Induction of Telomerase Activity During Development of Human Mast Cells from Peripheral Blood CD34+Cells: Comparisons with Tumor Mast-Cell Lines

Human mast cells are derived from CD34+ hematopoietic cells present in cord blood, bone marrow, and peripheral blood. However, little is known about the properties of the CD34+ cells. We demonstrated here that mast cell progenitors that have distinct phenotypes from other hematopoietic cell types are present in cord blood by culturing single, sorted CD34+ cells in 96-well plates or unsorted cells in methylcellulose. The CD34+ mast cell-committed progenitors often expressed CD38 and often lacked HLA-DR, whereas CD34+ erythroid progenitors often expressed both CD38 and HLA-DR and CD34+ granulocyte-macrophage progenitors often had CD33 and sometimes expressed CD38. We then cultured single cord blood-derived CD34+CD38+ cells under conditions optimal for mast cells and three types of myeloid cells, ie, basophils, eosinophils, and macrophages. Of 1,200 CD34+CD38+ cells, we were able to detect 13 pure mast cell colonies and 52 pure colonies consisting of either one of these three myeloid cell types. We found 17 colonies consisting of two of the three myeloid cell types, whereas only one colony consisted of mast cells and another cell type. These results indicate that human mast cells develop from progenitors that have unique phenotypes and that committed mast cell progenitors develop from multipotent hematopoietic cells through a pathway distinct from myeloid lineages including basophils, which have many similarities to mast cells.

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Targeting Glutamine Metabolism in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v124.21.1869.1869 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1869-1869

Author(s):

Huichun Zhan ◽

Geoffrey Girnun ◽

Katherine Dong ◽

Kristen Ciano ◽

Yupo Ma ◽

...

Keyword(s):

Stem Cell ◽

Cell Growth ◽

Cell Lines ◽

Peripheral Blood ◽

Myeloproliferative Neoplasms ◽

Mutant Cell ◽

Glutamine Metabolism ◽

Peripheral Blood Cd34 ◽

Cd34 Cells ◽

Mutant Cells

Abstract Introduction: The chronic Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal stem cell disorders characterized by unregulated stem cell expansion and overproduction of blood cells. The acquired mutation JAK2V617F plays a central role in the pathogenesis of MPN. Ruxolitinib was the first FDA-approved JAK2 inhibitor for the treatment of MPNs; however, its use is limited by various toxicities due to its suppression of normal marrow function. Because its toxicity is related to dosage, combinations of drugs may enhance the therapeutic effectiveness of Ruxolitinib and permit the administration of lower doses of Ruxolitinib. Cancer cells depend on a continued supply of glucose and glutamine for cell survival and proliferation. Glutaminase (GLS) is the key enzyme in glutamine metabolism. We report here that JAK2V617F mutation is associated with increased cellular metabolism and the GLS inhibitor BPTES (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide) improves the therapeutic efficacy of Ruxolitinib in both JAK2V617F-mutant cell lines and MPN patient samples. Methods: The JAK2V617F-mutant human erythroleukemia (HEL) cell line and the murine prolymphoid cell line Ba/F3 expressing wild-type or mutant JAK2 (BaF3-hEPOR-JAK2wt cell and BaF3-hEPOR-JAK2V617F cell) were maintained as previously described. Cells were treated with DMSO, BPTES (Sigma¨), and/or Ruxolitinib (Selleckchem¨) and cell proliferation was assessed by counting bright live cells in a hemocytometer using the trypan blue exclusion method. Mitochondrial respiration (oxygen consumption rate, OCR) and glycolysis (extracelluar acidification rate, ECAR) were measured using a Seahorse¨ XFe96 Bioanalyzer. We used 13C stable isotope analysis to determine differences in glucose and glutamine utilization. BaF3-hEPOR-JAK2wt cell and BaF3-hEPOR-JAK2V617F cell were grown in [U6]-13C glucose or [U5]-13C glutamine for 24 hours and metabolites extracted and prepared for GC-MS analysis. Data were analyzed using MassHunter software (Agilent Technologies¨). MPN patient specimens were obtained with informed consent and approval by the IRB of Northport VA Medical Center. Peripheral blood CD34+ cells from MPN patients were isolated using immunomagnetic beads (Miltenyi¨) as we previously did. MPN CD34+ cells were treated with DMSO, BPTES, and/or Ruxolitinib and assayed in methylcellulose semisolid medium (Stem Cell Technologies¨) with growth factors. Colonies were counted after 14 days of incubation. Results: OCR and ECAR were increased by 55% (p < 0.0001) and 2-fold (p < 0.0001), respectively, in BaF3-hEPOR-JAK2V617F cells compared to BaF3-hEPOR-JAK2wt cells. This indicated that the JAK2V617F-mutant cells have higher metabolic activity than JAK2WT cells. Stable isotope analysis using GC/MS showed that JAK2V617F-mutant cells had increased utilization of glucose and glutamine into the TCA cycle as well as increased lactate production from glucose. There was a higher glutaminase activity in the JAK2V617F-mutant cells than the JAK2WT cells. Next we determined the effect of the glutaminase inhibitor, BPTES, on Ruxolitinib-mediated inhibition of cell proliferation in JAK2V617F-mutant cell lines. While BPTES (5µM) had little effect on HEL cell growth, the combination of BPTES (5µM) and Ruxolitinib (1.5µM) significantly inhibited HEL cell growth by 66% while Ruxolitinib alone inhibited cell growth by only 35% (p = 0.04). Similar results were obtained with the BaF3-hEPOR-JAK2V617F cells. (Figure 1) Finally, we tested the combination therapy of BPTES and Ruxolitinib in peripheral blood CD34+ cells from 4 MPN patients (2 PMF and 2 ET). The combination of BPTES (2uM) and Ruxolitinib (125nM) suppressed total hematopoietic colony growth of MPN CD34+ cells by 91% in comparison to 68% inhibition by Ruxolitinib alone (p = 0.007). Both CFU-GM colony and BFUe colony were suppressed by the combination therapy more than by Ruxolitinib alone although it was only significant for CFU-GM colony formation. (Figure 2) Conclusion: Our study showed that JAK2V617F mutation was associated with increased cellular metabolism. Importantly, a glutaminase inhibitor enhanced the therapeutic efficacy of Ruxolitinib in both JAK2V617F-mutant cell lines and MPN patient peripheral blood CD34+ cells. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Blood CD34−c-Kit+ cell rate correlates with aggressive forms of systemic mastocytosis and behaves like a mast cell precursor

Blood ◽

10.1182/blood-2011-02-335950 ◽

2011 ◽

Vol 118 (19) ◽

pp. 5246-5249 ◽

Cited By ~ 18

Author(s):

Sophie Georgin-Lavialle ◽

Ludovic Lhermitte ◽

Cédric Baude ◽

Stéphane Barete ◽

Julie Bruneau ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Peripheral Blood ◽

Systemic Mastocytosis ◽

Cell Precursor ◽

Phenotypic Analysis ◽

Peripheral Blood Cd34 ◽

Healthy Donors ◽

Cellular Population ◽

Variation Rate

Abstract Mastocytosis is a heterogeneous disease characterized by the accumulation of mast cells in one or more organs. Our objective was to identify a peripheral mast cell precursor and assess its variation rate in mastocytosis. A peripheral blood phenotypic analysis was performed among 50 patients with mastocytosis who were enrolled in a prospective multicentric French study, and the phenotypic analysis results of the patients were compared with those of healthy donors. The rate of peripheral blood CD34−c-Kit+ cells correlated with the severity of mastocytosis. This cellular population was isolated from healthy donors as well as from patients with systemic mastocytosis. After 30 days of culture, the CD34−c-Kit+ cells gave birth to mature mast cells, indicating that this cellular population constitutes a mast cell circulating precursor. Monitoring peripheral CD34−c-Kit+ cells by flow cytometry could be a useful and low-invasive tool to determine the disease severity and the relapses and to assess treatment efficiency.

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Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/Fc epsilon RI- cell population

Blood ◽

10.1182/blood.v84.8.2489.2489 ◽

1994 ◽

Vol 84 (8) ◽

pp. 2489-2496 ◽

Cited By ~ 138

Author(s):

M Rottem ◽

T Okada ◽

JP Goff ◽

DD Metcalfe

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Cell Number ◽

Cd34 Cells ◽

Mast Cell Number ◽

Normal Controls ◽

Cells Cultured

Abstract Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL- 3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4- week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.

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Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/Fc epsilon RI- cell population

Blood ◽

10.1182/blood.v84.8.2489.bloodjournal8482489 ◽

1994 ◽

Vol 84 (8) ◽

pp. 2489-2496 ◽

Cited By ~ 1

Author(s):

M Rottem ◽

T Okada ◽

JP Goff ◽

DD Metcalfe

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Cell Number ◽

Cd34 Cells ◽

Mast Cell Number ◽

Normal Controls ◽

Cells Cultured

Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL- 3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4- week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.

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Faculty Opinions recommendation of A protocol for generating high numbers of mature and functional human mast cells from peripheral blood.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092083.545450 ◽

2007 ◽

Author(s):

Richard L Stevens

Keyword(s):

Mast Cells ◽

Peripheral Blood ◽

Human Mast Cells

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Successful transfer of ADA gene in vitro into human peripheral blood CD34+cells by transfecting EBV-based episomal vectors

FEBS Letters ◽

10.1016/s0014-5793(98)01489-6 ◽

1998 ◽

Vol 441 (1) ◽

pp. 39-42 ◽

Cited By ~ 25

Author(s):

Etsuko Satoh ◽

Hideyo Hirai ◽

Tohru Inaba ◽

Chihiro Shimazaki ◽

Masao Nakagawa ◽

...

Keyword(s):

Peripheral Blood ◽

Human Peripheral Blood ◽

Peripheral Blood Cd34 ◽

Cd34 Cells ◽

Episomal Vectors ◽

Successful Transfer

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Lower Hematopoietic Progenitor Cell Counts and Yields at Subsequent Donations Is Influenced By a Shorter Inter-Donation Interval between the First and Subsequent Mobilizations

Blood ◽

10.1182/blood-2019-123492 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1962-1962

Author(s):

Sandhya R. Panch ◽

Brent R. Logan ◽

Jennifer A. Sees ◽

Bipin N. Savani ◽

Nirali N. Shah ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Time Interval ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Peripheral Blood Cd34 ◽

Cell Counts ◽

Cd34 Cells

Introduction: Approximately 7% of unrelated hematopoietic stem cell (HSC) donors are asked to donate a subsequent time to the same or different recipient. In a recent large CIBMTR study of second time donors, Stroncek et al. incidentally found that second peripheral blood stem cell (PBSC) collections had lower total CD34+ cells, CD34+ cells per liter of whole blood processed, and CD34+ cells per kg donor weight. Based on smaller studies, the time between the two independent PBSC donations (inter-donation interval) as well as donor sex, race and baseline lymphocyte counts appear to influence CD34+ cell yields at subsequent donations. Our objective was to retrospectively evaluate factors contributory to CD34+ cell yields at subsequent PBSC donation amongst NMDP donors. Methods. The study population consisted of filgrastim (G-CSF) mobilized PBSC donors through the NMDP/CIBMTR between 2006 and 2017, with a subsequent donation of the same product. evaluated the impact of inter-donation interval, donor demographics (age, BMI, race, sex, G-CSF dose, year of procedure, need for central line) and changes in complete blood counts (CBC), on the CD34+ cell yields/liter (x106/L) of blood processed at second donation and pre-apheresis (Day 5) peripheral blood CD34+ cell counts/liter (x106/L) at second donation. Linear regression was used to model log cell yields as a function of donor and collection related variables, time between donations, and changes in baseline values from first to second donation. Stepwise model building, along with interactions among significant variables were assessed. The Pearson chi-square test or the Kruskal-Wallis test compared discrete variables or continuous variables, respectively. For multivariate analysis, a significance level of 0.01 was used due to the large number of variables considered. Results: Among 513 PBSC donors who subsequently donated a second PBSC product, clinically relevant decreases in values at the second donation were observed in pre-apheresis CD34+ cells (73.9 vs. 68.6; p=0.03), CD34+cells/L blood processed (32.2 vs. 30.1; p=0.06), and total final CD34+ cell count (x106) (608 vs. 556; p=0.02). Median time interval between first and second PBSC donations was 11.7 months (range: 0.3-128.1). Using the median pre-apheresis peripheral blood CD34+ cell counts from donation 1 as the cut-off for high versus low mobilizers, we found that individuals who were likely to be high or low mobilizers at first donation were also likely to be high or low mobilizers at second donation, respectively (Table 1). This was independent of the inter-donation interval. In multivariate analyses, those with an inter-donation interval of >12 months, demonstrated higher CD34+cells/L blood processed compared to donors donating within a year (mean ratio 1.15, p<0.0001). Change in donor BMI was also a predictor for PBSC yields. If donor BMI decreased at second donation, so did the CD34+cells/L blood processed (0.74, p <0.0001). An average G-CSF dose above 960mcg was also associated with an increase in CD34+cells/L blood processed compared to donors who received less than 960mcg (1.04, p=0.005). (Table 2A). Pre-apheresis peripheral blood CD34+ cells on Day 5 of second donation were also affected by the inter-donation interval, with higher cell counts associated with a longer time interval (>12 months) between donations (1.23, p<0.0001). Further, independent of the inter-donation interval, GCSF doses greater than 960mcg per day associated with higher pre-apheresis CD34+ cells at second donation (1.26, p<0.0001); as was a higher baseline WBC count (>6.9) (1.3, p<0.0001) (Table 2B). Conclusions: In this large retrospective study of second time unrelated PBSC donors, a longer inter-donation interval was confirmed to be associated with better PBSC mobilization and collection. Given hematopoietic stem cell cycling times of 9-12 months in humans, where possible, repeat donors may be chosen based on these intervals to optimize PBSC yields. Changes in BMI are also to be considered while recruiting repeat donors. Some of these parameters may be improved marginally by increasing G-CSF dose within permissible limits. In most instances, however, sub-optimal mobilizers at first donation appear to donate suboptimal numbers of HSC at their subsequent donation. Disclosures Pulsipher: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Honoraria. Shaw:Therakos: Other: Speaker Engagement.

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