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

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.

Mutant CXCR4-Triggered Apoptosis and Impaired Mobilization of Bone Marrow Cells in Myelokathexis Are Normalized upon Treatment with Specific Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1631-1631
Author(s):  
Andrew G. Aprikyan ◽  
Oscar Penate ◽  
Vahagn Makaryan
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Whim Syndrome ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells ◽  
Marrow Cells

Abstract Myelokathexis (MK) is a rare congenital disorder characterized by hypercellular marrow and severe chronic neutropenia. In some, but not all patients there is an association of Warts, Hypogammaglobulinemia, and Infections with Myelokathexis (WHIM syndrome). We and others reported that bone marrow myeloid cells in MK exhibit characteristic apoptotic features such as condensed chromatin, cytoplasmic blebbing, and cellular fragmentation. FACS analysis also demonstrated an impaired survival of myeloid cells in MK that was associated with reduced level of Bcl-X expression. Several heterozygous mutations in the CXCR4 gene have been identified in most of the families with autosomal dominant WHIM syndrome. CXCR4 is a G-protein coupled chemokine receptor and its interaction with SDF-1 ligand plays an important role in homing and mobilization of hematopoietic cells. Recently, it has been demonstrated that expression of mutant CXCR4 in human CD34+ cells results in reduced receptor internalization, enhanced calcium flux and enhanced migration of transduced CD34+ cells. However, the mechanism of mutant CXCR4-mediated neutropenia in MK remains largely unknown. We examined 6 patients representing 4 unrelated families with MK and found that all affected family members harbor heterozygous mutations in the CXCR4 gene including a previously reported R334ter mutation. We identified two novel mutations that result in frame shifts and a premature stop codon in the cytoplasmic domain of CXCR4. Expression of CXCR4 mutants in human promyelocytic HL-60 cells resulted in massive apoptosis similar to that reported for bone marrow-derived myeloid cells from MK patients. Specifically, more than 50% of HL-60 cells transfected with previously reported R334ter and novel truncation mutants were positive for annexin V, whereas 20±6% of apoptotic annexin V-positive cells were observed in control cells transfected with normal CXCR4. Impaired cell survival appeared to be due to aberrant dissipation of mitochondrial membrane potential as flow cytometry analysis revealed significantly increased DIOC6-staining in cells expressing CXCR4 mutants compared with control cells expressing normal CXCR4 (p<0.02). Moreover, the expression of CXCR4 mutants but not of the wild type form led to a significant increase in directional motility of myeloid cells towards SDF-1 (p<0.01), similar to the enhanced chemotaxis of blood mononuclear cells observed in our MK patients. These data indicate that our cellular model closely recapitulates the myelokathexis phenotype. Premature apoptosis, but not enhanced chemotaxis triggered by mutant CXCR4 was reduced to near-normal level by caspase-specific inhibitor zVAD-fmk. Interestingly, the mutant CXCR4-induced increase in directional motility to SDF-1, but not accelerated apoptosis was normalized upon treatment with protein kinase Cξ-specific inhibitor. These data suggest that accelerated apoptosis and increased chemotaxis are two independent pathways activated by mutant CXCR4. Importantly, treatment of primary blood mononuclear cells from 3 MK patients with PKCξ inhibitor restored the abnormal chemotactic properties to the levels comparable to that of treated control cells from healthy volunteers. Further studies needed for assessing the therapeutic potential of PKCξ-specific inhibitor for normalizing the impaired mobilization of bone marrow cells in MK. Our data also suggest that the PKCξ-specific inhibitor may be effective for mobilization of human hematopoietic stem 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 E-cadherin is functionally involved in the maturation of the erythroid lineage.

1995 ◽  
Vol 131 (1) ◽  
pp. 243-249 ◽  
Author(s):  
S Armeanu ◽  
H J Bühring ◽  
M Reuss-Borst ◽  
C A Müller ◽  
G Klein
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Unexpected Finding ◽  
Color Flow ◽  
Pcr Screening ◽  
Maturation Stages ◽  
Differentiation And Proliferation ◽  
E Cadherin

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E-cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E-cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.

Histological Parameters and “In Vitro” Cultures Identify Different Subsets of Myelofibrosis with Myeloid Metaplasia (MMM).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4952-4952
Author(s):  
Federica Servida ◽  
Claudia Vener ◽  
Viviana Tubazio ◽  
Umberto Gianelli ◽  
Paola Rafaniello Raviele ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cell ◽  
Who Classification ◽  
Mononuclear Cells ◽  
Cell Number ◽  
Myeloid Metaplasia ◽  
Semisolid Medium ◽  
Cd34 Cells ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder (CMPD) characterized by clonal hematopoietic proliferation, reactive marrow fibrosis and extramedullary hematopoiesis. Twenty CMPDs, previously diagnosed as chronic idiopathic myelofibrosis (CIMF), were reviewed according to the WHO morphological parameters (Vardiman et al., 2001) and classified as prefibrotic-CIMF (p-CIMF), fibrotic-CIMF (f-CIMF), polycythemia vera (PV) and post-polycythaemic myelofibrosis (PPMF). Patients median age was 67 yrs (range: 45–84), generally presenting cytogenetic abnormalities and treated with chemotherapy. Quantitation of peripheral blood (PB) CD34+ cells was carried out according to ISHAGE gating strategy. Clonogenic assays were performed both on bone marrow mononuclear cells (BM) and on cells extracted from the bone marrow biopsy (BMB) using either flushing and trypsin digestion. Myeloid progenitor cell number (CFU-GM) was evaluated in semisolid medium. Fibroblast progenitor cell number (CFU-F) was studied seeding cells in IMDM medium supplemented with 20% FBS. Microvessel density hot spots (MVD) was evaluated according to Weidner et. al., 1991. Data are summarized in Table 1. Circulating CD34+ cells were higher in all classes in comparison with normals but a clearcut decrease was observed from p-CIMF to f-CIMF and from PV to PPMF. A similar trend was observed in committed progenitor cells both in bone marrow aspirated (BM) and bone (BMB), suggesting that f-CIMF and PPMF may represent late stages of the disease in which hematopoietic cells have been progressively replaced with fibroblasts. Fibroblast precursors (CFU-F) were much lower in BM than in BMB (treated with flushing and trypsinization) in all group of patients even though these findings may be related to sampling artifacts. In any case, in both BM and BMB, CFU-F increased from p-CIMF to f-CIMF, suggesting an active progression on myelofibrosis; on the contrast, BMB CFU-F significantly decrease from PV to PPMF, indicating a progressive exhaustion of the fibroblast precursors. Moreover, MVD performed in a larger group of patients (n=50) showed higher median values than normal controls, particularly evident in PPMF, where an inverse correlation between fibrosis and angiogenesis was observed. These data suggest that f-CIMF and PPMF could be the fibrotic exit of two distinct MPDs, respectively p-CIMF and PV, indicating that the ethiopatogenesis of the two diseases may be intrinsically different. In conclusion, our “in vitro” data support the WHO classification and may have diagnostic and clinical relevance. Table 1 WHO Classification CD34+ PB cells/μl CFU-GM 5X10e5 cells (BM) CFU-GM 5X10e5 cells (BMB) CFU-F 1X10e6 cells (BM) CFU-F 1X10e6 cells (BMB) MVD (vessels number) Median (Range) p-CIMF (7pt) 110 (1.16–378) 75.5 (65–86) 12 (2–22) 1 (1–15) 67.5 (0–113) 30 (26–43) f-CIMF (3pt) 42 (15–165) 59 (40–60) nd 18 (18–32) 176 (27–325) 24 (20–44.6) PV (3pt) 393 (110–676) 41.5 (18–65) 111.5 (76–147) 12.5 (1–24) 167.5 (86–249) 18.6 (7.3–33) PPMF (7pt) 178 (0.04–376) 19 (10–28) 10 (0–81) 0 (2–4) 36 (10–214) 33.8 (15–40) Controls &lt;1 100 (60–120) nd 44 (18–78) 66 (40–108) 6.6 (4.6–16.3)

scholarly journals Characterization of Monoclonal Antibodies That Recognize Canine CD34

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1977-1986 ◽  
Author(s):  
Peter A. McSweeney ◽  
Katherine A. Rouleau ◽  
Philip M. Wallace ◽  
Benedetto Bruno ◽  
Robert G. Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Stem Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34− cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit–granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34− cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of ≤1.1 × 107 /kg that were 29% to 70% CD34+. Engraftment kinetics were similar to those of dogs previously transplanted with approximately 10- to 100-fold more unmodified autologous marrow cells. This suggests that CD34+ is a marker not only of canine bone marrow progenitors but also for cells with radioprotective or marrow repopulating function in vivo. MoAbs to CD34 will be valuable for future studies of canine hematopoiesis and preclinical studies concerning stem cell transplantation, gene therapy, and ex vivo progenitor cell expansion.

scholarly journals Characterization of Monoclonal Antibodies That Recognize Canine CD34

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1977-1986 ◽  
Author(s):  
Peter A. McSweeney ◽  
Katherine A. Rouleau ◽  
Philip M. Wallace ◽  
Benedetto Bruno ◽  
Robert G. Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Stem Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Cd34 Cells ◽  
Marrow Cells

Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34− cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit–granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34− cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of ≤1.1 × 107 /kg that were 29% to 70% CD34+. Engraftment kinetics were similar to those of dogs previously transplanted with approximately 10- to 100-fold more unmodified autologous marrow cells. This suggests that CD34+ is a marker not only of canine bone marrow progenitors but also for cells with radioprotective or marrow repopulating function in vivo. MoAbs to CD34 will be valuable for future studies of canine hematopoiesis and preclinical studies concerning stem cell transplantation, gene therapy, and ex vivo progenitor cell expansion.

Abstract 1856: Transplantation of Bone-marrow Mononuclear Cells into the Infarcted Heart has Favorable Engraftment Compared to Mesenchymal Stem Cells, Skeletal Myoblasts and Fibroblasts

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Koen E van der Bogt ◽  
Ahmad Y Sheikh ◽  
Sonja Schrepfer ◽  
Grant Hoyt ◽  
Feng Cao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cardiac Function ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Donor Cell ◽  
Cell Number ◽  
Left Ventricular ◽  
Skeletal Myoblasts

Introduction: A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study aimed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction. Methods: Bone-marrow cells (MN), mesenchymal cells (MSC), skeletal myoblasts (SkMb) and fibroblasts (Fibro) were isolated from male L2G transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Cells were characterized by flow cytometry, bioluminescence imaging (BLI), and luminometry. Female FVB mice (n=60) underwent LAD ligation and were randomized into 5 groups to intramyocardially receive one cell type (5 × 10 5 ) or PBS. Cell survival was measured in vivo by BLI and ex vivo by TaqMan PCR at week 6. Cardiac function was assessed by echocardiography and invasive hemodynamic measurements at week 6. Results: Fluc expression correlated with cell number in all groups (r 2 >0.93). In vivo BLI revealed donor cell death of MSC, SkMb, and Fibro within 3 weeks after transplantation. By contrast, cardiac signal was still present after 6 weeks in the MN group, as confirmed by PCR (p<0.01). Echocardiography showed significant preservation of fractional shortening in the MN group compared to controls (p<0.05). Measurements of left ventricular end-systolic/diastolic volume revealed the least amount of ventricular dilatation occurred in the MN group (p<0.05). Conclusion: This is the first study to directly compare a variety of cell candidates for myocardial therapy and indicates that MN exhibit a favorable survival pattern, which translates into preservation of cardiac function.

Failure of CD34+ Mobilization in AML Patients Is Associated to an Abnormally High Chemosensitivity of Non Leukemic CFU-GM.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2849-2849
Author(s):  
Giuseppe Milone ◽  
Benedetto Farsaci ◽  
Giuseppe Avola ◽  
Salvatore Mercurio ◽  
Aurora Strano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Chemotherapeutic Agents ◽  
Chemotherapy Drugs ◽  
In Vitro Incubation ◽  
Drug Concentrations ◽  
Cd34 Cells ◽  
Cell Mobilization ◽  
Normal Controls

Abstract Background: Low CD34+ cell mobilization in P.B. has been found in a quota of AML patients (10–30%). Contrary to what has been observed in CD34+ mobilization in other haematological afflictions, in AML no features pertaining to the disease or to the patient have been found to be predictive of CD34+ cell mobilization failure. A possible explanation for this particular aspect of CD34+ mobilization in AML patients could be an intrinsic abnormality of non leukemic hematopoietic cells determining an increased chemo-sensitivity to anti-neoplastic drugs. To test this hypothesis we assessed, in AML patients, frequency of various types of clonable precursors (CFUs) present in BM at the time of CR and their in vitro chemosensitivity. We also correlated this data with the efficiency of CD34+ cell mobilization in P.B. Methods: 31 consecutive patients, affected with AML and a group of 15 normal BM donors were prospectively studied. Baseline CFU-GEMM, BFU-E, CFU-GM, CFU-E as well as the sensitivity of these precursors to two chemotherapeutic agents (ASTA-Z and VP-16) were assayed on BM cells obtained in first CR after consolidation chemotherapy. Chemo-sensitivity (100 - normalized residual CFU) was studied after short term in vitro incubation of bone marrow precursors at various drug concentrations. All pts underwent a CD34+ mobilization attempt and, as measure of mobilization strength, peaks of CD34+ cells reached in P.B. were determined. Results: In AML patients, after induction and consolidation schedules, a reduced number of all types of CFUs were found in BM compared to normal controls. The frequency of any types of CFUs and the chemo-sensitivity of CFU-GEMM, BFU-E and CFU-E were not correlated to CD34+ peak reached in P.B. However, in AML patients, an inverse correlation was found between chemo-sensitivity of CFU-GM and maximum CD34+ cells peak reached in P.B. during mobilization (r= − 0.807 and p=0.0001, when ASTA-Z was used at 100 mcg/ml). In univariate and multivariate logistic regression, chemo-sensitivity to ASTA-Z of CFU-GM was the only factor significantly associated with mobilization failure (P=0.02), independently of age and cytogenetical risk. Chemosensitivity of CFU-GEMM, BFU-E and CFU-E after in vitro incubation with chemotherapeutic drugs was not different in AML patients compared to CFU obtained from normal control. The contrary was found for CFU-GM and, overall, CFU-GM from AML patients had a significantly higher chemosensitivity to ASTA-Z compared to CFU-GM of normal controls (p= 0.01 at 50 mcg/ml). Conclusions: We found that an abnormal high chemo-sensitivity of CFU-GM to some chemotherapy drugs in AML patients is associated with a high risk of CD34+ cell mobilization failure. This abnormality of non leukaemic bone marrow cells, present in CR, is restricted only to CFU-GM and is not evident in other CFUs. Figure Figure

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