Claude Bagnis

Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse

Here, we report the isolation of a human multipotent adipose-derived stem (hMADS) cell population from adipose tissue of young donors. hMADS cells display normal karyotype; have active telomerase; proliferate >200 population doublings; and differentiate into adipocytes, osteoblasts, and myoblasts. Flow cytometry analysis indicates that hMADS cells are CD44+, CD49b+, CD105+, CD90+, CD13+, Stro-1−, CD34−, CD15−, CD117−, Flk-1−, gly-A−, CD133−, HLA-DR−, and HLA-Ilow. Transplantation of hMADS cells into the mdx mouse, an animal model of Duchenne muscular dystrophy, results in substantial expression of human dystrophin in the injected tibialis anterior and the adjacent gastrocnemius muscle. Long-term engraftment of hMADS cells takes place in nonimmunocompromised animals. Based on the small amounts of an easily available tissue source, their strong capacity for expansion ex vivo, their multipotent differentiation, and their immune-privileged behavior, our results suggest that hMADS cells will be an important tool for muscle cell–mediated therapy.

Enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with forced expression of MyoD.

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose-derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2(-/-)gammaC(-/-) mice resulted in a substantial increase in the number of hMADS cell-derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.

Vanin-1 licenses inflammatory mediator production by gut epithelial cells and controls colitis by antagonizing peroxisome proliferator-activated receptor γ activity

Colitis involves immune cell–mediated tissue injuries, but the contribution of epithelial cells remains largely unclear. Vanin-1 is an epithelial ectoenzyme with a pantetheinase activity that provides cysteamine/cystamine to tissue. Using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis model we show here that Vanin-1 deficiency protects from colitis. This protection is reversible by administration of cystamine or bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor (PPAR)γ antagonist. We further demonstrate that Vanin-1, by antagonizing PPARγ, licenses the production of inflammatory mediators by intestinal epithelial cells. We propose that Vanin-1 is an epithelial sensor of stress that exerts a dominant control over innate immune responses in tissue. Thus, the Vanin-1/pantetheinase activity might be a new target for therapeutic intervention in inflammatory bowel disease.

Hyperthermia enhances CTL cross-priming.

Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8+ T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42°C before killing are more efficient in cross-priming of naive CD8+ T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8+ T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201+ melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay. Two mechanisms might explain the hyperthermia-induced enhanced cross-priming. First, heat-treated melanoma cells expressed increased levels of 70-kDa heat shock protein (HSP70), and enhanced cross-priming could be reproduced by overexpression of HSP70 in melanoma cells transduced with HSP70 encoding lentiviral vector. Second, hyperthermia resulted in the increased transcription of several tumor Ag-associated Ags, including MAGE-B3, -B4, -A8, and -A10. Thus, heat treatment of tumor cells permits enhanced cross-priming, possibly via up-regulation of both HSPs and tumor Ag expression.

p8 is critical for tumour development induced by rasV12 mutated protein and E1A oncogene

The p8 protein is involved in the cellular stress response of many tissues. Because p8 is overexpressed in many cancers, we investigated whether its expression was required for tumour development. Mouse embryo fibroblasts (MEFs) from p8+/+ and p8−/− animals were transformed with the pBabe‐rasV12/E1A retroviral vector, which expresses both the rasV12 mutated protein and the E1A oncogene. As expected, transformed p8+/+ MEFs could form colonies in soft agar. However, transformed p8−/− MEFs could not. In addition, subcutaneous or intraperitoneal injections of transformed p8+/+ MEFs always led to tumour formation in nude mice, but, again, no tumour was observed with transformed p8−/− MEFs. However, restoring p8 expression in transformed p8−/− MEFs before injection led to tumour formation. In the tumours, p8 expression was induced during tumour development. It was concluded that p8 expression in transformed MEFs is necessary for tumour formation, suggesting that the stress‐response mechanisms governed by p8 are required for tumour establishment.

Posttranscriptional deregulation of MYC via PTEN constitutes a major alternative pathway of MYC activation in T-cell acute lymphoblastic leukemia

Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down-regulation of PTEN, and that NOTCH1(m) might play a dual transcriptional and posttranscriptional role in this process. Altogether, our data lend further support to the significance of therapeutic targeting of MYC and/or the PTEN/AKT pathways, both in GSI-resistant and identified NOTCH1-independent/MYC-mediated T-ALL patients.

Isolation of a Highly Myogenic CD34-Negative Subset of Human Skeletal Muscle Cells Free of Adipogenic Potential†‡§

The differentiation of multipotent cells into undesirable lineages is a significant risk factor when performing cell therapy. In muscular diseases, myofiber loss can be associated with progressive fat accumulation that is one of the primary factors leading to decline of muscular strength. Therefore, to avoid any contribution of injected multipotent cells to fat deposition, we have searched for a highly myogenic but nonadipogenic muscle‐derived cell population. We show that the myogenic marker CD56, which is the gold standard for myoblast‐based therapy, was unable to separate muscle cells into myogenic and adipogenic fractions. Conversely, using the stem cell marker CD34, we were able to sort two distinct populations, CD34+ and CD34−, which have been thoroughly characterized in vitro and in vivo using an immunodeficient Rag2−/−γc−/− mouse model of muscle regeneration with or without adipose deposition. Our results demonstrate that both populations have equivalent capacities for in vitro amplification. The CD34+ cells and CD34− cells exhibit equivalent myogenic potential, but only the CD34− population fails to differentiate into adipocytes in vitro and in vivo after transplantation into regenerative fat muscle. These data indicate that the muscle‐derived cells constitute a heterogeneous population of cells with various differentiation potentials. The simple CD34 sorting allows isolation of myogenic cells with no adipogenic potential and therefore could be of high interest for cell therapy when fat is accumulated in diseased muscle. STEM CELLS 2010;28:753–764

Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: Optimized design and procedures for highly efficient transduction compatible with clinical constraints

Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector–mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75–85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14+ precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.

Gene transfer to hepatocellular carcinoma: Transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors

Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5′-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

Insights into Gene Expression Changes Impacting B-Cell Transformation: Cross-Species Microarray Analysis of Bovine Leukemia Virus Tax-Responsive Genes in Ovine B Cells

ABSTRACT Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor incidence, experimental infection of sheep leads to overt leukemia in the majority of infected animals, providing a model for studying the pathogenesis associated with BLV and HTLV-1. TaxBLV, the major oncoprotein, initiates a cascade of events leading toward malignancy, although the basis of transformation is not fully understood. We have taken a cross-species ovine-to-human microarray approach to identify TaxBLV-responsive transcriptional changes in two sets of cultured ovine B cells following retroviral vector-mediated delivery of TaxBLV. Using cDNA-spotted microarrays comprising 10,336 human genes/expressed sequence tags, we identified a cohort of differentially expressed genes, including genes related to apoptosis, DNA transcription, and repair; proto-oncogenes; cell cycle regulators; transcription factors; small Rho GTPases/GTPase-binding proteins; and previously reported TaxHTLV-1-responsive genes. Interestingly, genes known to be associated with human neoplasia, especially B-cell malignancies, were extensively represented. Others were novel or unexpected. The results suggest that TaxBLV deregulates a broad network of interrelated pathways rather than a single B-lineage-specific regulatory process. Although cross-species approaches do not permit a comprehensive analysis of gene expression patterns, they can provide initial clues for the functional roles of genes that participate in B-cell transformation and pinpoint molecular targets not identified using other methods in animal models.