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C De Bruyn

Publications and source records attributed to C De Bruyn.

12 recordsLinked to original sources
PubMed2003

Ex vivo expansion of neutrophil precursor cells from fresh and cryopreserved cord blood cells.

BACKGROUND: Neutropenia following cord blood (CB) transplantation may be abrogated by infusion of granulopoietic progenitor cells. The purpose of this study was to determine whether myeloid progenitors can be obtained by ex vivo expansion of cryopreserved cord blood aliquots, and whether these progenitors present the morphologic, biologic and functional properties of myeloid progenitors at various stages of differentiation. METHODS: The cells, plated for 7 days in serum-free medium with SCF, IL-3, G-CSF, Flt3-ligand and thrombopoietin in various combinations were assessed for the expression of CD34, CD38 and CD13. Maturation of cells into the myeloid lineage was evaluated by the expression of CD15, CD11b and CD16 and by the presence of primary (myeloperoxidase) and secondary granules (lactoferrin). The capacity of cells to phagocyte latex beads was evaluated to assess their functionality. RESULTS: We have shown that a). CD34+ cells isolated from thawed samples were able to produce expansions similar to fresh samples. b). The best combination for the expansion of neutrophil precursor cells was S3FG; c). in these conditions, all stages of myeloid progenitors were represented, but few mature cells were observed. d). However, when the cells were plated on a BM stroma to try to reproduce conditions occurring during transplant, they acquired rapidly the characteristics of mature segmented cells. e). The ex vivo generated granulocytes were able to phagocyte latex beads. DISCUSSION: In conclusion, it seems reasonable to systematically aliquot CB samples before cryopreservation. Some aliquots can then be thawed, enriched in CD34+ cells and ex vivo differentiated into myeloid lineage, while the other aliquots are conserved to be infused without manipulation.

Antigens, CD34↗
PubMed2003

Ex vivo myeloid differentiation of cord blood CD34+ cells: comparison of four serum-free media containing bovine or human albumin.

BACKGROUND: Infusion of ex vivo generated myeloid post-progenitor cells associated with unmanipulated cells appears to be a promising approach to reduce neutropenia following cord blood (CB) transplant. We compared four commercially available serum-free media, two containing BSA and two containing human albumin, on the in vitro differentiation of CB CD34+ cells into post-myeloid progenitor cells. METHODS: CB CD34+ cells were cultured for 7 days in CTM-H00 (Mabio-International), StemSpanH2000 (StemCell Technologies), RM-B00 (Mabio-International) and Stem(alpha)A (Stem Alpha). The cells were stimulated by SCF, G-SCF, IL3 and Flt3-ligand (FL) added once at Day 0. Expansion was evaluated as the increase of leucocytes, CD34+ cells and CD13+ cells. Maturation of cells into the myeloid lineage was evaluated by expression of CD15, CD11b and CD16 Ags and by the presence of primary (myeloperoxydase, MPO) and secondary granules (lacoferrin, LF). The capacity of cells to phagocyte latex beads was evaluated to assess their functionality. RESULTS: We observed that: a) the mononuclear cell and CD34+ cell expansions were significantly different according to the medium tested (respectively 61.5 +/- 7.7 and 15.5 +/- 3.4 for RM-B00, 37.3 +/- 5.4 and 10.3 +/- 1.6 for CTM-H00, 23.2 +/- 6.5 and 5.8 +/- 0.9 for StemSpanH2000 and 16.6 +/- 2.4 and 3.9 +/- 0.7 for Stem(alpha)A; b) the expansion of myeloid precursors is higher with RM-B00, similar with CTM-H00 and StemSpanH2000, and lower with Stem(alpha)A. This difference is essentially due to total leucocyte expansion, rather than to a selective expansion of myeloid cells, except for Stem(alpha)A, for which the percentages of neutrophil precursor cells [promyelocytes (CD15+ CD11b+), myelocytes (CD11b+ CD16-) and mature cells (CD11b+ CD16+)] are significantly decreased. DISCUSSION: It appears that during ex vivo differentiation into myeloid lineages, the medium is critical and should be systematically screened before use in preclinical protocols. The use of human rather than bovine albumin, seems to have neither a negative, nor positive impact on the effectiveness of the medium.

Albumins↗
PubMed2002

[Hematopoietic stem cells: source, indications and perspectives].

The haematopoietic stem cell (HSC) has been first described in the mouse and now identify in human as well. Exposed to a cocktail of growth factor, this HSC can self re-new and/or differentiate into the three lineages we have in the peripheral blood. These HSC are of major importance in the clinics since they can be used for some marrow (or stem cell) transplantation, and lead to the cure of a number of malignant and non malignant hemopathies. We have today three sources of HSC: the bone marrow, the mobilized peripheral blood stem cell and the cord blood. Bone marrow used to be the classical source of HSC after harvesting by aspirations in the iliac crest. However, this approach is now supplanted by the recovery of HSC in peripheral blood using a cell separation after four days of G-CSF administration. These are several advantages of this technique, but the most important one is the more rapid hematopoietic recovery after transplantation, reducing the risk of infection and transfusion. A recent source of HSC is the umbilical cord blood. At the moment of delivery, the cord blood is extremely enriched in HSC due to the migration of these cells from the liver to the bone marrow stroma, where they will persist after birth. We have learned that the marrow stroma display a major role in the regulation of hematopoiesis and the pathogenesis of several malignant hemopathies can be explained by disturbance in the function of stromal cell. We have particularly studied the patho-genesis of chronic lymphocytic leukaemia. We have also observed that a subpopulation of stromal cells, the mesenchymal cells are of major importance in the microenvironment. In addition, the plasticity of these cells is demonstrated in vitro and we have currently a research program investigating its differentiation in neural cells. All these observations bring new promises in the treatment of hemopathies but also in some other neurological degenerative diseases.

Animals↗
PubMed2000

Characterization of CD34+ subsets derived from bone marrow, umbilical cord blood and mobilized peripheral blood after stem cell factor and interleukin 3 stimulation.

We characterized CD34+ cells purified from bone marrow (BM), mobilized peripheral blood (PB) and cord blood (CB) and we tried to establish correlations between the cell cycle kinetics of the CD34+CD38- and CD34+CD38+ subpopulations, their sensitivity to SCF and IL-3 and their expression of receptors for these two CSFs. At day 0, significantly fewer immature CD34+CD38- cells from CB and mobilized PB are in S + G2M phases of the cell cycle (respectively 2.0 +/- 0.4 and 0.9 +/- 0.3%) than their BM counterpart (5.6 +/- 1.2%). A 48-h incubation with SCF + IL-3 allows a significant increase in the percentage of cycling CD34+CD38- cells in CB (19.2 +/- 2.2%, P < 0.0002) and PB (14.1 +/- 5.5%, P < 0.05) while the proliferative potential of BM CD34+CD38- progenitors remains constant (8.6 +/- 1.0%, NS). CD123 (IL-3 receptor) expression is similar in the three sources of hematopoietic cells at day 0 and after 48-h culture. CD117 (SCF receptor) expression, although very heterogeneous according to the subpopulations and the sources of progenitors evaluated, seems not to correlate with the difference of progenitor cell sensitivity to SCF nor with their proliferative capacity. Considering the importance of the c-kit/SCF complex in the adhesion of stem cells to the microenvironment, several observations are relevant. The density of CD117 antigen expression (expressed in terms of mean equivalent soluble fluorescence, MESF) is significantly lower on fresh PB cells than on their BM (P < 0.017) and CB (P < 0.004) counterparts, particularly in the immature CD34+CD38- population (560 +/- 131, 2121 +/- 416 and 1192 +/- 129 MESF respectively); moreover, when PB and BM CD34+CD38- cells are stimulated for 48 h with SCF + IL-3, the CD117 expression decreases by 1.5- and 1.66-fold, respectively. This reduction could modify the functional capacities of ex vivo PB and BM manipulated immature progenitor cells.

Antigens, CD34↗
PubMed1999

Adhesion to bone marrow stroma inhibits apoptosis of chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal long-lived B cells which are apparently resistant to normal apoptotic regulation. Since bone marrow stromal cells play an essential role in B lymphopoiesis, we have investigated whether stromal cells influence B-CLL cell survival. Our results indicate that intimate contact with stromal cells reduces B-CLL cell apoptosis and prevents the loss of bcl-2 protein expression. Binding of B-CLL cells to stromal cells requires simultaneous action of beta1 and beta2 integrins. The interaction between B-CLL cells and other cell types seems important for their survival and may represent an important mechanism underlying accumulation of malignant cells in B-CLL patients.

Apoptosis↗
PubMed1998

Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells.

The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 +/- 3% and 31 +/- 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 +/- 2%, P < . 0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 +/- 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by beta1 and beta2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

Aged↗
PubMed1997

Ex vivo expansion of CD34 + CD38- cord blood cells.

CD34+ cord blood (CB) cells were expanded in stromal cell-free long-term culture (LTC), in the presence of various combinations of interleukin-3 (IL-3), stem cell factor (SCF), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and anti-transforming growth factor-beta (anti-TGF-beta) antibody. The progenitor cell expansion was evaluated by monitoring the increase of CD34+ and CD34 + CD38- cells over a period of 21 days. The expansion of immature (B1-CFC, HPP-CFC) and of more committed progenitors (CFU-GM, CFU-GEMM, BFU-E) was also evaluated in specific samples. Our results show that (a) CD34+ cell expansion is highly variable depending on the cord blood samples studied, (b) significant correlations between B1-CFC and CD34 + CD38- and between total CFU and CD34+ cell expansion are observed, (c) SCF in combination with IL-3 appears to expand cell subsets that continue to express their CD34 + CD38- phenotype and that generate both immature and committed progenitors, and (d) the addition of IL-6, GM-CSF, or anti-TGF-beta does not significantly improve these expansions.

ADP-ribosyl Cyclase↗
PubMed1995

Comparison of the coexpression of CD38, CD33 and HLA-DR antigens on CD34+ purified cells from human cord blood and bone marrow.

Human umbilical cord blood (UCB) cells are currently considered as a potential source of stem cells for transplantation. However, it remains unclear whether a single collection of UCB contains enough progenitors to allow a successful engraftment in adult patients. We were interested in the comparison of the frequency of primitive progenitors in UCB and in human bone marrow (BM). UCB and BM CD34+ cells were purified and compared for their coexpression of CD38, CD33 and HLA-DR. UCB and BM mononuclear fractions were enriched in CD34+ cells using the CEPRATE LC system (CellPro, Bothell, WA). Double-labeling analysis with a flow cytometer showed that 67.9 +/- 7.2% of UCB CD34+ cells are CD38-, while in BM only 10.9 +/- 4.9% of CD34+ are CD38- (p < 0.001). Moreover, our study indicated that a significantly higher percentage of UCB CD34+ is CD33- (97.1 +/- 1.2%) compared to BM (61.8 +/- 8.6%) (p = 0.013). The coexpression of CD34 with HLA-DR was not significantly different in UCB and in BM (respectively, 86.3 +/- 2.7% and 92.7 +/- 5.1%). On the other hand, in vitro assays showed that the number of multipotent (colony-forming units granulocyte-erythroid-macrophage-megakaryocyte [CFU-GEMM]), myeloid (colony-forming units granulocyte-macrophage [CFU-GM]) and erythroid (burst-forming units-erythroid [BFU-E]) progenitors is lower in the CD34+ population from UCB than from BM. In conclusion, in UCB, we have found a significantly higher percentage of CD34+ cells which lacked the expression of CD38 and CD33 antigens suggesting that UCB contains higher proportions of immature progenitor cells (CD34+CD38- and CD34+CD33-) than BM. It seems thus likely that fewer UCB CD34+ cells than BM CD34+ cells would be required for sustained engraftment following transplantation.

Antigens, CD↗
PubMed1994

Modulation of human cord blood progenitor cell growth by recombinant human interleukin 3 (IL-3), IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF) and stem cell factor (SCF) in serum-supplemented and serum-free medium.

UNLABELLED: We evaluated the growth of cord blood myeloid progenitors or colony forming units granulocyte-macrophage (CFU-GM) and their response to various recombinant growth factors or colony stimulating factors (CSFs): interleukin 3 (IL-3), IL-6, granulocyte-macrophage CSF (GM-CSF) and stem cell factor (SCF). Using classical stimulant (human placenta conditioned medium or HPCM), we observed a significantly higher day-14/day-7 CFU-GM ratio in CB than in bone marrow (BM). The association of IL-3, IL-6, GM-CSF and SCF induced significantly more CB day-14 CFU-GM than HPCM. This effect is significantly greater in CB than in bone marrow. Since fetal calf serum (FCS) is known to contain inhibitors, we have compared the ability of CSFs to induce CFU-GM formation in FCS-supplemented and FCS-free culture. In CB, using HPCM, we obtained significantly more CFU-GM in FCS-free medium than in FCS-supplemented medium. This difference was corrected by the addition of anti-transforming growth factor-beta (TGF-beta) neutralizing antibody. However, with the association of the four CSFs, no significant difference between FCS and FCS-free culture was observed. IN CONCLUSION: a) day-14/day-7 CFU-GM ratio was higher in CB than in BM indicating that CB CFU-GM are more primitive than BM CFU-GM; b) FCS can be successfully replaced by serum-free medium; c) FCS contains inhibitors of day-14 CFU-GM and among them TGF-beta; and d) the association IL-3, SCF, GM-CSF and IL-6 seems able to totally overcome the inhibitory effect of FCS.

Bone Marrow↗
PubMed1984

Myoadenylate deaminase deficiency: an enzyme defect in search of a disease.

The frequency of MAD deficiency in cases with exercise intolerance compared with the frequency in series of consecutive muscle biopsies suggests a relation between the deficiency and exercise intolerance. Deficiency cases can be presumed by an impaired NH3 production during ischaemic exercise. The ischaemic exercise test also gives information concerning the familial character of the deficiency.

AMP Deaminase↗
PubMed1980

Determination of uric acid in serum using isotachophoresis.

An operational system is described for the isotachophoretic determination of uric acid in serum, making use of column coupling. The method has been compared with a standard enzymatic procedure. With the present technique small amounts of serum (ca. 3 microliter) can be applied without any pretreatment. Urate recovery was 99.0-100.5%. Under the non-physiological measuring conditions used, 12-28% of control serum uric acid was bound to macromolecules of molecular weight exceeding 25,000. The day-to-day variations of the isotachophoretic procedure were smaller than those of the enzymatic method, whereas standard deviations were comparable. The isotachophoretic procedure is less influenced by certain metabolites.

Adult↗