Prominin-1 (CD133) Defines Both Stem and Non-Stem Cell Populations in CNS Development and Gliomas

Prominin-1 (CD133) is a commonly used cancer stem cell marker in central nervous system (CNS) tumors including glioblastoma (GBM). Expression of Prom1 in cancer is thought to parallel expression and function in normal stem cells. Using RNA in situ hybridization and antibody tools capable of detecting multiple isoforms of Prom1, we find evidence for two distinct Prom1 cell populations in mouse brain. Prom1 RNA is first expressed in stem/progenitor cells of the ventricular zone in embryonic brain. Conversely, in adult mouse brain Prom1 RNA is low in SVZ/SGZ stem cell zones but high in a rare but widely distributed cell population (Prom1hi). Lineage marker analysis reveals Prom1hi cells are Olig2+Sox2+ glia but Olig1/2 knockout mice lacking oligodendroglia retain Prom1hi cells. Bromodeoxyuridine labeling identifies Prom1hi as slow-dividing distributed progenitors distinct from NG2+Olig2+ oligodendrocyte progenitors. In adult human brain, PROM1 cells are rarely positive for OLIG2, but express astroglial markers GFAP and SOX2. Variability of PROM1 expression levels in human GBM and patient-derived xenografts (PDX) – from no expression to strong, uniform expression – highlights that PROM1 may not always be associated with or restricted to cancer stem cells. TCGA and PDX data show that high expression of PROM1 correlates with poor overall survival. Within proneural subclass tumors, high PROM1 expression correlates inversely with IDH1 (R132H) mutation. These findings support PROM1 as a tumor cell-intrinsic marker related to GBM survival, independent of its stem cell properties, and highlight potentially divergent roles for this protein in normal mouse and human glia

CRX Is a Diagnostic Marker of Retinal and Pineal Lineage Tumors

Background: CRX is a homeobox transcription factor whose expression and function is critical to maintain retinal and pineal lineage cells and their progenitors. To determine the biologic and diagnostic potential of CRX in human tumors of the retina and pineal, we examined its expression in multiple settings. Methodology/Principal Findings: Using situ hybridization and immunohistochemistry we show that Crx RNA and protein expression are exquisitely lineage restricted to retinal and pineal cells during normal mouse and human development. Gene expression profiling analysis of a wide range of human cancers and cancer cell lines also supports that CRX RNA is highly lineage restricted in cancer. Immunohistochemical analysis of 22 retinoblastomas and 13 pineal parenchymal tumors demonstrated strong expression of CRX in over 95% of these tumors. Importantly, CRX was not detected in the majority of tumors considered in the differential diagnosis of pineal region tumors (n = 78). The notable exception was medulloblastoma, 40% of which exhibited CRX expression in a heterogeneous pattern readily distinguished from that seen in retino-pineal tumors. Conclusions/Significance: These findings describe new potential roles for CRX in human cancers and highlight the general utility of lineage restricted transcription factors in cancer biology. They also identify CRX as a sensitive and specific clinical marker and a potential lineage dependent therapeutic target in retinoblastoma and pineoblastoma

Calibrating genomic and allelic coverage bias in single-cell sequencing

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.National Cancer Institute (U.S.) (Grant P30-CA14051

Drug sensitivity of single cancer cells is predicted by changes in mass accumulation rate

Assays that can determine the response of tumor cells to cancer therapeutics could greatly aid the selection of drug regimens for individual patients. However, the utility of current functional assays is limited, and predictive genetic biomarkers are available for only a small fraction of cancer therapies. We found that the single-cell mass accumulation rate (MAR), profiled over many hours with a suspended microchannel resonator, accurately defined the drug sensitivity or resistance of glioblastoma and B-cell acute lymphocytic leukemia cells. MAR revealed heterogeneity in drug sensitivity not only between different tumors, but also within individual tumors and tumor-derived cell lines. MAR measurement predicted drug response using samples as small as 25 μl of peripheral blood while maintaining cell viability and compatibility with downstream characterization. MAR measurement is a promising approach for directly assaying single-cell therapeutic responses and for identifying cellular subpopulations with phenotypic resistance in heterogeneous tumors.United States. National Institutes of Health (R01 CA170592)United States. National Institutes of Health (R33 CA191143)National Cancer Institute (U.S.) (U54 CA143874)United States. National Institutes of Health (NIH/NIGMS T32 GM008334

DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape

Introduction: Transposable element (TE) derived sequences comprise half of our genome and DNA methylome, and are presumed densely methylated and inactive. Examination of the genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues revealed unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the tissue type and their expression correlated strongly with hypomethylation of the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks including H3K4me1 and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and exhibited evidence for evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type-specific regulatory networks, and have acquired tissue-specific epigenetic regulation

Fonction des facteurs de transcription Olig1 et Olig2 dans les cellules souches neurales du système nerveux central (Etude d'un modèle de souris transgéniques d'expression inductible)

L'objectif de ma thèse a été de définir l'impact de la sur-expression des facteurs de transcription Olig sur la différenciation des cellules souches neurales en oligodendrocyte dans un modèle de souris transgénique. L'expression des transgènes est induite par la doxycycline dans les cellules souches nestine+. Au cours du développement, l'expression forcée de Olig1 ou Olig2 induit une genèse ectopique d'oligodendrocytes. De plus, la sur-expression de Olig2 bloque la spécification des interneurones V3. En post-natal, la sur-expression de Olig2 dans les zones germinatives provoque une myélinisation précoce et une augmentation du nombre d'astrocytes dans le corps calleux. L'ensemble de mes résultats indique que la sur-expression des facteurs Olig pourrait constituer une stratégie thérapeutique intéressante pour promouvoir la régénération de la myéline dans des pathologies démyélinisantes, telle que la scérose en plaques.Olig1 and Olig2 are b-HLH transcription factors involved in oligodendrocyte development in the central nervous system. My project aims to analyse the effect of Olig gene over-expression in neural stem cells. Therefore, I designed and analyzed transgenic mice models with inducible expression of Olig genes in nestin+ neural stem/progenitor cells (Tet-On system). At embryonic stages, forced expression of Olig1 and Olig2 leads to ectopic expression of oligodendrocyte markers. Moreover, Olig2 over-expression decreased V3 interneuron specification. At postnatal stage Olig2 over-expression in germinative area induced earlier myelination and astrocyte specification in corpus callosum. These transgenic mice provide a useful model to test whether forced expression of Olig genes represents a possible strategy to enhance remyelination in demyelinating disease such as multiple sclerosis.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

CRISPR-to-Kill (C2K)–Employing the Bacterial Immune System to Kill Cancer Cells

CRISPR/Cas9 was described as a bacterial immune system that uses targeted introduction of DNA double-strand breaks (DSBs) to destroy invaders. We hypothesized that we can analogously employ CRISPR/Cas9 nucleases to kill cancer cells by inducing maximal numbers of DSBs in their genome and thus triggering programmed cell death. To do so, we generated CRISPR-to-kill (C2K) lentiviral particles targeting highly repetitive Short Interspersed Nuclear Element-Alu sequences. Our Alu-specific sgRNA has more than 15,000 perfectly matched target sites within the human genome. C2K-Alu-vectors selectively killed human, but not murine cell lines. More importantly, they efficiently inhibited the growth of cancer cells including patient-derived glioblastoma cell lines resistant to high-dose irradiation. Our data provide proof-of-concept for the potential of C2K as a novel treatment strategy overcoming common resistance mechanisms. In combination with tumor-targeting approaches, the C2K system might therefore represent a promising tool for cancer gene therapy

Doping of casted silk fibroin membranes with extracellular vesicles for regenerative therapy: a proof of concept

Abstract Bioactive material concepts for targeted therapy have been an important research focus in regenerative medicine for years. The aim of this study was to investigate a proof-of-concept composite structure in the form of a membrane made of natural silk fibroin (SF) and extracellular vesicles (EVs) from gingival fibroblasts. EVs have multiple abilities to act on their target cell and can thus play crucial roles in both physiology and regeneration. This study used pH neutral, degradable SF-based membranes, which have excellent cell- and tissue-specific properties, as the carrier material. The characterization of the vesicles showed a size range between 120 and 180 nm and a high expression of the usual EV markers (e.g. CD9, CD63 and CD81), measured by nanoparticle tracking analysis (NTA) and single-EV flow analysis (IFCM). An initial integration of the EVs into the membrane was analyzed using scanning and transmission electron microscopy (SEM and TEM) and vesicles were successfully detected, even if they were not homogeneously distributed in the membrane. Using direct and indirect tests, the cytocompatibility of the membranes with and without EVs could be proven and showed significant differences compared to the toxic control (p  0.05)