, lungs, and kidneys. This study's objective was to investigate the protective effects of ANP on cisplatin-induced granulocytopenia in mice. The mice were divided into two groups: cisplatin with vehicle and cisplatin with ANP. ANP (1.5μg/kg/min via osmotic pump, subcutaneously) or vehicle administration was started 1day before cisplatin injection until the mice were killed. At 0, 2, 4, 8, and 14days after cisplatin injection (16mg/kg, intraperitoneally as a single dose), the white blood cell, red blood cell, and platelet counts were measured in the peripheral blood in both groups. The numbers of total and live cells and colony-forming unit-granulocyte-macrophage (CFU-GM) colonies in the bone marrow of the mice were also examined. In addition, at 0, 0.5, 1, and 2days after cisplatin injection, serum granulocyte colony-stimulating factor (G-CSF) levels were measured. ANP significantly attenuated the white blood cell count decrease in the peripheral blood 2 and 4days after cisplatin injection. ANP also attenuated the decrease in the number of live cells and CFU-GM colonies in bone marrow 2, 4, and 8days after cisplatin injection. ANP significantly increased serum G-CSF levels 1day after cisplatin injection. ANP has protective effects in cisplatin-induced granulocytopenia, with increased G-CSF production.

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.

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.

Scorpion Venom Peptides Accelerate Hematopoietic Recovery of Myelosuppression in Irradiated Mice

Sublethally irradiated mice were administered with scorpion venom peptides (SVP) or with PBS in the saline control group, 3 days before and 7 consecutive days after irradiation. Hematopoietic recovery was assessed by bone marrow (BM) cell proliferation index (PI) and colony forming unit-granulocyte/macrophage (CFU-GM), spleen weight index (SI) and thymus weight index (TI), colony-forming unit-spleen (CFU-S) and peripheral leukocyte counts. In addition, IL-1alpha and SCF levels in BM, IL-6 and GM-CSF levels in serum were determined. In SVP treated groups, PI was improved dramatically versus control mice on day 22 after irradiation. The number of CFU-GM colonies in all SVP treated groups was higher than the control groups. The difference of the number of CFU-GM colonies between SVPV group (0.2 mg/kg) and the control was significant on day 5 and 10 after irradiation (p < 0.05). SVPIV (0.2 mg/kg) could activate the CFU-S formation on day 10 after irradiation. SI was in peak value on day 15 after irradiation in all groups and the SI value of SVPV treated group was higher than control group (p < 0.05). Our results suggest that SVP may be valuable natural peptides that relieve myelosuppression caused by radiation. The effect of SVP accelerating the hematopoietic recovery was potentially through a mechanism of stimulating the release of cytokines.

Effects of peritoneal lavage fluid from radiation or/and burn injured rats on the growth of hematopoietic progenitor cells

Purpose: To evaluate the effects of peritoneal lavage fluids from radiation injury, burn injury and combined radiation-burn injury on the growth of hematopoietic progenitor cells (HPC).Materials and methods: Rats were divided into four groups: A radiation group (RG), a burn group (BG), a combined radiation-burn group (CRBG) and normal control group (NG). RG and CRBG rats were irradiated with 12 Gy, and burns of 30% total body surface area were generated in group BG and group CRBG. Peritoneal lavage fluids were collected and tested for their effects on the growth of erythrocyte progenitor cells or granulocyte-macrophage progenitor cells of BALB/c mice in vitro.Results: The numbers of colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) formed after treatment with lavage fluids from BG or CRBG were significantly higher than those from NG. However, fewer CFU-E, BFU-E or CFU-GM colonies were found after treatment with lavage fluid from the RG. In lavage fluid from BG and CRBG, the concentration of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor α (TNFα) was increased in comparison to NG and RG. Treatment with these cytokines had similar promoting effects on the growth of hematopoietic colonies and neutralizing antibodies inhibited these effects significantly.Conclusions: Burns increase the responsiveness of the system and help the proliferation of hematipoietic progenitor cells, while radiation decreases all these responses relative to both the controls and the burn plus radiation group.

Evaluation of ‘out-of-specification’ CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products

Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Role of Downstream Effectors in Kras-Driven Myeloproliferative Disease.

Mutations that deregulate Ras signaling are highly prevalent in myeloid malignancies. Previous work has shown that expressing oncogenic KrasG12Din hematopoietic cells from the endogenous Kras promoter leads to growth factor-independent and hypersensitive myeloid progenitor colony formation, and causes a fatal myeloproliferative disorder (MPD) (Braun et al PNAS 101:597, 2004; Chan et al JCI 113:528, 2004). These characteristics mirror the human juvenile and chronic myelomonocytic leukemias (JMML and CMML) - diseases that are associated with Ras pathway mutations. Oncogenic Ras accumulates in its active GTP-bound form and constitutively activates a number of downstream effectors. However, it is unclear which effectors are necessary for the maintenance of disease as well as how they contribute to specific phenotypes such as enhanced proliferation and defective apoptosis. We constructed second site mutants - secondary mutations on a backbone of KrasG12D(G12D) that prevent K-RasG12D from binding to a subset of effectors - to begin to elucidate the individual contributions of downstream effector pathways to hematologic disease. Murine fetal liver cells engineered to express KrasG12D,E37G(E37G) and KrasG12D,Y64G(Y64G) second site mutants maintain a hypersensitive pattern of colony-forming unit granulocyte-macrophage (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), but are no longer able to grow in the absence of GM-CSF. Interestingly, both mutant proteins that display hypersensitivity also hyperactivate two major Ras effector pathways, as opposed to other second site mutants tested which only hyperactivate one major Ras effector pathway and do not show growth factor hypersensitivity. Whereas E37G activates both PI3 kinase and Ral-GDS but not Raf, Y64G stimulates Raf/MEK/ERK and PI3 kinase but not Ral-GDS. Consistent with the idea that activation of at least two effector cascades is required for aberrant in vitro growth, expression of activated effectors BrafV600E, p110a-CAAX, and RalGDS-CAAX did not confer GM-CSF hypersensitivity on CFU-GM colonies. We transplanted bone marrow cells transduced with MSCV-E37G-IRES-GFP or MSCV-Y64G-IRES-GFP retroviruses into lethally irradiated Balb/c mice. Seven of 8 mice that received Y64G cells died of T lineage acute lymphoid leukemia/lymphoma (T-ALL/L) with a median survival of 112 days. Diseased animals showed very high levels of GFP in the thymus, spleen, peripheral blood and bone marrow. Four of 9 mice injected with E37G-expressing cells mice also died of T-ALL/L with a median survival of 106 days and three of 9 died of anemia. None of the MIG mice (n=8) have died of leukemia/lymphoma. The Y64G and E37G T ALL/Ls studied to date are transplantable into sublethally irradiated recipients. We conclude that second site mutations in the KrasG12Doncogene that are defective for activation of either PI3 kinase or Raf/MEK/ERK are able to deregulate the growth of primary hematopoietic cells in vitro and in vivo. These data argue that targeting a single effector pathway downstream of oncogenic Ras may not be effective in many hematologic malignancies. We are interrogating Ras signaling networks in T-ALL/L cells and cloning MSCV integration sites to further characterize the effects of these second site mutant alleles.

Scoring CFU-GM colonies in vitro by data fusion: A first account

In vitro models of hematopoiesis used in investigative hematopathology and in safety studies on candidate drugs, involve clonogenic assays on colony-forming unit granulocyte macrophage (CFU-GM). These assays require live and unstained colonies to be counted. Most laboratories still rely on visual scoring, which is time-consuming and error-prone. As a consequence, automated scoring is highly desired. An algorithm that recognizes and scores CFU-GM colonies by data fusion has been developed. Some preliminary results are presented in this article. CFU-GM assays were carried out on hematopoietic progenitors (human umbilical cord blood cells) grown in methylcellulose. Colony images were acquired by a digital camera and stored. The classifier was designed to process images of layers sampled from a three-dimensional (3D) domain and forming a stack. Structure and texture information was extracted from each image. Classifier training was based on a 3D colony model applied to the image stack. The number of scored colonies (assigned class) was required to match the count supplied by the human expert (class of belonging). The trained classifier was validated on one more stack and then applied to a stack with overlapping colonies. Scoring in distortion- and caustic-affected border areas was also successfully demonstrated. Because of hardware limitations, compact colonies in some cases were missed. The industry's scoring methods all rely on structure alone and process 2D data. Instead, the classifier here fuses data from a whole stack and is capable, in principle, of high-throughput screening.

Involvement of Niemann-Pick Type C2 Protein in Hematopoiesis Regulation

Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease, an inherited neurodegenerative disorder. When analyzing gene expression profile, we noticed high expression of both NPC2 and its receptor, mannose 6-phosphate receptor (MPR), in murine hematopoietic stem cells. NPC2 protein, in the presence of thrombopoietin (TPO), causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells, a TPO-dependent hematopoietic leukemia cell line, NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly, these results are not observed under the standard 20% O(2) level of the standard incubator, but rather at 7% O(2), the physiological oxygen level of bone marrow. Furthermore, NPC2 protein upregulates hypoxia inducible factor 1-alpha protein level at 7% O(2), but not at 20% O(2). Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O(2).

HOXB4-Induced Self-Renewal of Hematopoietic Stem Cells Is Significantly Enhanced by p21 Deficiency

Enforced expression of the HOXB4 transcription factor and downregulation of p21(Cip1/Waf) (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self-renewal generated by overexpression of HOXB4 is enhanced in p21-deficient HSCs. HOXB4 was overexpressed in hematopoietic cells from wild-type (wt) and p21-/- mice. Bone marrow (BM) cells were transduced with a retroviral vector expressing HOXB4 together with GFP (MIGB4), or a control vector containing GFP alone (MIG) and maintained in liquid culture for up to 11 days. At day 11 of the expansion culture, the number of primary CFU-GM (colony-forming unit granulocyte-macrophage) colonies and the repopulating ability were significantly increased in MIGB4 p21-/- BM (p21B4) cells compared with MIGB4-transduced wt BM (wtB4) cells. To test proliferation of HSCs in vivo, we performed competitive repopulation experiments and obtained significantly higher long-term engraftment of expanded p21B4 cells compared with wtB4 cells. The 5-day expansion of p21B4 HSCs generated 100-fold higher numbers of competitive repopulating units compared with wtMIG and threefold higher numbers compared with wtB4. The findings demonstrate that increased expression of HOXB4, in combination with suppression of p21 expression, could be a useful strategy for effective and robust expansion of HSCs.

Essential Role of Synoviolin in Embryogenesis

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

Functional Analysis of Leukemia-Associated PTPN11 Mutations in Primary Hematopoietic Cells.

The PTPN11 gene encodes SHP-2, a non-receptor protein tyrosine phosphatase (PTPase) that relays signals from activated growth factor receptors to p21ras (Ras), Src family kinases, and other signaling molecules. Germ-line, missense mutations in PTPN11 account for approximately 50% of cases of the human developmental disorder Noonan Syndrome (NS). More recently, PTPN11 mutations have been identified in approximately 35% of children with juvenile myelomonocytic leukemia (JMML) without NS and have also been detected in other lymphoid and myeloid malignancies. Interestingly, almost all of these leukemia-associated mutations introduce amino acid substitutions within the N-SH2 domain of SHP-2 that are largely distinct from those found in NS. We have assessed the functional consequences of leukemia-associated PTPN11 mutations in primary hematopoietic cells. Expression of an E76K mutant SHP-2 in murine fetal liver and bone marrow cells confers a hypersensitive pattern of colony-forming unit granulocyte-macrophage (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). Specifically, cells expressing E76K mutant SHP-2 display enhanced colony growth at low concentrations of growth factor compared to cells expressing wild-type (WT) SHP-2 protein. Mutant colonies are significantly larger with an abnormal, spreading morphology. E76K-expressing cells also form CFU-GM colonies in the absence of growth factor. The catalytic activity of the E76K mutant is required for aberrant colony growth as expressing the E76K mutation in the context of defective phosphatase activity (C463S) abolishes hypersensitive CFU-GM growth. Mutant E76K expression also enhances the growth of immature progenitor cells with high repopulating potential (HPP-CFC and LPP-CFC) in response to GM-CSF and IL-3 and perturbs erythroid progenitor colony growth. In addition, expressing the E76K mutation results in more pronounced growth factor hypersensitivity than another leukemia-associated SHP-2 mutation (D61Y), and both of these mutations confer a stronger hematopoietic phenotype than the common N308S substitution found in patients with NS.

Osteoclastic Potential of Human CFU-GM: Biphasic Effect of GM-CSF

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

Inhibition of CFU-E/BFU-E and CFU-GM colony growth by Cyclophosphamide, 5-Fluorouracil and Taxol: development of a high-throughput in vitro method

The major side effect of anticancer drugs is damage to the hematopoietic system. These compounds may interfere with cell proliferation and differentiation in different blood lineages causing many diseases such as neutropenia, aplastic anaemia or trombocytopenia. The clonogenic assays are useful in vitro tools for evaluating and predicting acute xenobiotics myelotoxicity. A miniaturisation of these assays, in order to reduce costs and increase the number of compounds that could be tested, is under investigation. The in vitro sensitivity of human burst-forming unit erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) to three anticancer drugs: cyclophosphamide (CTX), 5-fluorouracil (5-FU) and taxol (TAX) was evaluated both in 35 mm plate and 96-well plate systems and the dose-response curves, IC50 values and IC90 values were compared. The correlation between in vitro data and clinical plasma levels confirms that severe hematotoxicity is the primary adverse effect of these drugs with an evident selectivity on erythroid progenitors for cyclophosphamide. IC50 and IC90 values, calculated on the basis of results obtained with the traditional assay, correlate with those obtained in microplate, as well as the dose-response curves, indicating that the 96 well plate assay could be a useful and reliable tool for high-throughput screening in early stages of drug development.

Expression of genes regulating angiogenesis in human circulating hematopoietic cord blood CD34+/CD133+ cells

Human CD34+ cells represent a heterogeneous population of immature cells which may differentiate to various cell types. The aim of the study was to determine angiogenesis regulating genes expression in CD34+ cells, their subpopulations, and during their differentiation induced by hematopoietic growth factors. We have measured the expression of angiogenesis regulating genes angiopoietin-1 (Ang-1), angiopoietin-1 (Ang-2) and their receptor Tie-2, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in sorted population of CD34+ and CD34+/CD133+ cells from human cord blood and bone marrow, and in their differentiating progeny, using real time reverse transcriptase polymerase chain reaction. The hematopoietic differentiation of CD34+ cells was induced in semisolid or liquid differentiation supporting media containing appropriate hematopoietic growth factors. A higher expression of Ang-1, Ang-2, and Tie-2 mRNAs was detected in CD34+/CD133+ cord blood cells as compared with CD34-/CD133- fraction, but no expression of these genes was detected in burst-forming unit erythroid (BFU-E) nor colony-forming unit granulocyte macrophage (CFU-GM) colonies. The level of Ang-1 and Tie-2 mRNAs, but not that of Ang-2 mRNA gradually decreased during a 14-d incubation of cord blood CD34+ cells in a liquid culture. A significantly higher expression of VEGF mRNA was in BFU-E as compared with CFU-GM cell colonies and CD34+/CD133+ cells. VEGFR-1 mRNA was equally expressed in CD34+/CD133+ and CD34-/CD133- cells as well as in BFU-E and CFU-GM colonies. Expression of VEGFR-2 mRNA was detected at the borderline of method sensitivity only in CD34+/CD133+ cells. CD34+/CD133+ cord blood cells express Ang-1, Ang-2 and VEGF as well as their receptor mRNAs, suggesting a role of these cells in regulation both angiopoiesis and hematopoiesis.

Expression of either NF-kappaB p50 or p52 in osteoclast precursors is required for IL-1-induced bone resorption.

Interleukin (IL)-1 is implicated in postmenopausal- and inflammation-mediated bone loss. Its expression is regulated by NF-kappaB and vice versa. To examine the role of NF-kappaB p50 and p52 (they are required for osteoclast formation during embryonic development) in IL-1-induced resorption, we used various NF-kappaB knockout (KO) mice, including p50-/- and p52-/- single KO, p50-/- and p52+/- (3/4KO), and p50-/- and p52-/- double KO (dKO) mice. IL-1 increased blood calcium and bone resorption in wild-type (wt), p50, and p52 single KO mice, but not in 3/4KO or dKO mice. Osteoclast formation was impaired in bone marrow cultures from 3/4KO compared with single KO and wt mice treated with IL-1. IL-1 receptor expression was similar in colony forming unit-granulocyte macrophage (CFU-GM) colony cells from wt and dKO mice. However, IL-1 promoted CFU-GM colony formation and survival as well as the formation, activity, and survival of osteoclasts generated from these colonies from wt mouse splenocytes, but not from dKO splenocytes. No difference in expression of the osteoclast regulatory cytokines, RANKL, and OPG, was observed in osteoblasts from wt and dKO mice. Thus, expression of either NF-kappaB p50 or p52 is required in osteoclasts and their precursors, rather than osteoblasts, for IL-1-mediated bone resorption.

NF-kappaB p50 and p52 expression is not required for RANK-expressing osteoclast progenitor formation but is essential for RANK- and cytokine-mediated osteoclastogenesis.

Expression of RANKL by stromal cells and of RANK and both NF-kappaB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF-KB signaling in this process, we used NF-kappaB p50-/- ;p52-/- double knockout (dKO) and wild-type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony-stimulating factor (M-CSF). NF-kappaB dKO splenocytes formed more colony-forming unit granulocyte macrophage (CFU-GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU-GM colonies. RANKL increased the number of CFU-GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from NF-kappaB dKO mice revealed a two-to threefold increase in the percentage of CD11b (Mac-1) and RANK double-positive cells compared with WT controls. Treatment of NF-kappaB dKO splenocytes with interleukin (IL)-1, TNF-alpha, M-CSF, GM-CSF, and IL-6 plus soluble IL-6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF-kappaB dKO or p50-/-;p52+/- (3/4KO) mice. Thus, NF-kappaB p50 and p52 expression is not required for formation of RANK-expressing osteoclast progenitors but is essential for RANK-expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.

Long-term engraftment stability of peripheral blood stem cells cryopreserved using the dump-freezing method in a -80 degrees C mechanical freezer with 10% dimethyl sulfoxide.

In this study, we summarize our long-term follow-up data of 24 patients who underwent autologous peripheral blood stem cell transplantation (PBSCT) using the dump-freezing method in a -80 degrees C freezer. Collected peripheral blood mononuclear cells were mixed with a cryoprotectant solution consisting of autologous plasma and 20% dimethyl sulfoxide, then placed in a -80 degrees C freezer. The recovery rate of mononuclear cells (MNCs), colony-forming unit-granulocyte/macrophage (CFU-GM) colonies, and CD34+ cells were calculated. Engraftment time (with neutrophil count > 0.5 x 10(9)/L, platelet count > 50 x 10(9)/L) and normal hemopoiesis (neutrophil count > 2 x 10(9)/L, platelet count > 100 x 10(9)/L) were evaluated. Median duration of cryopreservation was 76 days. The mean recovery rates of MNCs, CFU-GM colonies, and CD34+ cells were 93.4%, 78.4%, and 95.3%, respectively. The median engraftment times of neutrophils and platelets were 8 and 27 days, respectively. The median normal hemopoiesis times of neutrophil and platelet were 31 and 45 days, respectively. Nine patients are alive and in complete remission (CR). Seven patients in first CR sustained normal hemopoiesis with a median duration of 35 months. Two patients, who achieved second CR after salvage chemotherapy due to a leukemia relapse after PBSCT, maintained engraftment status for 24 and 28 months, and 1 reached normal hemopoiesis. These results demonstrate that PBSCT using the dump-freezing method in a -80 degrees C freezer leads to acceptable long-term engraftment stability.