Search PubMed⌕ Search

Biomedical subjects

Dominique Bonnet

Publications and source records attributed to Dominique Bonnet.

At least 19 recordsLinked to original sources
PubMed2026

Generation of TWO iPSC lines (CRICKi025-A and CRICKi026-A) from healthy donor bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are key components of the bone marrow (BM), providing structural support and paracrine signals that regulate haematopoietic stem cell maintenance, self-renewal and differentiation. However, primary BM MSCs are rare, heterogeneous, and subject to donor variability and have limited ex-vivo expansion capacity, restricting their utility. Here, we describe two human induced pluripotent stem cells lines, CRICKi0025-A and CRICKi0026-A, reprogrammed from adult BM-derived MSCs using non-integrating Sendai virus vectors. Both lines showcase grade-A morphology, are genomically stable, upregulate essential pluripotent markers and can differentiate into the three germ layers. These lines are a well-characterised resource for generating MSCs.

Journal Article↗
PubMed2007

Convenient method to access new 4,4-dialkoxy- and 4,4-diaryloxy-diaza-s-indacene dyes: Synthesis and spectroscopic evaluation.

A straightforward method for the synthesis of original 4,4-dialkoxy- or 4,4-diaryloxy-diaza-s-indacenes (BODIPY) derivatives obtained by treatment of BODIPY 1 with various alcohols in the presence of AlCl3 is described. The novel compounds are characterized by spectroscopic properties similar to those of the parent BODIPY 1, absorption and emission spectra with similar band shapes, high molar absorption coefficients (epsilon lambda max approximately 80,000 M(-1) cm(-1)), and for most of them high fluorescence quantum yields (Phi exp from 0.52 to 0.71). Among all of the new compounds synthesized, the dye 2 h exhibits higher fluorescence quantum yield (0.71) and lifetime (4.09 ns) than compound 1 and a good chemical stability toward conditions compatible with biological cell-based assays.

Aza Compounds↗
PubMed2006

Age-dependent increase in side population distribution within hematopoiesis: implications for our understanding of the mechanism of aging.

It is thought that, as we age, damage to our stem cells may lead to diminished stem cell pool function and, consequently, a reduced organ regeneration potential that contributes to somatic senescence. Stem cells have evolved many antitoxicity mechanisms, and certain mechanisms may be utilized to isolate hematopoietic stem cells. One method exploits the activity of the ATP-binding cassette/G2 transporter to efflux Hoechst 33,342 and results in a stem cell population known as the side population (SP). The SP subset represents a remarkable enrichment for hematopoietic stem cells and provides an opportunity to re-evaluate age-based changes in hematopoietic stem cells. We report here that the frequency of SP cells steadily increases with age, as does the proportion of Lin(-)/Sca-1(+)/c-kit(+) cells that is capable of Hoechst efflux. Phenotyping, progenitor, and long-term repopulation assays have indicated that SP cells in older mice are still stem cells, albeit with a lower homing efficiency than SP cells from younger mice. Analysis of apoptosis within SP cells has revealed an apoptosis-resistant population in SP cells from old mice. Gene expression analysis has determined that SP cells from old mice have a reduced expression of apoptosis-promoting genes than SP cells from young mice. This increase in SP cells with age seems to be an intrinsic property that may be independent of the age of the microenvironment (niche), and our data might provide some clues as to how this alteration in the proportion of stem/progenitor cells occurs. A possible selection-based mechanism of stem cell pool aging is discussed.

Aging↗
PubMed2006

Use of a fluorescent polarization based high throughput assay to identify new calmodulin ligands.

In order to develop a fluorescence polarization (FP) assay for calcium binding proteins, a fluorescent peptides based library of 1328 compounds has been synthesized. The use of this library has been validated by setting up a FP-high-throughput screening (FP-HTS) assay for calmodulin using the synthetic gene product (synCaM). With this assay, a set of 880 FDA approved compounds was screened. Besides the promazine class, we discovered two new classes of compounds that interact with calmodulin in a calcium dependent manner. One class has compounds with anti-histaminic/spasmolytic activities, and the other one are detergents with antibacterial activities.

Calcium-Binding Proteins↗
PubMed2006

Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment.

It is believed that a primitive cell type that maintains the mesenchymal compartment exists in the bone marrow. However, this putative mesenchymal stem/progenitor cell is yet to be identified and isolated. We are reporting the identification, isolation, and detailed characterization of the most primitive mesenchymal progenitor cells in the adult murine bone marrow, based on the expression of stage-specific embryonic antigen-1 (SSEA-1). This primitive subset can be identified in mesenchymal cell cultures and also directly in the bone marrow, thus ascertaining for the first time their existence in an adult organism. Characterization of SSEA-1+ mesenchymal cells revealed that upon purification these cells gave rise to SSEA-1- mesenchymal cells, whereas the reverse could not be observed. Also, these SSEA-1+ cells have a much higher capacity to differentiate than their negative counterparts, not only to several mesenchymal cell types but also to unconventional cell types such as astrocyte-, endothelial-, and hepatocyte-like cells in vitro. Most importantly, a single-cell-derived population was capable of differentiating abundantly into different mesenchymal cell types in vivo. Altogether we are proposing a hierarchical organization of the mesenchymal compartment, placing SSEA-1+ cells at the apex of this hierarchy.

Animals↗
PubMed2006

Implications of the expression of myeloid markers on normal and leukemic stem cells.

We have recently described the expression of CD33 and other antigens previously thought to be myeloid specific on both acute myeloid leukemia-initiating cells (AML-IC) and normal hematopoietic stem cells (HSC) that are capable of repopulating immuno-deficient mice. Here, we discuss that the presence of myeloid markers on AML-ICs and HSCs makes identification of an antigen for targeted therapy extremely difficult. Xenotransplantation assays should be used wherever possible to test the suitability of candidate antigens for targeted therapy, including the assessment of the effect on normal cells.

Aminoglycosides↗
PubMed2006

Out of the blue: a comparison of Hoechst side population (SP) analysis of murine bone marrow using 325, 363 and 407 nm excitation sources.

BACKGROUND: The discrimination of side population (SP) cells is a useful flow cytometric tool for the study of putative stem cells in many tissue types. Previous studies on murine bone marrow (BM) SP cells and concurrent surface immunophenotyping provide a good model for comparing Hoechst bivariate profiles. Here we compare the results of SP cells from the same specimens generated on three different instruments. METHODS: Concurrent SP analysis and immunophenotyping was performed on murine BM. The data acquisition was performed on a DakoCytomation MoFlo, a Becton Dickinson LSR and a Becton Dickinson FACSAria to compare the ability of each instrument to discriminate putative stem cells. Further experiments examined the effect of apoptotic changes, assessed by Annexin V binding, and the presence of nucleated erythroid precursors on the Hoechst profile. RESULTS: Identifiable and equivalent side populations were generated on all three instruments despite differences in appearance of the Hoechst profiles. Differences in laser type, collection filter combinations and the presence of erythroid precursors and apoptotic cells all contributed to this effect. CONCLUSIONS: In our hands, murine SP cells with the expected surface immunophenotype can be identified on a MoFlo, LSR and a factory standard FACSAria.

Animals↗
PubMed2005

In vivo formation of unstable heterokaryons after liver damage and hematopoietic stem cell/progenitor transplantation.

Following reports of lineage plasticity in human hematopoietic stem cells (HSCs), we investigated the potential of human cord blood HSC-enriched cells to create hepatocytes in hosts after inducing liver damage. Carbon tetrachloride induces severe liver damage and subsequent repair via mitosis of resident hepatocytes. It additionally leads to a threefold increase in homing of human mononuclear cells to bone marrow and liver and subsequently to a substantial enhancement of bone marrow engraftment. Eight weeks after liver damage and infusion of an enhanced green fluorescent protein (eGFP) lentivirus-transduced human HSC-enriched cell population, we observed eGFP-positive cells with clear hepatocyte morphology in the livers of animals. These eGFP-positive cells co-expressed human albumin, and reverse-transcription polymerase chain reaction (PCR) analysis demonstrated the presence of human albumin and alpha-anti-trypsin mRNA. However, two antibodies against human mitochondria and human nuclei failed to mark eGFP-positive hepatocyte-like cells but did give clear staining of donor-derived hematopoietic cells. Subsequent fluorescent in situ hybridization (FISH) analysis revealed the presence of mouse Y chromosome in eGFP-positive hepatocyte-like cells. To resolve this discrepancy, we performed single-cell PCR analysis of microdissected eGFP-positive hepatocyte-like cells and found that they contained mostly mouse and little human genomic material. FISH analysis highlighting the centromeres of all human chromosomes revealed only few human chromosomes in these cells. From these results, we conclude that similar to their murine counterparts, human hematopoietic cells have the potential to fuse with resident host hepatocytes. Because no selective pressure is applied to retain the human genomic material, it is gradually lost over time, leading to a variable phenotype of the chimeric cells and making their detection difficult.

Animals↗
PubMed2005

AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) assay is the current model for assessment of human normal and leukemic stem cells. We explored why 51% of 59 acute myeloid leukemia (AML) patients were unable to initiate leukemia in NOD/SCID mice. Increasing the cell dose, using more permissive recipients, and alternative tissue sources did not cause AML engraftment in most previously nonengrafting AML samples. Homing of AML cells to the marrow was the same between engrafters and nonengrafters. FLT3 internal tandem duplication (ITD) and nucleophosmin mutations occurred at a similar frequency in engrafters and nonengrafters. The only variable that was related to engraftment ability was the karyotypically defined risk stratification of individual AML cases. Of interest, follow-up of younger patients with intermediate-risk AML revealed a significant difference in overall survival between NOD/SCID engrafting and nonengrafting AMLs. Hence, the ability of AML to engraft in the NOD/SCID assay seems to be an inherent property of AML cells, independent of homing, conditioning, or cell frequency/source, which is directly related to prognosis. Our results suggest an important difference between leukemic initiating cells between engrafting and nonengrafting AML cases that correlates with treatment response.

Age Factors↗
PubMed2005

Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia.

Human hematopoietic stem cells (HSCs) are generally regarded as being devoid of the markers expressed by differentiated blood cells, the lineage-specific antigens. However, recent work suggests that genes associated with the myeloid lineage are transcribed in mouse HSCs. Here, we explore whether myeloid genes are actually translated in human HSCs. We show that CD33, CD13, and CD123, well-established myeloid markers, are expressed on human long-term repopulating cells from cord blood and bone marrow. In addition, we demonstrate that nonobese diabetic/severe combined immunodeficiency (NOD/SCID) leukemia-initiating cells (SL-ICs) are restricted to the CD33+ fraction in 11 of 12 acute myeloid leukemia (AML) samples studied, indicating that leukemic stem cells (LSCs) express this antigen. This study changes our view of HSCs and the process of differentiation. Furthermore, based on the phenotypic similarity of HSCs and LSCs, our data provide support for the hypothesis that AML derives from an HSC. Our findings also provide a challenge to contemporary attempts to improve the outcome of AML using myeloid antigen-targeted therapies, given the potential for HSC killing.

Animals↗
PubMed2005

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Animals↗
PubMed2005

Development of a human acute myeloid leukaemia screening panel and consequent identification of novel gene mutation in FLT3 and CCND3.

A study was undertaken to develop an acute myeloid leukaemia (AML) screening panel to uncover novel recurring gene mutations. Analysis was performed on six genes known to be mutated in AML (RUNX1, FLT3, KIT, CEBPA, PTPN11 and NRAS) and an additional two candidate genes (CCND3 and FES) in a panel of 175 primary human AML samples that included all French-American-British types except M3, and all cytogenetic risk groups. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with two mutations and two patients (1%) with three mutations. Fifty-five of 88 (63%) patients with normal karyotype AML had at least one mutation. Correlation was observed between KIT mutation and 'favourable risk' cytogenetics (P <0.001), CEBPA mutation and 'intermediate risk' cytogenetics (P=0.045), and PTPN11 mutation and 'poor risk' disease (P <0.001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 were detected (Y589D, D839G, Y842H) that would have been overlooked by conventional gel electrophoresis. A 51-bp deletion was detected in CCND3 in a patient with normal karyotype AML. This validated panel now provides an important tool to evaluate other candidate genes in the genesis of myeloid malignancy.

Acute Disease↗
PubMed2005

Normal and leukaemic stem cells.

The blood-related cancer leukaemias were the first diseases where human cancer stem cells, or leukaemic stem cells (LSC), were isolated. The haematopoietic system is one of the best tissues for investigating cancer stem cells, because the developmental hierarchy of normal blood formation is well defined. Leukaemias can now be viewed as aberrant haematopoietic processes initiated by rare LSC that have maintained or reacquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of LSC and the mechanisms responsible for their emergence in the course of the disease. This report will review our current knowledge on normal and LSC development and examine the impact of these discoveries may have clinically and in our understanding of the leukaemogenic process.

Acute Disease↗
PubMed2005

Leukemic stem cells show the way.

The blood-related cancer leukemia was the first disease where human cancer stem cells (CSCs), or leukemic stem cells (LSCs), were isolated. The hematopoietic system is one of the best tissues for investigating cancer stem cells, since the developmental hierarchy of normal blood formation is well defined. Leukemia can now be viewed as aberrant hematopoietic processes initiated by rare leukemic stem cells (LSC) that have maintained or reacquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of LSC and the mechanisms responsible for their emergence in the course of the disease. This report will review our current knowledge on leukemic stem cell development and finally demonstrate how these discoveries provide a paradigm for identification of Cancer Stem Cell (CSC) from solid tumors.

Acute Disease↗
PubMed2004

In vivo contribution of murine mesenchymal stem cells into multiple cell-types under minimal damage conditions.

Murine mesenchymal stem cells are capable of differentiating in vitro into different lineages under stimulation with certain cytokines, growth factors and chemicals. However, the true capacity of these cells to contribute to different cell-types in vivo is still unclear, especially under minimal injury conditions. In this study, we describe a method of purifying murine mesenchymal stem cells from bone marrow and efficiently transducing them using a lentivirus vector expressing the eGFP reporter gene. Lentivirus-transduced mesenchymal stem cells retained their in vitro ability to differentiate into adipocytes, osteocytes and chondrocytes as well as into myocyte- and astrocyte-like cells. eGFP-mesenchymal stem cells were delivered systemically into minimally injured syngeneic mice. Tracking and tissue-specific differentiation were determined by PCR and immunohistochemistry, respectively. We found donor-derived hepatocytes, lung epithelial cells, myofibroblasts, myofibers and renal tubular cells in some of the recipient mice. Our data indicate that even in the absence of substantial injury, phenotypically defined murine mesenchymal stem cells could acquire tissue specific morphology and antigen expression and thus contribute to different tissue cell-types in vivo.

Animals↗
PubMed2003

Multiparameter analysis of murine bone marrow side population cells.

We describe the multiparameter flow cytometric analysis of the relationship between side population (SP) formation and well-characterized, antigen-defined stem cell subsets. We also compared the competitive repopulation ability of subsets defined by the SP profile to those identified by antigenic markers. The vast majority of SP cells possessed a primitive cell phenotype (c-kit+, SCA-1+, Thy-1+, CD31+, CD135neg, lineage neg), but only a minority of antigen-defined subsets were SP cells. Hence, although SP cells are identified independently of antigenic markers, they are not distinct from established stem cell phenotypes, but are a small subset of them. Approximately half of SP cells expressed CD34 at readily detectable levels, and one-third of SP cells possessed the primitive c-kit+, SCA-1+, lineage neg, CD34neg cell phenotype. Since most SP cells are a subset of c-kit+, Thy-1+, lineage neg, SCA-1+ cells (KTLS), we determined whether the SP cell subset represents a further enrichment in long-term repopulating cell content. The SP+ subset of KTLS+ cells was more enriched for competitive repopulation units than the SP- fraction of KTLS+ cells. Hence, the SP cell fraction highlights a subset of the long-term repopulating cells found within the already highly purified KTLS fraction.

Animals↗
PubMed2003

Solid-phase functionalization of peptides by an alpha-hydrazinoacetyl group.

A novel procedure for the preparation of alpha-hydrazinoacetyl peptides is reported on the basis of the solid-phase coupling of partially or fully Boc-protected hydrazino acetic acid derivatives. The degree of unwanted polymerization of the activated ester during both activation and coupling was found to be significant for the monoprotected derivative BocNHNHCH(2)CO(2)H but could be minimized with the diprotected derivative BocNHNH(Boc)CH(2)CO(2)H and suppressed with the fully protected acid. Despite the instability of the imidocarbonate group toward acids and bases, a low-cost and effective route was sought for the preparation of the tris(Boc)-protected derivative. The N,N,N'-tris(Boc)hydrazinoacetic acid could be introduced on the solid phase after or before peptide elongation using Fmoc/tert-butyl chemistry. In this latter case, HR MAS NMR analysis of model solid supports demonstrated the partial loss of one Boc group during the repetitive piperidine treatments. Despite this slight instability, N,N,N'-tris(Boc)hydrazinoacetic acid was found to be a highly convenient reagent for the robust and easily scalable preparation of hydrazinopeptides in good yield and high purity.

Amino Acid Sequence↗