Diderik Tirefort

Rapid Generation of Stable Transgenic Embryonic Stem Cell Lines Using Modular Lentivectors

Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time‐consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV‐1 element and woodchuck hepatitis virus post‐transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue‐specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research.

A Sox1 to Pax6 Switch Drives Neuroectoderm to Radial Glia Progression During Differentiation of Mouse Embryonic Stem Cells

The transcription factors Sox1 and Pax6 are expressed sequentially during early mouse embryonic neurogenesis. Sox1 expression starts upon formation of neuroectoderm, whereas Pax6 is subsequently expressed in radial glial cells, the latter giving rise to most neurons of the cerebral cortex. Here we used mouse embryonic stem (ES) cells to study the role of Sox1 and Pax6 in regulating differentiation of neural progenitors. For this purpose, we investigated the effect of overexpression and knockdown of Sox1 and Pax6, using three differentiation protocols. We show that (a) expression of Sox1 or Pax6 in uncommitted ES cells favored neuroectodermal lineage choice; (b) continuous Sox1 expression maintained cells at the neuroepithelial stage and prevented expression of Pax6 and other radial glial cell markers; (c) Sox1 knockdown facilitated exit from the progenitor stage, whereas Pax6 knockdown decreased formation of radial glia; (d) forced Pax6 expression in neuroepithelial cells triggered their differentiation into radial glia and neurons; and (e) Pax6 expression induced cell migration, a feature typical of radial glia‐derived early neurons. We conclude that Sox1 enhances neuroectodermal commitment and maintenance but blocks further differentiation. In contrast, Pax6 is involved in the progression of neuroectoderm toward radial glia. STEM CELLS 2009;27:49–58

Neural progenitors derived from human embryonic stem cells are targeted by allogeneic T and natural killer cells.

Neural progenitor cells (NPC) of foetal origin or derived from human embryonic stem cells (HESC) have the potential to differentiate into mature neurons after transplantation into the central nervous system, opening the possibility of cell therapy for neurodegenerative disorders. In most cases, the transplanted NPC are genetically unrelated to the recipient, leading to potential rejection of the transplanted cells. Very few data provide reliable information as to the potential immune response of allogeneic neural progenitors derived from HESC. In this study, we analyzed in vitro the allogeneic immune response of T lymphocytes and natural killer (NK) cells to NPC derived from HESC or of foetal origin. We demonstrate that NPC induce T‐cell stimulation and a strong NK cytotoxic response. NK‐cell activity is unrelated to MHC‐I expression but driven by the activating NKG2D receptor. Cyclosporine and dexamethasone previously used in clinical studies with foetal NPC did not only fail to prevent NK alloreactivity but strongly inhibited the terminal maturation from NPC into mature neurons. We conclude that allogenic transplantation of NPC in the central nervous system will most likely require an immunosuppressive regimen targeting allogenic T and NK cells, whereas possible interference with the differentiation of NPC needs to be carefully evaluated.

Development of Human Nervous Tissue upon Differentiation of Embryonic Stem Cells in Three‐Dimensional Culture

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air‐liquid interface‐based culture of human ESC. This culture system allows three‐dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3‐month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural‐like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air‐liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural‐like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three‐dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues. STEM CELLS 2009;27:509–520

Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non‐neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells.

Comprehensive metagenomic analysis of glioblastoma reveals absence of known virus despite antiviral‐like type I interferon gene response

Glioblastoma is a deadly malignant brain tumor and one of the most incurable forms of cancer in need of new therapeutic targets. As some cancers are known to be caused by a virus, the discovery of viruses could open the possibility to treat, and perhaps prevent, such a disease. Although an association with viruses such as cytomegalovirus or Simian virus 40 has been strongly suggested, involvement of these and other viruses in the initiation and/or propagation of glioblastoma remains vague, controversial and warrants elucidation. To exhaustively address the association of virus and glioblastoma, we developed and validated a robust metagenomic approach to analyze patient biopsies via high‐throughput sequencing, a sensitive tool for virus screening. In addition to traditional clinical diagnostics, glioblastoma biopsies were deep‐sequenced and analyzed with a multistage computational pipeline to identify known or potentially discover unknown viruses. In contrast to the studies reporting the presence of viral signatures in glioblastoma, no common or recurring active viruses were detected, despite finding an antiviral‐like type I interferon response in some specimens. Our findings highlight a discrete and non‐specific viral signature and uncharacterized short RNA sequences in glioblastoma. This study provides new insights into glioblastoma pathogenesis and defines a general methodology that can be used for high‐resolution virus screening and discovery in human cancers.

Cellular diversity within embryonic stem cells: pluripotent clonal sublines show distinct differentiation potential

Embryonic stem cells (ESC), derived from the early inner cell mass (ICM), are constituted of theoretically homogeneous pluripotent cells. Our study was designed to test this concept using experimental approaches that allowed characterization of progenies derived from single parental mouse ESC. Flow cytometry analysis showed that a fraction of ESC submitted to neural differentiation generates progenies that escape the desired phenotype. Live imaging of individual cells demonstrated significant variations in the capacity of parental ESC to generate neurons, raising the possibility of clonal diversity among ESC. To further substantiate this hypothesis, clonal sublines from ESC were generated by limit dilution. Transcriptome analysis of undifferentiated sublines showed marked differences in gene expression despite the fact that all clones expressed pluripotency markers. Sublines showed distinct differentiation potential, both in phenotypic differentiation assays and with respect to gene expression in embryoid bodies. Clones generated from another ESC line also showed individualities in their differentiation potential, demonstrating the wider applicability of these findings. Taken together, our observations demonstrate that pluripotent ESC consist of individual cell types with distinct differentiation potentials. These findings identify novel elements for the biological understanding of ESC and provide new tools with a major potential for their future in vitro and in vivo use.

The relationship between brain tumor cell invasion of engineered neural tissues and in vivo features of glioblastoma

Glioblastoma is an aggressive brain tumor characterized by its high propensity for local invasion, formation of secondary foci within the brain, as well as areas of necrosis. This study aims to (i) provide a technical approach to reproduce features of the disease in vitro and (ii) characterize the tumor/host brain tissue interaction at the molecular level. Human engineered neural tissue (ENT) obtained from pluripotent stem cells was generated and co-cultured with human glioblastoma-initiating cells. Within two weeks, glioblastoma cells invaded the nervous tissue. This invasion displayed features of the disease in vivo: a primary tumor mass, diffuse migration of invading single cells into the nervous tissue, secondary foci, as well as peritumoral cell death. Through comparative molecular analyses, this model allowed the identification of more than 100 genes that are specifically induced and up-regulated by the nervous tissue/tumor interaction. Notably the type I interferon response, extracellular matrix-related genes were most highly represented and showed a significant correlation with patient survival. In conclusion, glioblastoma development within a nervous tissue can be engineered in vitro, providing a relevant model to study the disease and allows the identification of clinically-relevant genes induced by the tumor/host tissue interaction.

Metagenomics analysis of red blood cell and fresh-frozen plasma units

Although the risk of transmitting infectious agents by blood transfusion is dramatically reduced after donor selection, leukoreduction, and laboratory testing, some could still be present in donors blood. A description of metagenomes in blood products eligible for transfusion represents relevant information to evaluate the risk of pathogen transmission by transfusion.

Human tissue engineering allows the identification of active miRNA regulators of glioblastoma aggressiveness.

Glioblastoma multiforme (GBM) is among the most aggressive cancers associated with massive infiltration of peritumoral parenchyma by migrating tumor cells. The infiltrative nature of GBM cells, the intratumoral heterogeneity concomitant with redundant signaling pathways likely underlie the inability of conventional and targeted therapies to achieve long-term remissions. In this respect, microRNAs (miRNAs), which are endogenous small non-coding RNAs that play a role in cancer aggressiveness, emerge as possible relevant prognostic biomarkers or therapeutic targets for treatment of malignant gliomas. We previously described a tissue model of GBM developing into a stem cell-derived human Engineered Neural Tissue (ENT) that allows the study of tumor/host tissue interaction. Combined with high throughput sequencing analysis, we took advantage of this human and integrated tissue model to understand miRNAs regulation. Three miRNAs (miR-340, -494 and -1293) active on cell proliferation, adhesion to extracellular matrix and tumor cell invasion were identified in GBM cells developing within ENT, and also confirmed in GBM biopsies. The components of miRNAs regulatory network at the transcriptional and the protein level have been also revealed by whole transcriptome analysis and Tandem Mass Tag in transfected GBM cells. Notably, miR-340 has a clinical relevance and modulates the expression of miR-494 and -1293, emphasizing its biological significance. Altogether, these findings demonstrate that human tissue engineering modeling GBM development in neural host tissue is a suitable tool to identify active miRNAs. Collectively, our study identified miR-340 as a strong modulator of GBM aggressiveness which may constitute a therapeutic target for treatment of malignant gliomas.