Biomedical subjects

W Vainchenker

Publications and source records attributed to W Vainchenker.

At least 37 records · Page 2Linked to original sources

PubMed2001

Genesis of clone size heterogeneity in megakaryocytic and other hemopoietic colonies: the stochastic model revisited.

OBJECTIVE: We previously showed that the distributions of the numbers of doublings (NbD) undergone by individual megakaryocyte progenitors before commitment to polyploidization are markedly skewed and can consistently be fitted to straight lines when plotted on semilogarithmic coordinates. The slope of such lines, which yields the probability of polyploidization per doubling, is made less steep by stimulators of megakaryocyte colony formation and is less steep in mixed erythroid-megakaryocyte than in pure megakaryocyte colonies. Therefore, megakaryocytopoiesis provides a unique model for the study of clonal heterogeneity in a hemopoietic lineage, which is the subject of this review. DATA SOURCES: Articles relevant to the interpretation of these data were selected from the authors' and public databases. DATA SYNTHESIS: Exponential NbD distributions were first explained by postulating that following the assembly of thrombopoiesis-specific regulators, megakaryocyte progenitors require only a single random event to arrest proliferation and commit to polyploidization. However, this stochastic model was refuted by data indicating that intrinsic properties of individual progenitors affect the NbD they achieve. We suggest that the unequal repartition of critical compounds (including receptors, signaling molecules, and gene regulators) inherent in the stem cell-progenitor transition causes a heritable heterogeneity in megakaryocyte progenitor responsiveness to polyploidization inducers. This model would be compatible with 1) the evidence for intraclonal synchronization in megakaryocyte and other hemopoietic clones generated by committed progenitors; 2) the low probability of polyploidization of the relatively insensitive bipotent megakaryocyte progenitors; and 3) the thesis that stimulators act in part by recruiting megakaryocyte progenitor cells endowed with lesser responsiveness to polyploidization inducers and higher proliferative potential. CONCLUSION: The responsiveness of individual megakaryocyte progenitors to polyploidization inducers may be a major determinant of the exponential shape of NbD distributions.

Animals↗

PubMed2001

Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette.

The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2-3 weeks. A single dose of doxycycline added to the medium (1 microg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60-90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 x 10(6) Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 microg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin- bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.

Animals↗

PubMed2001

Efficient ex vivo expansion of NOD/SCID-repopulating cells with lympho-myeloid potential in hematopoietic grafts of children with solid tumors.

INTRODUCTION: The ex vivo expansion of hematopoietic grafts could be an important therapeutic tool for accelerating hematopoietic recovery after administration of high-dose chemotherapy regimens. The fate of the long-term repopulating cells during the ex vivo manipulation of grafts is a critical issue and will ultimately define the clinical applicability of this technology to hematopoietic transplantation. MATERIALS AND METHODS: To study the effects of a clinically applicable ex vivo expansion protocol in the proliferative potential of the most primitive human hematopoietic cells, both LTC-IC and NOD/SCID-RC assays were used to determine LTC-IC and NOD/SCID-RC contents of hematopoietic grafts, both before and after expansion (SCF, IL-3, PEG-MGDF Flt3-L and 5% AB serum), in four children with non-hematological malignancies. RESULTS: The mean percentage of CD34+ cells after expansion was 16%. The numbers of nucleated cells increased 20-fold with a mean three-fold increase in the numbers of CD34+ cells during the expansion period. The CFC content of the samples showed a mean 11-fold increase (range: 5-17) after ex vivo expansion. The primitive hematopoietic stem cell content of the expanded cell fraction evaluated by LTC-IC assays was found to be increased in two patients out of three, with maintenance of the LTC-IC frequency in the third patient. The NOD/SCID-RC potential, evaluated in five experiments from four patients using 109 mice injected 5-6 weeks earlier with human hematopoietic cells, increased from a mean percentage of 36% (range: 7-75%) before expansion, to a mean percentage of 70% (range: 37-100%) after expansion (P < 0.00001). The frequency of NOD/SCID-RC calculated with pooled data from all patients was 1/80,000 at day 0 and 1/40,000 after seven days of culture. The full phenotypic analysis of human hematopoietic cells obtained in NOD/SCID mice injected with expanded cells showed the presence of significant numbers of CD34+, CD19+ and CD15+ cells, suggesting the persistent lympho-myeloid potential of the expanded hematopoietic cells. CONCLUSION: Our results suggest that efficient expansion of NOD/SCID-RC with lympho-myeloid potential can be achieved not only in cord blood or normal marrow as previously reported, but also in hematopoietic grafts obtained from children exposed to high-dose chemotherapy.

Animals↗

PubMed2001

The granulocyte colony-stimulating factor receptor supports erythroid differentiation in the absence of the erythropoietin receptor or Stat5.

To evaluate the functional conservation of signal transduction mechanisms between haematopoietic receptors and to characterize the molecules activated in this phenomenon, we introduced granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into mouse fetal liver cells using a retroviral vector. In semi-solid medium assays, G-CSFR-infected cells gave rise to all types of colonies [granulocyte-macrophage (GM), megakaryocyte (MK) and mixed lineage (GEMM) colony-forming units (CFU) and erythroid burst-forming units (BFU-E)] in the presence of G-CSF alone. The direct effect of G-CSF on erythroid differentiation of G-CSFR-transduced erythroid progenitors was demonstrated by the development of erythroid colonies using G-CSFR-expressing Lin- cells cloned at one cell per well in liquid culture in the presence of G-CSF. Interestingly, while Stat5, but not Stat3, was activated in erythroid cells in response to erythropoietin (EPO), both were activated in erythroid and granulocytic cells stimulated by G-CSF. Furthermore, G-CSF induced the growth of erythroid colonies from G-CSFR-expressing fetal liver cells from EPO receptor-/- (EPO-R-/-) or Stat5a-/- Stat5b-/- mice, demonstrating that erythroid differentiation can occur in the absence of EPO-R or Stat5. These data show that forced expression of G-CSFR allows G-CSF-dependent multilineage proliferation and differentiation of haematopoietic progenitors and rescues EPO-R-/- erythroid cells. While G-CSF induces Stat5 activation in G-CSFR-expressing erythroid cells, this activation is not necessary for the terminal erythroid differentiation induced by G-CSF.

Animals↗

PubMed2001

Early and persistent bone marrow hematopoiesis defect in simian/human immunodeficiency virus-infected macaques despite efficient reduction of viremia by highly active antiretroviral therapy during primary infection.

The hematological abnormalities observed in human immunodeficiency virus (HIV)-infected patients appear to be mainly due to bone marrow dysfunction. A macaque models of AIDS could greatly facilitate an in vivo approach to the pathogenesis of such dysfunction. Here, we evaluated in this model the impact of infection with a pathogenic simian/human immunodeficiency virus (SHIV) on bone marrow hematopoiesis. Three groups of macaques were inoculated with 50 50% median infective doses of pathogenic SHIV 89.P, which expresses env of dual-tropic HIV type 1 (HIV-1) 89.6 primary isolate. During the primary phase of infection, animals were treated with either a placebo or highly active antiretroviral therapy (HAART) combining zidovudine, lamivudine, and indinavir, initiated 4 or 72 h postinfection (p.i.) and administered twice a day until day 28 p.i. In both placebo-treated and HAART-treated animals, bone marrow colony-forming cells (CFC) progressively decreased quite early, during the first month p.i. One year p.i., both placebo- and HAART-treated animals displayed decreases in CFC to about 56% of preinfection values. At the same time, a dramatic decrease (greater than 77%) of bone marrow CD34(+) long-term culture-initiating cells was noted in all animals were found. No statistically significant differences between placebo- and HAART-treated monkeys were found. These data argue for an early and profound alteration of myelopoiesis at the level of the most primitive CD34(+) progenitor cells during SHIV infection, independently of the level of viremia, circulating CD4(+) cell counts, or antiviral treatment.

Animals↗

PubMed2001

BCR-ABL fails to inhibit apoptosis in U937 myelomonocytic cells expressing a carboxyl-terminal truncated STAT5.

Recent experimental data suggest that one of the major effects of BCR-ABL gene expression in hematopoietic cells is the inhibition of apoptosis. Although the exact mechanisms of this phenomenon are not clear, it is thought to be related to the fact that BCR-ABL induces several signalling pathways also activated by growth factors. In order to determine the anti-apoptotic role of BCR-ABL in a hematopoietic cell line and to by-pass the influence of cytokine-dependence, BCR-ABL gene was expressed in the autonomously growing myelomonocytic U937 cell line using retroviral vectors. There was no resistance to apoptosis induced by either serum deprivation or different doses of etoposide in any U937 clones expressing BCR-ABL protein. In addition to serum deprivation and etoposide, BCR-ABL-expressing clones were not protected from apoptosis induced by TNF, ceramide-C2 and FAS-cross-linking. BCL2 expression was absent in U937 cells and BAX levels were identical between Neo and BCR-ABL clones. To further investigate the mechanisms of this phenomenon, band-shift assays were performed to detect activation of STAT molecules. No constitutive activation of STATs was detected in either NeoR or BCR-ABL-U937 cells, although both IFN-gamma and GM-CSF activated STAT1 and STAT5, respectively, with similar kinetics in both NeoR and BCR-ABL-U937 cells. In addition, the GM-CSF-induced-STAT5 activation was found to be weakened in all clones expressing BCR-ABL. In both control NeoR and BCR-ABL-transfected clones, band-shift assays revealed the presence of an abnormal truncated STAT5 recognized only by an anti-N-terminal but not by an anti-C-Terminal STAT5 antibody. These findings suggest a possible link between the absence of anti-apoptotic potential of BCR-ABL and abnormalities of the STAT5 pathway, including, absence of constitutive activation of STAT5, inhibition of GM-CSF-induced STAT5 activation and expression of a carboxyl-terminal-truncated STAT5.

Apoptosis↗

PubMed2000

The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells.

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34(+) hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).

Adult↗

PubMed2000

Regulation of Id gene expression during embryonic stem cell-derived hematopoietic differentiation.

To elucidate the role of helix-loop-helix (HLH) Id proteins in hematopoietic differentiation, we used a model of embryonic stem (ES) cell differentiation in vitro which gives access not only to hematopoietic myeloid progenitor cells but also to the more primitive blast colony-forming cell (BL-CFC), the in vitro equivalent of the hemangioblast that gives rise to blast cell colonies in the presence of VEGF. We first demonstrated that ES cell-derived blast cell colonies could be used as a model to study hematopoietic differentiation and maturation. We next established the expression profile of Id genes in this model. Transcripts of the four Id genes were present in ES cells. Id1, Id3 and Id4 gene expression was down-regulated during the development of blast cell colonies while that of Id2 was maintained. Thus, Id1, Id3, and Id4 proteins are candidates for being negative regulators of hematopoiesis in the model of hematopoietic ES cell differentiation in vitro.

Animals↗

PubMed2000

Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.

Injuries to the vessel wall and subsequent exposure of collagen from the subendothelial matrix result in thrombus formation. In physiological conditions, the platelet plug limits blood loss. However, in pathologic conditions, such as rupture of atherosclerotic plaques, platelet-collagen interactions are associated with cardiovascular and cerebral vascular diseases. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets, and people who are deficient in GPVI suffer from bleeding disorders. Based on the fact that GPVI is coupled to the Fc receptor (FcR)-gamma chain and thus should share homology with the FcR chains, the genes encoding human and mouse GPVI were identified. They belong to the immunoglobulin (Ig) superfamily and share 64% homology at the protein level. Functional evidence demonstrating the identity of the recombinant protein with GPVI was shown by binding to its natural ligand collagen; binding to convulxin (Cvx), a GPVI-specific ligand from snake venom; binding of anti-GPVI IgG isolated from a patient; and association to the FcR-gamma chain. The study also demonstrated that the soluble protein blocks Cvx and collagen-induced platelet aggregation and that GPVI expression is restricted to megakaryocytes and platelets. Finally, human GPVI was mapped to chromosome 19, long arm, region 1, band 3 (19q13), in the same region as multiple members of the Ig superfamily. This work offers the opportunity to explore the involvement of GPVI in thrombotic disease, to develop alternative antithrombotic compounds, and to characterize the mechanism involved in GPVI genetic deficiencies. (Blood. 2000;96:1798-1807)

Amino Acid Sequence↗

PubMed2000

Radiation-induced expression of functional Fas ligand in EBV-positive human nasopharyngeal carcinoma cells.

Ionizing radiation remains a major therapeutic tool against human cancers, especially epithelial tumors, which account for the majority of human malignancies. Although Fas and Fas-L are essential determinants of apoptosis, few data support their role in the cytotoxic effect of ionizing radiation. Epstein-Barr-virus (EBV)-positive nasopharyngeal carcinoma (NPC) were chosen to address this question owing to their known sensitivity to ionizing radiation and their constitutive expression of the Fas-receptor. We here report that, in xenografted NPC cells, Fas-L expression, which was very low in basal conditions, was dramatically increased by tumor irradiation. Both the Fas receptor and the Fas ligand were found to be functional in this model, and a high proportion of irradiated NPC cells underwent apoptosis following tumor irradiation. Induction of Fas-L expression and apoptosis were observed for doses as low as 2 Gy. These data show an increase in Fas-L expression upon irradiation exposure, and strongly suggest that, in some epithelial malignancies, Fas-mediated apoptosis can play a major role in the anti-tumor effect of ionizing radiation, in the range of doses used for therapeutic applications.

Animals↗

PubMed2000

Existence of a differentiation blockage at the stage of a megakaryocyte precursor in the thrombocytopenia and absent radii (TAR) syndrome.

The thrombocytopenia and absent radii (TAR) syndrome is a rare disease associating bilateral radial agenesis and congenital thrombocytopenia. Here, we investigated in vitro megakaryocyte (MK) differentiation and expression of c-mpl in 6 patients. Using blood or marrow CD34(+) cells, the colony-forming unit (CFU)-MK number was markedly reduced. CD34(+) cells were also cultured in liquid medium in the presence of a combination of 3 cytokines (stem cell factor, interleukin-3, and interleukin-6) or megakaryocyte growth and development factor (PEG-rHuMGDF) with or without SCF. In the presence of PEG-rHuMGDF, the majority of mature megakaryocytes (CD41 high, CD42 high) underwent apoptosis. This phenomenon was also observed in cultures stimulated by three cytokines. However, this last combination of cytokines allowed a more complete terminal MK differentiation. Surprisingly, a homogeneous population of CD34(-)CD41(+)CD42(-) cells accumulated during the cultures. This population was unable to differentiate along the myeloid pathways. This result suggests that a fraction of MK cells is unable to differentiate in the TAR syndrome. We subsequently investigated whether this could be related to an abnormality in c-mpl. No mutation or rearrangement in the c-mpl gene was found by Southern blots or by sequencing of the c-mpl coding region and its promoter in any of the patients. Using Western blot analysis, a decreased level of Mpl was found in patient platelets. A decreased level of c-mpl messenger RNA in TAR platelets was also detected with a lower c-mpl-P to c-mpl-K ratio in comparison to adult platelets. Altogether, these results demonstrate that the thrombocytopenia of the TAR syndrome is associated with a dysmegakaryocytopoiesis characterized by cells blocked at an early stage of differentiation. (Blood. 2000;95:1633-1641)

Adolescent↗

PubMed2000

Embryonic stem cell differentiation to hematopoietic cells: A model to study the function of various regions of the intracytoplasmic domain of cytokine receptors in vitro.

To examine whether the in vitro model of embryonic stem (ES) cell hematopoietic differentiation is suitable to study the function of intracytoplasmic regions of cytokine receptors, we used the thrombopoietin receptor Mpl as a typical cytokine receptor.ES cells deficient in c-mpl (mpl(-/)-) were transfected with genes encoding the full-length or two mutated forms of the intracytoplasmic domain of Mpl using the pEF-BOS expression vector. The mutated forms lack box1 or box2.pEF-BOS was able to maintain protein production during ES cell differentiation. Reintroduction of full-length-c-mpl into mpl(-/)- ES cells restored the response of megakaryocyte progenitors to a truncated form of human Mpl-ligand conjugated to polyethylene glycol (PEG-rhuMGDF) and the formation of platelets, for which mpl(-/)- ES cells are defective. In addition, enforced expression of Mpl resulted in the development of all myeloid progenitors and mature cells in the presence of PEG-rhuMGDF. Blast colony-forming cells, the in vitro equivalent of the hemangioblast, also generated blast cell colonies with a hematopoietic potential equivalent to that of the wild type in the presence of PEG-rhuMGDF, although its growth is normally dependent on vascular endothelial cell growth factor (VEGF). Thus, Mpl acts as a substitute for other cytokine receptors and for a tyrosine kinase receptor, Flk-1, indicating that Mpl has no instructive role in hematopoietic cell commitment and differentiation. The Mpl mutant forms lacking box1 or box2 prevented response of ES cell-derived blast colony-forming cells or progenitors to PEG-rhuMGDF. Therefore, these two regions, essential for signaling by cytokine receptors, are required for the responses of ES cell-derived hematopoietic cells to PEG-rhuMGDF.These results show that the in vitro hematopoietic differentiation of ES cells is suitable for studying the role of various intracytoplasmic regions of cytokine receptors.

Animals↗

PubMed2000

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7.

There is currently no satisfactory model allowing analysis of dose-effect relationships of BCR-ABL proteins in human hematopoietic cells. To study comparatively the proliferative, differentiative and anti-apoptotic actions of different levels of BCR-ABL proteins in the context of the same cellular background, we have introduced the BCR-ABL gene into the GM-CSF-dependent pluripotent human cell line UT-7. Individual clones expressing BCR-ABL were analyzed by Western blots. After normalization to equivalent levels of endogenous ABL protein, 14 clones always grown in GM-CSF were found to express low but variable levels of BCR-ABL whereas two clones selected in the absence of GM-CSF expressed very high levels of BCR-ABL. All low-level BCR-ABL expressing clones exhibited a behavior similar to that of the GM-CSF-dependent parental cells as they ceased to proliferate upon growth factor deprivation and showed a strong proliferative response upon GM-CSF addition. One out of 14 clones showed progressive GM-CSF independence during culture over several weeks and was found to have a significant increase of BCR-ABL expression at that time. The resistance of this clone (E8-2) to different apoptotic stimuli was found to be increased as compared to its low BCR-ABL-expressing counterpart (E8-1) and similar to that observed in clones with very high levels of BCR-ABL (UT-7/9 and UT-7/11) which were totally resistant to apoptotic stimuli. When injected into nude mice, parental UT-7 cells and clones with low-level of BCR-ABL were not tumorigenic over 10 weeks of observation whereas UT-7 clones with high levels of BCR-ABL (UT-7/9, UT-7/11 and UT-7/E8-2) induced aggressive tumors in 2-4 weeks with a significant correlation between the amount of BCR-ABL protein and the rate of tumor growth. In conclusion, the establishment of an in vitro and in vivo CML model using UT-7 cells suggests for the first time in human cells, that the fully transformed phenotype induced by BCR-ABL requires high levels of BCR-ABL expression. These findings suggest that variable levels of BCR-ABL in primary patient cells could also be responsible for the different phenotypic features seen in chronic and acute phases of CML, such as the differentiation ability induced by growth factors.

Animals↗

PubMed2000

Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate haematological recovery in a baboon model of autologous transplantation.

To address the value of ex vivo expanded haematopoietic cells for shortening cytopenia in autologous haematopoietic transplantation, we designed an ex vivo expansion protocol based on a cocktail of early acting cytokines and short-term culture and tested it in a baboon model. Expansion involved enriched CD34+ peripheral blood haematopoietic cells cultured for 6 d with a combination of FLT3-L, stem cell factor (SCF), thrombopoietin (TPO) and interleukin (IL)-3 (50 ng/ml each); CD34+ cells, granulocyte-macrophage colony-forming units (GM-CFU) and megakaryocytic colony-forming units (MK-CFU) were amplified, respectively, 10.5-, 20.5- and 17.9-fold. Baboons were submitted to a myeloablative regimen consisting of cyclophosphamide plus total body irradiation (TBI; 6 Gy) and were then grafted with either 2 x 106/kg unmanipulated CD34+ cells (control group, n = 4) or cells cultured from 2 x 106/kg CD34+ cells (expansion group, n = 4). No cytokines were administered after transplantation. All the animals engrafted. The mean times to white blood cell (WBC), granulocyte and platelet recovery were significantly shorter in the expansion group than in the control group: WBC (> 1 x 109/l) and neutrophil (> 0.5 x 109/l) recovery occurred on days 8 (range 6-9) and 9 (range 6-11), respectively, compared with days 12 (range 10-15) and 14 (range 11-16); platelets recovered (> 20 x 109/l) on day 9 (range 7-12) compared with day 13 (range 11-15) in the control group (P < 0.05). No toxicity was observed after reinfusion. No secondary hypoplasia was observed during more than 12 months of follow-up. Functions of both neutrophils and platelets produced from expanded cells were normal in terms of oxidative metabolism, chemotaxis and the bleeding time. This study shows that in comparison with unmanipulated cells peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells, under the conditions defined here, accelerated both neutrophil and platelet recovery without impairing long-term haematopoiesis.

Animals↗

PubMed2000

Thromboxane synthase has the same pattern of expression as platelet specific glycoproteins during human megakaryocyte differentiation.

Regulation of the platelet formation process is poorly understood. It has been shown that p45NF-E2 deficient mice have a profound defect in platelet formation and recently the first platelet/megakaryocytic gene regulated by NF-E2, thromboxane synthase (TXS), has been identified. In this study, we investigated TXS expression as a model of a gene regulated by NF-E2 during MK differentiation. Megakaryocytic cells derived from blood CD34+ cells were purified according to their stage of maturation on the basis of expression of CD34, CD41a and CD42a, permitting to define different stages in MK differentiation. By means of real-time quantitative RT-PCR, we could determine that the level of TXS increased during differentiation in parallel with the expression of c-mpl and GPIIb (CD41). However, amounts of TXS transcripts increased about 1.6-fold more than that of GPIIb or c-mpl transcripts during maturation. Expression of TXS and MK specific proteins such as CD41a, CD42a and vWF was also correlated in maturing MKs. In addition, staining by anti-TXS antibody of proplatelet bearing MKs was not increased in comparison to that observed in mature MK, suggesting that TXS is not upregulated during platelet formation. In addition, we investigated whether TXS and cyclooxygenase could be involved in platelet formation by adding aspirin into the cultures. No significant decrease of platelet production was observed. In conclusion, this study shows that TXS is coordinately expressed with the other platelet proteins during MK differentiation but is not directly involved in platelet formation.

Antigens, CD34↗

PubMed1999

The thrombocytopenia of Wiskott Aldrich syndrome is not related to a defect in proplatelet formation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34(+) cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1alpha (SDF-1alpha), and actin normally polymerized after SDF-1alpha or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.

Blood Platelets↗

PubMed1999

Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34(+)CD38(low/neg) human bone marrow cells.

Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, such as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the properties of primitive progenitor cells exposed to cytokines. Single CD34(+)CD38(low) and CD38(neg) cells were incubated 10 days in serum-containing or serum-free medium in the presence or in the absence of murine marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cell factor (SCF), pegylated-megakaryocyte growth and differentiation factor (PEG-MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were evaluated in each clone after 10 days. A striking and consistent observation was the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alone in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wells containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days. We further showed by analyzing multiple replicates of a single clone at day 10 that MS-5 cells promoted a net increase in the LTC-IC compartment through self-renewal divisions. Interestingly, these primitive LTC-IC were equally distributed among small and large clones, as counted at day 10, indicating that active proliferation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation events triggered by cytokines and promoted self-renewal divisions. Furthermore, the almost identical distribution of the size of the clones with or without MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines.

ADP-ribosyl Cyclase↗

PubMed1999

Identification of lymphomyeloid primitive progenitor cells in fresh human cord blood and in the marrow of nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice transplanted with human CD34(+) cord blood cells.

Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In this work, we used single cell cultures to show that human cord blood (CB) contains totipotent CD34(+) cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34(+) CD19(-)Thy1(+) (or CD38(-)) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on murine stromal feeder layers. 10% of the clones individually analyzed produced CD19(+), CD56(+), and CD15(+) cells in stromal cocultures and CD4(+)CD8(+) T cells in FTOCs, identifying totipotent progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34(+) cells. These results provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34(+) cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.

Animals↗