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See detailAdhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture.
Huygen, Sandra; Giet, Olivier ULg; Artisien, Vincent et al

in Blood (2002), 100(8), 2744-52

Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto ... [more ▼]

Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of alpha4 and alpha5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34(+) cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-alpha4 but not by anti-alpha5 antibodies. In mitotically activated CD34(+) cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on alpha5 integrin and to a lesser extent on alpha4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant alpha4 to a preferential alpha5 integrin-dependent interaction with Fn. [less ▲]

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See detailIncreased binding and defective migration across fibronectin of cycling hematopoietic progenitor cells.
Giet, Olivier ULg; Van Bockstaele, Dirk R; Di Stefano, Ivano et al

in Blood (2002), 99(6), 2023-31

Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood ... [more ▼]

Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood CD34+ cells. Migration toward medium conditioned by the stromal-derived factor-1-producing cell line MS-5 was studied in bovine serum albumin- and fibronectin (Fn)-coated transwells. Migration was reduced in cycling CD34+ cells and long-term culture-initiating cells (LTC-ICs) compared with their noncycling counterparts across Fn but not across bovine serum albumin. Conversely, Fn binding was higher in cycling CD34+ cells and LTC-ICs compared with noncycling progenitor cells, while adhesion of both subsets to bovine serum albumin was undetectable. The contribution of alpha4 and alpha5 integrins in mediating adhesion and migration of activated CD34+ cells onto Fn was analyzed by neutralization experiments. While alpha4-mediated Fn binding decreased during G(2)/M, alpha5 integrin-mediated adhesion increased during transit from G(0)/G(1) to S and G(2)/M phases. As for migration, the contribution of alpha4 integrin was similar in all phases, whereas alpha5-directed migration was lower in G(2)/M compared with G(0)/G(1) and S phases. Defective migration of cycling CD34+ cells was not due to differences in alpha5 integrin expression. In conclusion, chemotaxis across Fn is less efficient in cycling progenitor cells in correlation with an increased Fn binding capacity. In addition, alpha4 and alpha5 integrin functions are independently modulated during cell cycle transit. [less ▲]

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See detailP2X(1)-mediated activation of extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation induced by collagen.
Oury, Cécile ULg; Toth-Zsamboki, Emese; Vermylen, Jos et al

in Blood (2002)

This study shows that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules ... [more ▼]

This study shows that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed. [less ▲]

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See detailDoes the P(2X1del) variant lacking 17 amino acids in its extracellular domain represent a relevant functional ion channel in platelets?
Oury, Cécile ULg; Toth-Zsamboki, Emese; Vermylen, Jos et al

in Blood (2002)

In this letter, we questionned the existence of a ADP responsive P2X1 protein variant in platelets.

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See detailEnhanced Platelet Reactivity to Collagen and Shear Stress in Transgenic Mice Overexpressing the Platelet P2X1 Ion Channel
Oury, Cécile ULg; Kuijpers, Marijke; Toth-Zsamboki, Emese et al

in Blood (2002), 100

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See detailLeukemic target susceptibility to natural killer cytotoxicity: relationship with BCR-ABL expression.
Baron, Frédéric ULg; Turhan, Ali G; Giron-Michel, Julien et al

in Blood (2002), 99(6), 2107-13

Chronic myeloid leukemia is a clonal myeloproliferative expansion of transformed primitive hematopoietic progenitor cells characterized by high-level expression of BCR-ABL chimeric gene, which induces ... [more ▼]

Chronic myeloid leukemia is a clonal myeloproliferative expansion of transformed primitive hematopoietic progenitor cells characterized by high-level expression of BCR-ABL chimeric gene, which induces growth factor independence. However, the influence of BCR-ABL expression on cell-mediated cytotoxicity is poorly understood. In the present study, we asked whether BCR-ABL expression interferes with leukemic target sensitivity to natural killer (NK) cell cytolysis. Our approach was based on the use of 2 BCR-ABL transfectants of the pluripotent hematopoietic cell line UT-7 expressing low (UT-7/E8, UT-7/G6) and high (UT-7/9) levels of BCR-ABL. As effector cells, we used CD56(bright), CD16-, CD2- NK cells differentiated in vitro from CD34 cord blood progenitors. We demonstrated that BCR-ABL transfectants UT-7/9 were lysed by NK cells with a higher efficiency than parental and low UT-7/E8.1 and UT-7/G6 transfectants. This enhanced susceptibility to lysis correlated with an increase in expression of intercellular adhesion molecule 1 (ICAM-1) by target cells. Treatment of UT-7/9 cells by STI571 (a specific inhibitor of the abl kinase) resulted in a decrease in NK susceptibility to lysis and ICAM-1 down-regulation in target cells. Furthermore, the constitutive activation of nuclear factor-kappaB (NF-kappaB) detected in BCR-ABL transfectant UT-7/9, was significantly attenuated when cells were treated by STI571. Interestingly, inhibition of NF-kappaB activation by BAY11-67082 (a specific NF-kappaB inhibitor) resulted in down-regulation of ICAM-1 expression and a subsequent decrease in NK-induced killing of UT-7/9 transfectants. Our results show that oncogenic transformation by BCR-ABL may increase susceptibility of leukemic progenitors to NK cell cytotoxicity by a mechanism involving overexpression of ICAM-1 as a consequence of NF-kappaB activation. [less ▲]

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See detailEffects of BM-573, a dual thromboxane A(2) receptor antagonist and thromboxane synthase inhibitor, on osteogenic sarcoma cell-induced platelet aggregation
de Leval, X.; David, Jean-Louis ULg; Neven, P. et al

in Blood (2001, November 16), 98(11, Part 2), 43

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See detailAlkaline phosphatase positive reticular cell network recovery after radiation-induced marrow aplasia in mice
Almohamad, K.; Thiry, Anne-Marie ULg; Boniver, Jacques ULg et al

in Blood (2001, November 16), 98(11, Part 2), 141

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See detailCessation of intensive treatment with recombinant human erythropoietin is followed by secondary anemia.
Piron, Maude ULg; Loo, Martine; Gothot, André ULg et al

in Blood (2001), 97(2), 442-8

Little information is available on the evolution of erythropoiesis after interruption of recombinant human erythropoietin (rHuEpo) therapy. Iron-overloaded rats received 20 daily injections of rHuEpo ... [more ▼]

Little information is available on the evolution of erythropoiesis after interruption of recombinant human erythropoietin (rHuEpo) therapy. Iron-overloaded rats received 20 daily injections of rHuEpo. During treatment, reticulocytes, soluble transferrin receptor (sTfR), and hematocrit increased progressively. This was accompanied by a substantial expansion of spleen erythropoiesis but a decrease in the bone marrow. Five weeks after treatment, rats developed a significant degree of a regenerative anemia. Erythropoietic activity, as assessed by reticulocytes, sTfR, erythroid cellularity, iron incorporation into heme, and the number of erythroid colonies, was severely depressed 3 weeks after cessation of rHuEpo. This was followed by regeneration of erythroblasts and reticulocytes at weeks 6 to 7 post-Epo, but erythroid progenitors recovered only partially by that time. The anemia was definitely corrected 2 months after cessation of rHuEpo treatment. Serum Epo levels remained elevated for several weeks, but the sensitivity of marrow erythroid precursors to Epo was preserved. No rat antibodies to rHuEpo were detected, and serum from post-Epo animals did not exert any inhibitory activity on erythropoiesis. In conclusion, after cessation of intensive rHuEpo therapy, there was a strong inhibition of erythropoietic activity with secondary anemia followed by late recovery. This was not due to antibodies or other soluble inhibitory factors, a defect in endogenous Epo production, or a loss of sensitivity to Epo. This may rather represent intrinsic erythroid marrow exhaustion, mostly at the level of erythroid progenitors but also at later stages of erythropoiesis. [less ▲]

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See detailDistinct behavior of mutant triosephosphate isomerase in hemolysate and in isolated form: molecular basis of enzyme deficiency
Orosz, F.; Olah, J.; Alvarez, M. et al

in Blood (2001), 98(10), 3106-12

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line-identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu ... [more ▼]

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line-identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu and Glu145stop codon) mutations. The kinetic, thermodynamic, and associative properties of the recombinant human wild-type and Phe240Leu mutant enzymes were compared with those of TPIs in normal and deficient erythrocyte hemolysates. The specific activity of the recombinant mutant enzyme relative to the wild type was much higher (30%) than expected from the activity (3%) measured in hemolysates. Enhanced attachment of mutant TPI to erythrocyte inside-out vesicles and to microtubules of brain cells was found when the binding was measured with TPIs in hemolysate. In contrast, there was no difference between the binding of the recombinant wild-type and Phe240Leu mutant enzymes. These findings suggest that the missense mutation by itself is not enough to explain the low catalytic activity and "stickiness" of mutant TPI observed in hemolysate. The activity of the mutant TPI is further reduced by its attachment to inside-out vesicles or microtubules. Comparative studies of the hemolysate from a British patient with Glu104Asp homozygosity and with the platelet lysates from the Hungarian family suggest that the microcompartmentation of TPI is not unique for the hemolysates from the Hungarian TPI-deficient brothers. The possible role of cellular components, other than the mutant enzymes, in the distinct behavior of TPI in isolated form versus in hemolysates from the compound heterozygotes and the simple heterozygote family members is discussed. [less ▲]

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See detailThe effect of recombinant human erythropoietin on platelet counts is strongly modulated by the adequacy of iron supply.
Loo, M.; Beguin, Yves ULg

in Blood (1999), 93(10), 3286-93

The effect of recombinant human erythropoietin (rHuEpo) on megakaryopoiesis remains controversial. Treatment with rHuEpo in renal failure patients has been associated with a slight elevation of platelet ... [more ▼]

The effect of recombinant human erythropoietin (rHuEpo) on megakaryopoiesis remains controversial. Treatment with rHuEpo in renal failure patients has been associated with a slight elevation of platelet counts. In animal studies, high doses of rHuEpo produced an increase of platelet counts followed by a gradual return to normal after 7 to 15 days or even a substantial degree of thrombocytopenia. However, because iron deficiency is also known to be associated with thrombocytosis, (functional) iron deficiency during rHuEpo could be contributing to these observations. We investigated the impact of iron supply on changes in platelet counts induced by rHuEpo. Rats were either fed normal food (normal rats) or received 1% carbonyl iron for 2 weeks or 3 months, as well as during the experiment, to achieve iron supplementation or overload, respectively. Rats of all three categories then received daily intravenous injections of rHuEpo (10, 50, or 150 U) or normal saline (0 U) for 20 days. With 0 to 10 U rHuEpo, platelets remained stable. In normal rats receiving 50 to 150 U rHuEpo, platelets increased to 120% to 140% of baseline at 4 to 12 days to level off at 120% at 16 to 20 days. This response was less sustained in splenectomized animals. Iron-supplemented rats receiving 50 to 150 U rHuEpo also increased platelets initially, but the peak was at day 4, followed by a gradual return to baseline and even a moderate thrombocytopenia later on. Iron-overloaded rats receiving 50 to 150 U rHuEpo also had increased platelets at day 4, but the duration of platelet increase was shorter, and they experienced a more pronounced degree of thrombocytopenia in proportion to the dose of rHuEpo. Because the early elevation of platelets was of larger magnitude than hematocrit changes, it is unlikely that it could be accounted for by shrinkage of plasma volume. Because it was observed in all three iron conditions, there appears to be some direct positive effect of rHuEpo on platelet production. However, after this transient effect, expanded erythropoiesis appears to exert a negative impact upon platelet production. Secondary thrombocytopenia was not related to splenic pooling, and its very slow correction after cessation of rHuEpo therapy is not compatible with changes in platelet survival. Rather, it is consistent with stem cell competition between erythroid and megakaryocytic development. However, this secondary thrombocytopenia is masked by (functional) iron deficiency in rats not receiving an adequate iron supply from food or stores. [less ▲]

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See detailA dominant negative mutation in the P2X1 receptor causes a severe bleeding disorder.
Oury, Cécile ULg; Toth-Zsamboki, Emese; Van Geet, Chris et al

in Blood (1999)

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See detailWhole-body positron emission tomography using 18F-fluorodeoxyglucose for posttreatment evaluation in Hodgkin's disease and non-Hodgkin's lymphoma has higher diagnostic and prognostic value than classical computed tomography scan imaging.
Jerusalem, Guy ULg; Beguin, Yves ULg; Fassotte, Marie-France ULg et al

in Blood (1999), 94(2), 429-33

A residual mass after treatment of lymphoma is a clinical challenge, because it may represent vital tumor as well as tissue fibrosis. Metabolic imaging by 18F-fluorodeoxyglucose (18F-FDG) positron ... [more ▼]

A residual mass after treatment of lymphoma is a clinical challenge, because it may represent vital tumor as well as tissue fibrosis. Metabolic imaging by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) offers the advantage of functional tissue characterization that is largely independent of morphologic criteria. We compared 18F-FDG PET to computed tomography (CT) in the posttreatment evaluation of 54 patients with Hodgkin's disease (HD) or intermediate/high-grade non-Hodgkin's lymphoma (NHL). Residual masses on CT were observed in 13 of 19 patients with HD and 11 of 35 patients with NHL. Five of 24 patients with residual masses on CT versus 1 of 30 patients without residual masses presented a positive 18F-FDG PET study. Relapse occurred in all 6 patients (100%) with a positive 18F-FDG PET, 5 of 19 patients (26%) with residual masses on CT but negative 18F-FDG PET, and 3 of 29 patients (10%) with negative CT scan and 18F-FDG PET studies (P </=.0001). We observed a higher relapse and death rate in patients with residual masses at CT compared with patients without residual masses at CT (progression-free survival at 1 year: 62 +/- 10 v 88 +/- 7%, P =. 0045; overall survival at 1 year: 77 +/- 5 v 95 +/- 5%, P =.0038). A positive 18F-FDG PET study was even more consistently associated with poorer survival: compared with patients with a negative 18F-FDG PET study, the 1-year progression-free survival was 0% versus 86% +/- 5% (P <.0001) and the 1-year overall survival was 50% +/- 20% versus 92% +/- 4% (P <.0001). The detection of vital tumor by 18F-FDG PET after the end of treatment has a higher predictive value for relapse than classical CT scan imaging (positive predictive value: 100% v 42%). This could help identify patients requiring intensification immediately after completion of chemotherapy. However, 18F-FDG PET mainly predicts for early progression but cannot exclude the presence of minimal residual disease, possibly leading to a later relapse. [less ▲]

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See detailTreatment of anemia in myelodysplastic syndromes with granulocyte colony-stimulating factor plus erythropoietin: results from a randomized phase II study and long-term follow-up of 71 patients.
Hellstrom-Lindberg, E.; Ahlgren, T.; Beguin, Yves ULg et al

in Blood (1998), 92(1), 68-75

Treatment with erythropoietin (epo) may improve the anemia of myelodysplastic syndromes (MDS) in approximately 20% of patients. Previous studies have suggested that treatment with the combination of ... [more ▼]

Treatment with erythropoietin (epo) may improve the anemia of myelodysplastic syndromes (MDS) in approximately 20% of patients. Previous studies have suggested that treatment with the combination of granulocyte colony-stimulating factor (G-CSF) and epo may increase this response rate. In the present phase II study, patients with MDS and anemia were randomized to treatment with G-CSF + epo according to one of two alternatives; arm A starting with G-CSF for 4 weeks followed by the combination for 12 weeks, and arm B starting with epo for 8 weeks followed by the combination for 10 weeks. Fifty evaluable patients (10 refractory anemia [RA], 13 refractory anemia with ring sideroblasts [RARS], and 27 refractory anemia with excess blasts [RAEB]) were included in the study, three were evaluable only for epo as monotherapy and 47 for the combined treatment. The overall response rate to G-CSF + epo was 38%, which is identical to that in our previous study. The response rates for patients with RA, RARS, and RAEB were 20%, 46%, and 37%, respectively. Response rates were identical in the two treatment groups indicating that an initial treatment with G-CSF was not neccessary for a response to the combination. Nine patients in arm B showed a response to the combined treatment, but only three of these responded to epo alone. This suggests a synergistic effect in vivo by G-CSF + epo. A long-term follow-up was made on 71 evaluable patients from both the present and the preceding Scandinavian study on G-CSF + epo. Median survival was 26 months, and the overall risk of leukemic transformation during a median follow-up of 43 months was 28%. Twenty patients entered long-term maintenance treatment and showed a median duration of response of 24 months.The international prognostic scoring system (IPSS) was effective to predict survival, leukemic transformation, and to a lesser extent, duration of response, but had no impact on primary response rates. [less ▲]

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See detailRed blood cell precursor mass as an independent determinant of serum erythropoietin level.
Cazzola, M.; Guarnone, R.; Cerani, P. et al

in Blood (1998), 91(6), 2139-45

Serum erythropoietin (sEpo) concentration is primarily related to the rate of renal production and, under the stimulus of hypoxia, increases exponentially as hemoglobin (Hb) decreases. Additional factors ... [more ▼]

Serum erythropoietin (sEpo) concentration is primarily related to the rate of renal production and, under the stimulus of hypoxia, increases exponentially as hemoglobin (Hb) decreases. Additional factors, however, appear to influence sEpo, and in this work, we performed studies to evaluate the role of the red blood cell precursor mass. We first compared the relationship of sEpo with Hb in patients with low versus high erythroid activity. The first group included 27 patients with erythroid aplasia or hypoplasia having serum transferrin receptor (sTfR) levels < 3 mg/L (erythroid activity < 0.6 times normal), while the second one included 28 patients with beta-thalassemia intermedia having sTfR levels > 10 mg/L (erythroid activity > 2 times normal). There was no difference between the two groups with respect to Hb (8.3 +/- 1.6 v 8.0 +/- 1.3 g/dL, P > .05), but sEpo levels were notably higher in patients with low erythroid activity (1,601 +/- 1,542 v 235 +/- 143 mU/mL, P < . 001). In fact, multivariate analysis of variance (ANOVA) showed that, at any given Hb level, sEpo was higher in patients with low erythroid activity (P < .0001). Twenty patients undergoing allogeneic or autologous bone marrow transplantation (BMT) were then investigated. A marked increase in sEpo was seen in all cases at the time of marrow aplasia, disproportionately high when compared with the small decrease in Hb level. Sequential studies were also performed in five patients with iron deficiency anemia undergoing intravenous (IV) iron therapy. Within 24 to 72 hours after starting iron treatment, marked decreases in sEpo (up to one log magnitude) were found before any change in Hb level. Similar observations were made in patients with megaloblastic anemia and in a case of pure red blood cell aplasia. These findings point to an inverse relationship between red blood cell precursor mass and sEpo: at any given Hb level, the higher the number of red blood cell precursors, the lower the sEpo concentration. The most likely explanation for this is that sEpo levels are regulated not only by the rate of renal production, but also by the rate of utilization by erythroid cells. [less ▲]

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See detailAnemia in children with cancer is associated with decreased erythropoietic activity and not with inadequate erythropoietin production.
Corazza, Francis; Beguin, Yves ULg; Bergmann, Pierre et al

in Blood (1998), 92(5), 1793-8

A defect in erythropoietin (EPO) production has been advocated as being the main cause of anemia presented at time of diagnosis or during treatment by adults with solid tumors. On the basis of this defect ... [more ▼]

A defect in erythropoietin (EPO) production has been advocated as being the main cause of anemia presented at time of diagnosis or during treatment by adults with solid tumors. On the basis of this defect, anemic cancer patients, both adults and children, have been treated with recombinant human EPO (rHuEPO). To further elucidate the pathophysiology of anemia in children with cancer, we measured serum soluble transferrin receptor (sTfR), a quantitative marker of erythropoiesis, and serum EPO at time of diagnosis and during chemotherapy in children suffering from solid tumor or leukemia. We determined serum EPO in 111 children (55 leukemia, 56 solid tumors) at time of diagnosis. In the last 44 patients (23 leukemia and 21 solid tumors), sTfR levels were also measured. Serum EPO together with sTfR levels were also determined in 60 children receiving chemotherapy (29 leukemia, 31 solid tumors). These results were compared with those obtained from appropriate control groups. In all patients, we found a highly significant correlation between the logarithm of EPO (log[EPO]) and the hemoglobin (Hb) level. In all subsets of patients, sTfR levels were inappropriately low for the degree of anemia. Neither leukemic nor solid tumor groups showed a significant inverse relationship between log(sTfR) and the Hb level as would be expected in anemic patients with appropriate marrow response. Thus, in children with cancer, anemia is associated with a decreased total bone marrow erythropoietic activity which, in contrast to what has been reported in anemic cancer adults, is not related to defective EPO production. [less ▲]

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See detailCell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34(+) cells in non-obese diabetic/severe combined immune-deficient mice.
GOTHOT, André ULg; Van der loo, J. C. M.; Clapp, D. W. et al

in Blood (1998), 92(8), 2641-9

Most primitive hematopoietic progenitor cells reside in vivo within the G0/G1 phase of the cell cycle. By simultaneous DNA/RNA staining it is possible to distinguish G0 and G1 states and to isolate cells ... [more ▼]

Most primitive hematopoietic progenitor cells reside in vivo within the G0/G1 phase of the cell cycle. By simultaneous DNA/RNA staining it is possible to distinguish G0 and G1 states and to isolate cells in defined phases of the cell cycle. We report here the use of cell cycle fractionation to separate human mobilized peripheral blood (MPB) CD34(+) cells capable of repopulating the bone marrow (BM) of non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice. In freshly isolated MPB, repopulating cells were predominant within the G0 phase, because transplantation of CD34(+) cells residing in G0 (G0CD34(+)) resulted on average in a 16.6- +/- 3.2-fold higher BM chimerism than infusion of equal numbers of CD34(+) cells isolated in G1. We then investigated the effect of ex vivo cell cycle progression, in the absence of cell division, on engraftment capacity. Freshly isolated G0CD34(+) cells were activated by interleukin-3 (IL-3), stem cell factor (SCF), and flt3-ligand (FL) for a 36-hour incubation period during which a fraction of cells progressed from G0 into G1 but did not complete a cell cycle. The repopulating capacity of stimulated cells was markedly diminished compared with that of unmanipulated G0CD34(+) cells. Cells that remained in G0 during the 36-hour incubation period and those that traversed into G1 were sorted and assayed separately in NOD/SCID recipients. The repopulating ability of cells remaining in G0 was insignificantly reduced compared with that of unstimulated G0CD34(+) cells. On the contrary, CD34(+) cells traversing from G0 into G1 were largely depleted of repopulating capacity. Similar results were obtained when G0CD34(+) cells were activated by the combination of thrombopoietin-SCF-FL. These studies provide direct evidence of the quiescent nature of cells capable of repopulating the BM of NOD/SCID mice. Furthermore, these data also demonstrate that G0-G1 progression in vitro is associated with a decrease in engraftment capacity. [less ▲]

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See detailOrderly process of sequential cytokine stimulation is required for activation and maximal proliferation of primitive human bone marrow CD34+ hematopoietic progenitor cells residing in G0.
Ladd, A. C.; Pyatt, R.; GOTHOT, André ULg et al

in Blood (1997), 90(2), 658-68

Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor ... [more ▼]

Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0/G1, unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation. [less ▲]

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See detailFunctional heterogeneity of human CD34(+) cells isolated in subcompartments of the G0 /G1 phase of the cell cycle.
GOTHOT, André ULg; Pyatt, R.; McMahel, J. et al

in Blood (1997), 90(11), 4384-93

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34(+) cells were isolated in subcompartments of the G0 /G1 phase of the ... [more ▼]

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34(+) cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34(+) cells isolated in G0 (G0CD34(+) cells) than in those residing in G1 (G1CD34(+) cells). However, as MPB CD34(+) cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34(+) cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34(+) cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34(+) cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34(+) cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2(dim) population whereby LTHC-IC frequency was higher for CD34(+) cells reselected in G0 after in vitro division than for CD34(+) cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2(bright) CD34(+) cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture. [less ▲]

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