[show abstract][hide abstract] ABSTRACT: Mesenchymal Stem Cells (MSC) play a fundamental role in allograft rejection and Graft-versus-Host Disease (GvDH) through their immunosuppressive abilities. Recently, Toll like Receptors (TLR) have been shown to modulate MSC functions. The aim of this study was to investigate the effects of several TLR ligands on the interaction between MSC and NK cells. Our results show that TLR-primed adult bone marrow and embryonic MSC are more resistant than unprimed MSC to IL2-activated NK-induced killing. Such protection can be explained by the modulation of NKG2D ligands MICA and ULBP3 and DNAM1 ligands by TLR-primed MSC. These results indicate that MSC are able to adapt their immuno-behaviour in an inflammatory context, decreasing their susceptibility to NK killing. In addition, TLR3 but not TLR4-primed MSC enhance their suppressive functions against NK cells. However, the efficiency of this response is heterogeneous, even if the phenotypes of different analyzed MSC are rather homogeneous. The consequences could be important in MSC-mediated cell therapy, since the heterogeneity of adult MSC responders may be explored in order to select the more efficient responders. Stem Cells 2013;
[show abstract][hide abstract] ABSTRACT: X-chromosome inactivation (XCI) in mammals relies on XIST, a long noncoding transcript that coats and silences the X chromosome in cis. Here we report the discovery of a long noncoding RNA, XACT, that is expressed from and coats the active X chromosome specifically in human pluripotent cells. In the absence of XIST, XACT is expressed from both X chromosomes in humans but not in mice, suggesting a unique role for XACT in the control of human XCI initiation.
[show abstract][hide abstract] ABSTRACT: Background. Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathologic erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods. We addressed the question whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathologic induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation.Results. We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch of fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathologic induced pluripotent stem cells into mice.Conclusions. These results demonstrate that human induced pluripotent stem cells (i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation and (ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.
[show abstract][hide abstract] ABSTRACT: Because many of the genes used to produce induced pluripotent stem cells (iPSCs) from somatic cells are either outright established oncogenes, such as c-myc and Klf4, or potentially related to tumorigenesis in various cancers, both the safety and the risks of tumorigenesis linked to iPSC generation require evaluation. In this work, we generated, by lentivirus-mediated gene transfer of Oct4, Sox2, Nanog, and Lin28, two types of iPSCs from human mesenchymal stem cells and human amniotic fluid-derived cells: fully reprogrammed iPSCs with silencing of the four transgenes and partially reprogrammed iPSCs that still express one or several transgenes. We assessed the behavior of these cells during both their differentiation and proliferation using in vivo teratoma assays in nonobese diabetic mice with severe combined immunodeficiency. In contrast to fully reprogrammed iPSCs, 43% of partially reprogrammed iPSC cases (6 of 14 teratomas) generated major dysplasia and malignant tumors, with yolk sac tumors and embryonal carcinomas positive for α-fetoprotein, cytokeratin AE1/AE3, and CD30. This correlated with the expression of one or several transgenes used for the reprogramming, down-regulation of CDK 1A mRNA (p21/CDKN1A), and up-regulation of antiapoptotic Bcl-2 mRNA. Therefore, the oncogenicity of therapeutically valuable patient-specific iPSC-derived cells should be scrupulously evaluated before they are used for any clinical applications.
American Journal Of Pathology 03/2012; 180(5):2084-96. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.
PLoS ONE 01/2012; 7(4):e30743. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cationic lipid-based nonviral gene delivery is an attractive approach for therapeutic gene transfer. Basically, gene transfection can be achieved by using synthetic vectors that compact DNA, forming cationic lipoplexes which can interact with the cell plasma membrane by electrostatic interactions. Among the basic components of any cationic lipid, the type of cationic headgroup has been shown to have a major role in transfection efficiency. We have previously reported the DNA transfection potential of vectors characterized by a kanamycin A headgroup. The encouraging transfection results obtained with these compounds prompted us to evaluate the potential of cationic lipids bearing headgroups based on other aminoglycosides. Thus, we herein report the synthesis and gene transfection properties of novel cationic lipids consisting of cholesteryl or dioleyl moieties linked, via various spacers, to paromomycin or neomycin B headgroups. Our results confirm that these new aminoglycoside-based cationic lipids are efficient for gene transfection both in vitro and into the mouse airways in vivo. We also investigated physico-chemical properties of the DNA complexes formed by this particular type of synthetic vectors in order to better understand their structure-activity relationships.
Journal of Controlled Release 12/2011; 158(3):461-9. · 7.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: A major issue in immunosuppressive biotherapy is the use of mesenchymal stem cells (MSCs) that harbor regulatory capacity. However, currently used bone marrow-derived MSCs (BM-MSCs) are short-lived and cannot assure long lasting immunoregulatory function both in vitro and in vivo. Consequently, we have generated MSCs from human induced pluripotent stem (IPS-MSCs) cells that share similar properties with embryonic stem cells (ES-MSCs). Herein, we compared the immunoregulatory properties of ES/IPS-MSCs with those of BM-MSCs and showed, for the first time, that IPS-derived MSCs display remarkable inhibition of NK-cell proliferation and cytolytic function in a similar way to ES-MSCs. Both MSCs disrupt NK-cell cytolytic machinery in the same fashion that BM-MSCs, by down-regulating the expression of different activation markers and ERK1/2 signaling, leading to an impairment to form immunologic synapses with target cells and, therefore, secretion of cytotoxic granules. In addition, they are more resistant than adult BM-MSCs to preactivated NK cells. IPS-MSCs could represent an attractive alternative source of immunoregulatory cells, and their capacity to impair NK-cell cytotoxicity constitutes a complex mechanism to prevent allograft rejection.
[show abstract][hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT-PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.
Journal of Molecular Cell Biology 10/2010; 2(5):291-8.
[show abstract][hide abstract] ABSTRACT: In utero tracheal occlusion (TO) has been developed to improve the lung hypoplasia associated with congenital diaphragmatic hernia (CDH). However, although TO stimulates fetal lung growth, it results in a decrease of alveolar type II cells (ATII) and surfactant production. Because keratinocyte growth factor (KGF) is a potent stimulus of ATII proliferation and maturation, we evaluated, in a fetal lamb model of CDH, a gene therapy strategy combining TO and ovine KGF transfection into the fetal airways using bisguanidinium-tren-cholesterol/dioleoyl-phosphatidylethanolamine (BGTC/DOPE) cationic liposomes.
Three groups of sheep fetuses with CDH and a group of normal fetuses were studied. The fetuses of the three groups with CDH (KGF, Medium and Hernia groups) underwent surgery at 85 days of gestation to create a diaphragmatic hernia. The KGF and medium group fetuses underwent a second surgery step at day 125 to perform TO associated with injection of the KGF transfection mixture (KGF group) or control medium (Medium group), whereas the fetuses of the Hernia group were left untreated. Normal fetuses were used as a control (Normal group). All fetuses were euthanized at 132 days of gestation and various analytical studies [lung weight, radial alveolar count (RAC), KGF and surfactant protein B (SPB) expression, number of ATII cells] were performed to assess the efficiency of KGF transfection and its effects on fetal lung development.
TO was associated with lung hyperplasia and increased RAC in the Medium and KGF groups versus the Hernia group. Expression of KGF was increased in the KGF group compared to all other groups and was associated with an increased synthesis of SPB by alveolar cells and an ectopic synthesis of SPB by bronchiolar cells compared to TO treatment alone.
Thus, BGTC/DOPE liposomes can mediate efficient KGF transfection into the airways in a fetal sheep model of CDH. Furthermore, combining KGF transfection and TO resulted not only (as did TO alone) in the correction of the CDH-associated lung hypoplasia and decreased RAC, but also in increased SPB synthesis, suggesting a better maturation of the re-growing lung (compared to TO alone). Additional studies are required to further explore the therapeutic potential of such a combined strategy; in particular, studies evaluating the lung function of in utero-treated CDH lamb newborns.
The Journal of Gene Medicine 05/2010; 12(5):413-22. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Photodynamic therapy combines a photosensitizer, localised preferentially in malignant cells with light activation. Hypocrellin A (HA), a lipid-soluble peryloquinone, is considered as a high potential photosensitizer. We report dose and light irradiation effects of HA on HeLa, Calu and K562 cell lines, the latter including a subclone resistant to Imatinib mesylate (IM, Gleevec). All cell lines and subclones tested are sensitive to HA PDT. In the epithelial tumour cell lines, we observe a significant photosensitizing effect in the presence of HA. In the leukemic K562 cells, HA exposure led to an inhibitory effect, which was not seen in the K562 cells resistant to Imatinib mesylate. However, experiments using IM and HA led to a reversal of IM-resistant phenotype in this cell line, with evidence of a major sensitizing effect of photodynamic therapy. Overall our results suggest a phototoxicity of HA in epithelial cell lines and demonstrate for the first time, a synergy between IM and photodynamic therapy to circumvent IM-resistance.
Journal of photochemistry and photobiology. B, Biology 03/2010; 99(2):100-4. · 1.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Various pulmonary disorders, including cystic fibrosis, are potentially amenable to a treatment modality in which a therapeutic gene is directly delivered to the lung. Current gene delivery systems, either viral or nonviral, need further improvement in terms of efficiency and safety. We reported that nonionic amphiphilic block copolymers hold promise as nonviral gene delivery systems for transfection of muscular tissues. To evaluate the efficiency of these vectors in the lung, intratracheal instillation or aerosolization of reporter genes complexed with Lutrol or PE6400 was performed. Lutrol-DNA and, to a lesser extent, PE6400-DNA complexes promoted efficient gene transfection into mouse airways in a dose-dependent manner. This improvement over naked DNA was observed irrespective of the reporter gene. Lutrol enabled us to deliver significantly higher DNA amounts than current nonviral vectors, with even greater increases in gene expression and without the formation of colloidally unstable complexes. Time course studies showed that Lutrol-DNA complexes permitted prolonged gene expression for up to 5 days whereas with poly(ethylenimine) (PEI)-DNA polyplexes, expression peaked on days 1-2 postinstillation, was strongly reduced by day 5, and reached background levels on day 7. Aerosolized delivery of Lutrol-DNA complexes, a less invasive approach to deliver genes to the lung, gave 5- to 15-fold higher reporter gene expression compared with PEI-DNA polyplexes administered via the same delivery route. After intratracheal instillation of Lutrol-DNA complexes, histochemical staining for beta-galactosidase expression showed the presence of large blue areas. Histopathological analysis showed that Lutrol alone did not elicit inflammation, and that the inflammatory response after intratracheal instillation of Lutrol-DNA complexes was reversible and was observed only with the highest amounts of DNA. We also found that Lutrol can efficiently deliver genes to the airways of cystic fibrosis mice. Thus, we conclude that Lutrol is a highly promising vector for gene delivery to the lung.
Human Gene Therapy 08/2005; 16(7):821-9. · 4.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cationic lipids nowadays constitute a promising alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a new class of lipids based upon the use of aminoglycosides as cationic polar headgroups. The encouraging results obtained with a first cholesterol derivative of kanamycin A prompted us to investigate this family of vectors further, by modulating the constituent structural units of the cationic lipid. For this study, we have investigated the transfection properties of a series of new derivatives based on a kanamycin A scaffold. The results primarily confirm that aminoglycoside-based lipids are efficient vectors for gene transfection both in vitro and in vivo (mouse airways). Furthermore, a combination of transfection and physicochemical data revealed that some modifications of the constitutive subunits of kanamycin A-based vectors were associated with substantial changes in their transfection properties.
[show abstract][hide abstract] ABSTRACT: Synthetic gene delivery vectors are gaining increasing importance in gene therapy as an alternative to recombinant viruses. Among the various types of non-viral vectors, cationic lipids are especially attractive as they can be prepared with relative ease and extensively characterised. Further, each of their constituent parts can be modified, thereby facilitating the elucidation of structure-activity relationships. In this forward-looking review, cationic lipid-mediated gene delivery will mainly be discussed in terms of the structure of the three basic constituent parts of any cationic lipid: the polar headgroup, hydrophobic moiety and linker. Particular emphasis will be placed on recent advances in the field as well as on our own original contributions. In addition to reviewing critical physicochemical features (such as headgroup hydration) of monovalent lipids, the use of headgroups with known nucleic-acid binding modes, such as linear and branched polyamines, aminoglycosides and guanidinium functions, will be comprehensively assessed. A particularly exciting innovation in linker design is the incorporation of environment-sensitive groups, the intracellular hydrolysis of which may lead to more controlled DNA delivery. Examples of pH-, redox- and enzyme-sensitive functional groups integrated into the linker are highlighted and the benefits of such degradable vectors can be evaluated in terms of transfection efficiency and cationic lipid-associated cytotoxicity. Finally, possible correlations between the length and type of hydrophobic moiety and transfection efficiency will be discussed. In conclusion it may be foreseen that in order to be successful, the future of cationic lipid-based gene delivery will probably require the development of sophisticated virus-like systems, which can be viewed as "programmed supramolecular systems" incorporating the various functions required to perform in a chronological order the different steps involved in gene transfection.
Current Pharmaceutical Design 02/2005; 11(3):375-94. · 3.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cationic lipid-mediated gene transfection involves uptake of the lipid/DNA complexes via endocytosis, a cellular pathway characterized by a significant drop in pH. Thus, in the present study, we aimed to explore the impact on transfection efficiency of the inclusion of an acid-sensitive acylhydrazone function in the cationic lipid structure. We synthesized and evaluated the transfection properties of a series of four cationic steroid derivatives characterized by an acylhydrazone linkage connecting a guanidinium-based headgroup to a saturated cholestanone or an unsaturated cholest-4-enone hydrophobic domain. Acid-catalyzed hydrolysis was confirmed for all lipids, its rate being highest for those with a cholestanone moiety. The compound bis-guanidinium bis(2-aminoethyl)amine hydrazone (BGBH)-cholest-4-enone was found to mediate efficient gene transfection into various mammalian cell lines in vitro and into the mouse airways in vivo. In vitro transfection studies with BGBH-cholest-4-enone formulations also showed that incorporation of a degradable acylhydrazone bond led to low cytotoxicity and impacted the intracellular trafficking of the lipoplexes. Thus, our work allowed us to identify a cationic lipid structure with an acid-cleavable acylhydrazone linker capable of mediating efficient gene transfection in vitro and in vivo and it thereby provides a basis for further development of related acid-sensitive gene delivery systems.
Journal of Medicinal Chemistry 11/2004; 47(21):5210-23. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Over the last several years, we have developed a novel class of cationic lipids, cholesterol derivatives characterized by polar head groups with guanidinium functions. We have in particular shown that bis(guanidinium)-tren-cholesterol/dioleoylphosphatidylethanolamine (BGTC/DOPE) cationic liposomes can mediate efficient gene transfection into the mouse airways in vivo via direct intratracheal administration or intranasal instillation. As prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we have explored in the present work the feasibility of BGTC-mediated gene transfection into the respiratory tract of fetal sheep in utero.
Thus, BGTC/DOPE liposomes were complexed with plasmids expressing the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene and the resulting lipoplexes were administered to fetal sheep at 70 days of gestation via surgical replacement of the airway fluid by the transfection mixture followed by tracheal occlusion. The fetal lungs and tracheas were harvested at 72 h and examined for CAT expression and evidence of toxicity.
CAT expression was detected in both lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked plasmid DNA. Immunohistochemical analysis showed that airway epithelial cells and some mesenchymal cells were transfected. Pulmonary histopathology of varied severity was however observed under our transfection conditions and manifested as focal epithelial and mesenchymal lesions.
These results show that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium. They also invite the development of optimized BGTC-based formulations and administration conditions with a view to future prenatal gene transfer experiments involving therapeutic genes.
The Journal of Gene Medicine 04/2004; 6(3):328-36. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Over previous years, problems associated with virus-mediated gene delivery have stimulated the synthesis and biological evaluation of non-viral vectors as a possible alternative for gene therapy applications. Of the various non-viral vectors, cationic lipids have come forward as effective gene delivery agents, although it is clear that their transfection efficiency must be increased in order for them to become of real therapeutic value. This can be achieved by overcoming both the intracellular and extracellular barriers they encounter while conveying the transgene towards the nucleus of the target cells. The purpose of this review is to highlight the advances made to date in facing these challenges by paying particular attention to the design of the cationic lipid itself and the complexes (termed lipoplexes) formed on interacting with DNA. Because the structures of all three parts of a cationic lipid – the cationic headgroup, the hydrophobic moiety and the connecting linker – are important determinants of transfection efficiency, each will be considered here in turn, with special attention focused on recent studies including our own work. In addition, the stability of the lipoplex in the extracellular medium and the features of its intracellular trafficking towards the cell nucleus will be assessed from both chemical and biological viewpoints. In conclusion, the future will probably see the development of sophisticated modular self-assembling gene delivery systems incorporating various functional elements to face the various biological barriers encountered. Such vectors can be envisaged as ‘virus-like’ systems which share the levels of gene delivery efficiency of their viral counterparts, but coupled with the safety of purpose-made organic molecules.
[show abstract][hide abstract] ABSTRACT: Artificial self-assembling systems are currently widely investigated as an alternative approach to recombinant viruses for gene transfection in vitro and in vivo. Cationic lipids are particularly attractive, as a great variety of well-characterized reagents can be synthesized from there. Over the last few years, numerous cationic lipid systems have been developed and shown to be efficient for in vitro transfection. However, although some promising results have been reported in the in vivo setting (even in clinical gene therapy trials in man), the in vivo use of cationic lipid-based systems is still problematic, especially when considering the systemic route of administration. Herein, we summarize our own research on a particular class of cationic lipids, cholesterol derivatives characterized by polar headgroups with guanidinium functions, in order to illustrate the basic principles of and the positive results already obtained by cationic lipid-mediated gene delivery as well as the remaining problems that need to be urgently resolved, particularly as regards the systemic administration. In this forward-looking review, we also discuss the present efforts to develop modular systems for improved in vivo transfection. Indeed, lipid-based vectors offer the possibility to create sophisticated modular gene delivery systems capable of self-assembly via hydrophobic interaction between their components, the role of the different functional elements being to help in overcoming the distinct extracellular and cellular barriers to in vivo gene transfection into the various somatic target tissues.
Current Drug Targets 03/2002; 3(1):1-16. · 3.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.
We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino-sugar-based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin-cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.
These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo.
The Journal of Gene Medicine 01/2002; 4(5):517-26. · 2.16 Impact Factor