Myelodysplastic Syndrome with Bone Marrow Hypoplasia Is Associated with Much Higher Frequency of Apoptosis in CD 34+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2370-2370
Author(s):  
Tatsuo Oyake ◽  
Shigeki Ito ◽  
Shugo Kowata ◽  
Kazunori Murai ◽  
Yoji Ishida
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Immunosuppressive Therapy ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Cells ◽  
Dramatic Improvement ◽  
Color Flow ◽  
Cd34 Cells ◽  
The Difference ◽  
Complete Blood Counts

Abstract Myelodysplastic Syndrome (MDS) is a clonal disorder characterized by dysplastic changes and ineffective hematopoiesis. The ineffective hematopoiesis is considered as the results of excessive apoptosis of bone marrow (BM) hematopoietic cells. Recently, immunosuppressive therapy is effective in some hypoplastic MDS patients, resulting in the dramatic improvement of complete blood counts. To elucidate the difference between MDS patients with hypoplasia and normo/hyperplasia in BM, we measured the frequency of apoptosis in each lineage CD34+ BM cells in 35 MDS (12 RA patients with hypoplastic BM, 9 RA, 7 RAEB, and 7 RAEB-t patients with normo/hyperplastic BM) at diagnosis by three color flow cytometric analysis. Apototic cells were analyzed by PE labeled AnnexinV. The lineage cell population was identified as CD34+/GlycophorineA+, CD34+/CD33+, CD34+/CD41+, CD34−/GlycophorineA+, CD34−/CD33+ and CD34−/CD41+. In this study, the higher frequency of apoptosis was observed in each lineage CD34+ cells in all MDS patients (n=35, median: 32.2% (range: 6.3–80.5%) in erythroid, 38.0% (8.8–93.3%) in myeloid, 41.4% (10.2–78.4%) in megakaryocytic lineage, p<0.05, respectively), compared to that in normal controls (n=10, 8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineage, respectively). While much higher frequency of apoptosis was observed in each lineage CD34+ cells in hypoplastic MDS patients (n=12, 49.1% (31.2–80.5%) in erythroid, 66.0% (37.8–93.3%) in myeloid, 68.5% (43.4–78.4%) in megakaryocytic lineage, p<0.05, respectively), compared to that in normo/hyperplastic MDS patients (n=23, 28.7% (6.3–69.4%) in erythroid, 30.0% (8.8–61.7%) in myeloid, 29.8% (10.2–58.1%) in megakaryocytic lineage, respectively). The increased frequency of apoptosis in each lineage CD34+ cells decreased after immunosuppressive therapy (n=5, 25.3% (19.8–36.4%) in erythroid, 35.2% (20.1–42.0%) in myeloid, 38.5% (30.6–47.9%) in megakaryocytic lineage, respectively). Our findings suggested that the excessive apoptosis occurred mainly in CD34+ cells in hypoplastic MDS as well as in non-hypoplastic MDS. Much more increased frequency of excessive apoptosis in CD34+ cells resulted in BM hypoplasia in hypoplastic MDS patients. This method is useful to evaluate quantitatively the ineffective hematopoiesis in BM hematopoietic cells.

Vitamin K2 Supports Hematopoiesis through Acting on Bone Marrow Mesenchymal Stromal/Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1192-1192 ◽  
Author(s):  
Aya Fujishiro ◽  
Yasuo Miura ◽  
Masaki Iwasa ◽  
Sumie Fujii ◽  
Akihiro Tamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Cells ◽  
Vitamin K2 ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Cd34 Cells

Abstract [Background] Myelodysplastic syndrome is an intractable disorder characterized by ineffective hematopoiesis. Although allogeneic hematopoietic stem cell transplantation is the only curative therapy for eligible patients, hematopoiesis-supportive pharmacotherapy is practically important for transplant-ineligible patients to overcome transfusion dependency and infections. Vitamin K2 (VK2, menatetrenone) is a drug used to aim at improvement of hematopoiesis in MDS patients (Leukemia 14: 1156, 2000). However, the exact mechanism how VK2 improves hematopoiesis remains largely unknown. It was reported that VK2 induces MDS cells to undergo apoptosis (Leukemia 13: 1399, 1999). Here, we investigated our hypothesis that VK2 exerts its hematopoiesis-supportive effects through acting on mesenchymal stem/stromal cells (BM-MSCs) in the bone marrow microenvironment. [Methods] Normal bone marrow (BM) samples from healthy adult volunteers were purchased from AllCells (Emeryville, CA). BM-CD34+ cells were isolated from BM-mononuclear cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Human BM-MSCs were isolated according to our previously published methods (Stem Cells 32:2245, 2014). In co-culture experiments, BM-MSCs with or without VK2 treatment were seeded on a 24-well culture plate. BM-CD34+ cells were applied on the MSC-grown plate and co-cultured in SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 100 ng/mL SCF, 100 ng/mL Flt-3 ligand, 50 ng/mL TPO and 20 ng/mL IL-3. After 10 days of co-culture, the number and surface marker expression of the expanded hematopoietic cells were examined by flow cytometric analysis. [Results] We first tested the direct effect of VK2 on BM-CD34+ cells. BM-CD34+ cells were treated with VK2 at various concentrations ranged from 0 µM to 10 µM for 24 hours and then cultured in SFEM in combinations with cytokines. Surprisingly, viable hematopoietic cells were hardly detected in the expansion culture of BM-CD34+ cells treated with 10 µM VK2. Even with 1 µM treatment, the number of CD45+ cells was decreased, as compared to that of expansion culture of untreated BM-CD34+ cells. The apoptosis analysis showed that the percentage of AnnexinV+ PI+ cells in the expanded hematopoietic cells is increased by VK2 treatment. We next examined the effect of VK2 on the hematopoiesis-supportive capability of BM-MSCs. BM-MSCs were pretreated with VK2 at various concentrations and then co-cultured with BM-CD34+ cells. The numbers of CD34+ cells and CD45+ cells were increased in a VK2 dose-dependent manner. These results demonstrated that VK2 shows different effects on distinct stem/progenitor cells: the induction of apoptosis in BM-CD34+ cells and the enhancement of hematopoiesis-supportive capability of BM-MSCs. We then investigated whether apoptosis-related cell death of BM-CD34+ cells by VK2 treatment is ameliorated in the presence of BM-MSCs. Both BM-CD34+ cells and BM-MSCs were treated with VK2 for 24 hours, and then co-cultured. The number of CD34+ cells was not decreased significantly in contrast to its severe decrease in single culture of VK2-treated BM-CD34+ cells. We further analyzed the effect of VK2 on BM-MSCs. Subpopulation analysis in co-culture of CD34+ cells with VK2-treated BM-MSCs showed that the expansion efficacy of CD34+CD38+ cells is higher in comparison to that of CD34+CD38- cells. In addition, the percentages of CD34-CD33+ cells and CD34-CD13+ cells were higher than those in co-cultures with untreated BM-MSCs. Therefore, VK2-treated BM-MSCs supported the expanded CD34+ cells to skew their phenotype toward myeloid lineage. The presence of a transwell in the co-culture system was unrelated to the expansion pattern of CD34+ cells, which suggested the involvement of soluble factors with respect to the underlining mechanism. We therefore compared the levels of hematopoiesis-supporting cytokine mRNA expression in VK2-treated and untreated BM-MSCs: VK2-treated BM-MSCs showed lower expression of CXCL12/SDF-1 mRNA and a trend toward higher expression of GM-CSF mRNA. [Summary] VK2 acted on BM-MSCs to support their ability to enhance expansion and myeloid differentiation of BM-CD34+ cells probably via altered GM-CSF and CXCL12/SDF-1 expression in MSCs. These findings may help to identify the mechanisms of therapeutic effects of VK2 in patients with MDS (Figure). Disclosures No relevant conflicts of interest to declare.

Ineffective Hematopoiesis in Megaloblastic Anemia Is Associated with Higher Frequency of Apoptosis in Each Lineage Immature and Mature Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3601-3601
Author(s):  
Tatsuo Oyake ◽  
Shigeki Ito ◽  
Shugo Kowata ◽  
Kazunori Murai ◽  
Yoji Ishida
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Megaloblastic Anemia ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Cell Number ◽  
Color Flow ◽  
Cd34 Cells ◽  
Normal Controls

Abstract A deficiency of Vitamin B12 is a major cause of megaloblastic anemia (MBA). Ineffective hematopoiesis is observed in MBA, characterized by cytopenia, bone marrow cells with dysplastic change and normal to hypercellularity. We reported that excessive apoptosis of each lineage CD34(+) cells was observed in myelodysplastic syndrome (MDS) in the last ASH meeting. In this study, we investigated the hypothesis that excessive apoptosis induced the ineffective hematopoiesis in MBA. We performed the three color flow cytometric analysis of bone marrow mononuclear cells in 12 MBA patients using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-each lineage antibody (anti-glycophorin A (GPA) antibody, anti-CD33 antibody and anti-CD41 antibody). The frequency of apoptosis in subpopulations of immature (CD34(+)) and each lineage (+) cells or those of mature (CD34(−)) and each lineage (+) cells were calculated as the ratio (%) of (cell number with Annexin V(+)) divided by (cell number in the subpopulation). The subpopulations include CD34(+)GPA(+) (immature erythroid), CD34(+)CD33(+) (immature myeloid), CD34(+)CD41(+) (immature megakaryocytic), CD34(−)GPA(+) (mature erythroid), CD34(−)CD33(+) (mature myeloid) and CD34(−)CD41(+) (mature megakaryocytic) cells. Much higher frequency of apoptosis was observed in each lineage CD34(+) cells in MBA (median: 23.8% (range: 10.8–43.6%) in erythroid, 43.5% (12.7–67.3%) in myeloid, 50.1% (21.0–64.1%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineages, respectively, n=10). While, the relatively higher frequency of apoptosis was observed in each lineage CD34(−) cells in MBA patients (median: 15.9% (range: 5.1–20.6%) in erythroid, 16.4% (5.6–23.2%) in myeloid, 16.1% (10.2–24.8%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (4.8% (1.3–6.6%) in erythroid, 2.2% (0.6–4.4%) in myeloid, 3.3% (1.5–7.1%) in megakaryocytic lineages, respectively, n=10). These results suggest that the excessive apoptosis occurs not only in CD34(+) but also in CD34(−) cells, which induces ineffective hematopoiesis in MBA. Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 2. The frequency of apoptosis in CD34− BM cells Figure 2. The frequency of apoptosis in CD34− BM cells

Alpha-Catenin, ARHGAP21 and β-Catenin Are Abnormally Expressed in Bone Marrow Cells From Patients with Myelodysplatic Syndromes and Are a Possible Target for Decitabine Treatment.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1778-1778
Author(s):  
Karin Spat Barcellos ◽  
Sheila Maria Winnischofer ◽  
Mariana Lazarini ◽  
Adriana Silva Santos Duarte ◽  
Carolina Louzao Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Membrane ◽  
Myelodysplastic Syndrome ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Refractory Anemia ◽  
Beta Catenin ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Abstract 1778 Poster Board I-804 Introduction Myelodysplastic syndrome (MDS) encloses a group of clonal hematopoietic disorders clinically and morphologically characterized by ineffective hematopoiesis. The gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with MDS del(5q). Thus, loss of alpha-catenin tumor suppressor expression in hematopoietic cells may provide a growth advantage that collaborates MDS pathogenesis. ARHGAP21, a negative regulator of RhoGTPase signaling pathways, is a partner of alpha-catenin that controls its recruitment to the adherens junctions. ARHGAP21 is upregulated during myeloid differentiation, and could be involved in the malignant process of hematopoietic cells. In addition, alpha-catenin is a target for decitabine (DAC) treatment, a demethylating agent with potent antitumorigenic properties against MDS. The aim of this work was to evaluate the expression of alpha-catenin, ARHGAP21 and beta-catenin (gene CTNNB1) in bone marrow cells from MDS patients with or without del(5q) and to analyze CTNNA1, ARHGAP21 and CTNNB1 expression after DAC treatment. PATIENTS AND METHODS cells were isolated from bone marrow of 6 MDS patients, including 5 refractory anemia (RA), being two with del(5q), and 1 refractory anemia with excess blasts (RAEB), based on the French-American-British classification, and 4 control subjects (normal hematopoietic tissues were obtained from healthy donors). The study was approved by the National Ethical Committee Board and bone marrow samples were collected at the Hematology and Hemotherapy Center, University of Campinas, after all participants provided informed written consent. Alpha-catenin, ARHGAP21 and beta-catenin localization in CD34+ cells was obtained using confocal microscopic analysis. ARHGAP21 localization was also analyzed in HS-5 stromal cells that were submitted to a CTNNA1 RNA interference (RNAi) approach. Leukemia cells lines (HL-60 and P-39) and bone marrow mononuclear cells obtained from 7 MDS patients, 5 RA and 2 refractory anemia with ringed sideroblasts (RARS), were treated with DAC for 72 hours; then mRNA expression of CTNNA1, ARHGAP21 and CTNNB1 was analyzed by Real-time PCR (normalized by GAPDH and beta-actin). RESULTS alpha-catenin, ARHGAP21 and beta-catenin are preferentially localized in the nucleus of CD34+ cells from MDS patients in contrast to the preferential cytoplasm and membrane localization in healthy donors and in MDS patients with del(5q). In del(5q) patients and healthy donors, ARHGAP21 and alpha-catenin co-localizated in the cell membrane. ARHGAP21 was abnormally expressed in cells with decreased CTNNA1 expression: in HS-5 stromal cells, ARHGAP21 was localized at the cytoplasm (mainly in the perinuclear region) and at the nucleus, in contrast, ARHGAP21 was poorly detectable in the nucleus of CTNNA1-RNAi treated cells. DAC treatment of MDS cells and leukemia cell lines induced CTNNA1, ARHGAP21, and CTNNB1 expression in a dose-dependent way. In HL60 and P39 cells, ARHGAP21 relocate to the cell membrane after DAC treatment. CONCLUSION The abnormal localization of alpha-catenin, ARHGAP21 and beta-catenin in MDS may compromise the reorganization of actin dynamics at sites of cell–cell contact that stabilizes cadherin-mediated cell–cell adhesion; moreover, these results also suggest a deficient recruitment of alpha-catenin to the cell membrane and an aberrant signaling in the Wnt pathway. In addition, ARHGAP21, alpha-catenin and beta-catenin are a target for DAC treatment in MDS. Supported by: FAPESP. Keywords: alpha-catenin, ARHGAP21, beta-catenin, myelodysplastic syndrome, Rho-GAP, decitabine Disclosures No relevant conflicts of interest to declare.

Disturbed Regulation and Activity of PI 3-Kinase Activity Due to Enhanced Lyn Kinase but Decreased Ship-1 and Gab2 Levels in Patients with Myelodysplastic Syndrome.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3423-3423
Author(s):  
Daniel W. Lee ◽  
Quan-Sheng Zhu ◽  
Gus Zamora ◽  
Ogonna Nwawka ◽  
Elizabeth J. Shpall ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Kinase Activity ◽  
Mononuclear Cells ◽  
Hematopoietic Cells ◽  
Sh2 Domain ◽  
Cd34 Cells

Abstract Myelodysplastic Syndrome (MDS) results from ineffective hematopoiesis characterized by peripheral cytopenia(s) and hypercellular bone marrow. Both enhanced proliferation and accelerated apoptosis are hallmarks of the early phases of MDS, while the latter phases are characterized by diminished apoptosis and the accumulation of blasts. The genetic and biochemical basis of this clonal disorder is poorly understood; cell lines and mouse models are few and incomplete. We propose that patients with Severe Congenital Neutropenia who have a ten thousand-fold risk of developing MDS/AML provide a well-defined entity to study the signaling dysfunction in MDS. Almost all of these patients have a truncated G-CSF Receptor. We then compared Ba/F3 cells expressing either the full-length or the truncated G-CSF Receptor. Stimulation with G-CSF led to sustained Lyn tyrosine kinase activity. Altered PI 3-kinase signaling was evidenced by enhanced and sustained Akt serine kinase activity with diminished tyrosine phosphorylation of the adaptor molecule Gab2 and the inositol phosphatase SHIP-1. Furthermore, an in vitro lipid phosphatase assay showed decreased SHIP activity in cells expressing truncated G-CSF Receptor following G-CSF stimulation. Both Gab2 and SHIP-1 critically regulate PI 3-kinase activity in hematopoietic cells. We next sought to corroborate these findings in primary hematopoietic cells from adult patients with MDS. Biochemical analysis of CD3/CD19 depleted bone marrow mononuclear cells from patients with mid- to late-stage MDS revealed similar findings: increased tyrosine phosphorylation of the activated form of Lyn (of 13 specimens, 10 demonstrated 2 to 30-fold increase by western blotting when compared to CD34+ cells from umbilical cord blood), increased serine and threonine phosphorylation of Akt (8/10 specimens), and decreased tyrosine phosphorylation of Gab2 (6/6) and SHIP-1 (9/9). Importantly, decreased total protein levels of Gab2 and SHIP-1 accounted for their decreased tyrosine phosphorylation. Real-time PCR studies confirmed expression of Gab2 and SHIP-1 in MDS patients, but the level of SHIP-1 transcripts was ~40% less than that in CD34+ cells. Short-SHIP (SIP110), a 104 kDa form of SHIP-1 that lacks the SH2 domain and is expressed only in embryonic stem cells and hematopoietic stem cells, was not identified by RT-PCR in MDS mononuclear cells, and hence, it could not substitute for SHIP-1 deficiency. Ongoing studies are examining hypermethylation state of these genes and their sensitivity to azacytidine. Altogether, these findings suggest that enhanced proliferation and diminished apoptosis may be due to changes in PI 3-kinase regulation and activity in some patients with MDS, which is consistent with both cell lines expressing the truncated G-CSF Receptor and the SHIP−/ −, PTEN+/− mouse model for MDS (Blood103:4503, 2004).

Decreased Megakaryocytepoiesis and No Excess of Platelet Destruction in Myelodysplastic Syndrome Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4616-4616
Author(s):  
Kazunori Murai ◽  
Tatsuo Oyake ◽  
Shugo Kowata ◽  
Mamiko Ishiguro ◽  
Shigeki Ito ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Refractory Anemia ◽  
Double Positive ◽  
Platelet Destruction ◽  
Color Flow ◽  
Normal Controls

Abstract Myelodysplastic syndrome (MDS) is a malignant disorder of hematopoietic progenitor cells and characterize by peripheral blood cytopenias with normo- to hyper-cellular bone marrow (BM) and morphologically dysplastic changes. Thrombocytopenia is observed in approximately 50% of MDS. The underlying pathophysiology is not fully understood. We analyzed the megakaryocytopoiesis and thrombocytopoiesis state by several parameters, including % reticulated platelet (%RP), which indicated platelet production state, glycoalicine index (GCI), which indicated platelet destruction state, and serum thromopoietin (TPO) levels in 47 refractory anemia in myelodysplastic syndrome (MDS-RCMD) patients with platelet counts less than 100 x 109/L. Furthermore, apoptosis frequency of megakaryocyte progenitors was analyzed in several patients. In all patients, dysmegakaryocytopoiesis findings such as hypolobulated micromegakaryocyte, non-lobulated nuclei in all sizes, and multiple, widely-separated nuclei were observed. The megakaryocytes in BM was normal to decreased in number in 32 patients (70%). Plasma TPO levels were significantly higher (718.7±746.0 pg/ml, n=50) in MDS-RCMD, while they were less than 205.0 pg/ml in normal volunteers (68.3 ±65.3 pg/ml, n=32)(p< 0.01). The %RPs in MDS-RCMD and normal controls were similar (MDS-RCMD 1.7±0.9% vs control 1.2±0.6%), indicating that increased thrombocytopoiesis was not observed in MDS-RCMD, regardless of high TPO levels. GCI was similar to normal controls (MDS-RCMD, 1.5±1.3% vs controls, 1.6±0.3%), indicating no excess of platelet destruction. There was no correlation between %RP and GCI. These data strongly suggested that platelet life span be not shortend in MDS-RCMD. We have reported that excessive apoptosis of CD34(+) cells was observed in MDS patients in the previous ASH meetings. The three-color flow cytometric analysis of bone marrow mononuclear cells using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-CD41 antibody were carried out in 17 MDS-RCMD patients. Much higher frequency of apoptosis was observed in double positive cells for CD34 and CD41 (48.1%:13.4∼78.4%, median: range) in MDS-RCMD, compared to that in normal controls (7.7%: 4.4∼17.2%, median: range) (P<0.05). The frequency of apoptosis was not increased significantly in CD34(−)CD41(+) cells in MDS-RCMD patients (median: 6.9% ; 1.3∼21.5%), n=12), compared to those in normal controls (3.3%: 1.5∼8.1%, n=10). These data strongly suggested that the main cause of thrombocytopenia in MDS-RCMD should be apoptosis in megakaryocytes progenitors, followed by the decreased megakaryocytopoiesis.

scholarly journals Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2301-2309 ◽  
Author(s):  
G Fritsch ◽  
P Buchinger ◽  
D Printz ◽  
FM Fink ◽  
G Mann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Color Flow ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Myeloid Progenitor Cells ◽  
Macrophage Colony ◽  
Rapid Discrimination

Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45- RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU- GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst- forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.

scholarly journals Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2301-2309 ◽  
Author(s):  
G Fritsch ◽  
P Buchinger ◽  
D Printz ◽  
FM Fink ◽  
G Mann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Color Flow ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Myeloid Progenitor Cells ◽  
Macrophage Colony ◽  
Rapid Discrimination

Abstract Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45- RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU- GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst- forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.

scholarly journals Expression of FLIPLong and FLIPShort in bone marrow mononuclear and CD34+ cells in patients with myelodysplastic syndrome: correlation with apoptosis

Leukemia ◽  
2003 ◽  
Vol 17 (12) ◽  
pp. 2460-2466 ◽  
Author(s):  
M Benesch ◽  
U Platzbecker ◽  
J Ward ◽  
H J Deeg ◽  
W Leisenring
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Cd34 Cells

scholarly journals Augmented Production of Interleukin-6 by Normal Human Osteoblasts in Response to CD34+ Hematopoietic Bone Marrow Cells In Vitro

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1165-1172 ◽  
Author(s):  
Russell S. Taichman ◽  
Marcelle J. Reilly ◽  
Rama S. Verma ◽  
Stephen G. Emerson
Keyword(s):  
Bone Marrow ◽  
Interleukin 6 ◽  
Transforming Growth Factor ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Colony Stimulating Factor ◽  
Cd34 Cells ◽  
Stimulating Factor ◽  
Marrow Cells

Abstract Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-β1 , but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% ± 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1β or tumor necrosis factor-α, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.

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