Stefan Wild

Absolute quantification of microRNAs by using a universal reference

MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(-) hematopoietic progenitor cells.

Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells.

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor‐matched CD133+ cells with the more differentiated CD34+CD133− and CD34−CD133− cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing‐based profiling, we found that between 109 (CD133+) and 216 (CD34−CD133−) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR‐142‐3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34+CD133− cells. Eighteen miRNAs were significantly differentially expressed between CD133+ and CD34+CD133− cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase‐based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR‐142‐3p as well as tropomyosin 1 and frizzled homolog 5 by miR‐29a. Overexpression of miRNAs in CD133+ cells demonstrated that miR‐142‐3p has a negative influence on the overall colony‐forming ability. In conclusion, the miRNAs expressed differentially between the CD133+ and CD34+CD133− cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell‐based therapies with CD133+ cells and delineate for the first time how the stem cell character of CD133+ cells is defined by the expression of specific miRNAs. STEM CELLS 2011;29:847–857

Overlapping gene coexpression patterns in human medullary thymic epithelial cells generate self-antigen diversity

Significance The ability of the immune system to distinguish self from foreign (“self-tolerance”) is largely established in the thymus, a primary lymphoid organ where T cells develop. Intriguingly, T cells encounter most tissue-specific constituents already in the thymus, thus imposing a broad scope of tolerance before T cells circulate through the body. This preemption of the “immunological self” is afforded by the “promiscuous” expression of numerous tissue-specific antigens in medullary thymic epithelial cells. Here, we identified principles by which promiscuous gene expression at the single-cell level adds up to the full diversity of self-antigens displayed at the population level. Promiscuous expression of numerous tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential to safeguard self-tolerance. A distinct feature of promiscuous gene expression is its mosaic pattern (i.e., at a given time, each self-antigen is expressed only in 1–3% of mTECs). How this mosaic pattern is generated at the single-cell level is currently not understood. Here, we show that subsets of human mTECs expressing a particular TRA coexpress distinct sets of genes. We identified three coexpression groups comprising overlapping and complementary gene sets, which preferentially mapped to certain chromosomes and intrachromosomal gene clusters. Coexpressed gene loci tended to colocalize to the same nuclear subdomain. The TRA subsets aligned along progressive differentiation stages within the mature mTEC subset and, in vitro, interconverted along this sequence. Our data suggest that single mTECs shift through distinct gene pools, thus scanning a sizeable fraction of the overall repertoire of promiscuously expressed self-antigens. These findings have implications for the temporal and spatial (re)presentation of self-antigens in the medulla in the context of tolerance induction.

Highly sensitive profiling of CD44+/CD24− breast cancer stem cells by combining global mRNA amplification and next generation sequencing: Evidence for a hyperactive PI3K pathway

We performed next generation sequencing- and microarray-based gene expression profiling of CD44(+)/CD24(-)/CD45(-) breast CSCs (cancer stem cells) isolated from primary ERα-positive breast cancer. By combining semi-automated dissociation of human tumor tissue, magnetic cell sorting and cDNA amplification less than 500 CSCs were required for transcriptome analyses. Besides overexpressing genes involved in maintenance of stemness, the CSCs showed higher levels of genes that drive the PI3K pathway, including EGFR, HB-EGF, PDGFRA/B, PDGF, MET, PIK3CA, PIK3R1 and PIK3R2. This suggests that, in CSCs of ERα-positive breast cancer, the PI3K pathway which is involved in endocrine resistance is hyperactive.

HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection

Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvβ3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c+ CD14+ cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity.

Dynamic expression of the pro-dopaminergic transcription factors Pax6 and Dlx2 during postnatal olfactory bulb neurogenesis

Olfactory bulb (OB) neurogenesis generates neurons that use GABA or dopamine as their neurotransmitters throughout life. Regionalized stem cell populations in the periventricular zone (PVZ) of the lateral ventricles (LVs) have been shown to be at the basis of neuronal diversity in the system. For example dopaminergic neurons arise predominantly from neural stem cells (NSCs) residing in the dorsal PVZ and depend on the expression of the transcription factors Pax6 and Dlx2 for their specification. In addition, Dlx2 is required for neurogenesis in general. Using targeted in vivo electroporation combined with immuno-fluorescence imaging and microarray analysis, we provide here detailed spatial and temporal expression data with cellular resolution in this system. We find that all along the neurogenic process Pax6 expression remains restricted to the dorsal PVZ, whereas nearly all neuroblasts express Dlx2, including those of the dorsal lineage, which are switched on for Dlx2 when they enter the rostral migratory stream (RMS). These data allow to explain and precise the functions of these two genes in postnatal OB neurogenesis.

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.

Melanoma Affects the Composition of Blood Cell-Derived Extracellular Vesicles

Extracellular vesicles (EVs) are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of EVs reflects the type and status of the originating cell and EVs in melanoma patient’s plasma could be indicative for the tumor. Likewise, EVs might influence tumor progression by regulating immune responses. We performed a broad protein characterization of EVs from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer (NK) cells, monocytes, monocyte-derived dendritic cells (moDCs), and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on EVs. Hierarchical clustering of protein intensity patterns grouped EVs according to their originating cell type. The analysis of EVs from stimulated B cells and moDCs revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of EVs from platelets, antigen-presenting cells and NK cells as shown by platelet markers, co-stimulatory proteins, and a NK cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers, indicating a changed vesicle secretion or protein loading of EVs by platelets and a lower CD8 signal that might be associated with a diminished activity of NK cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of EVs in melanoma plasma, but rather argue for an indirect influence of melanoma cells on the vesicle secretion or vesicle protein loading by blood cells.

Zic-proteins are repressors of dopaminergic forebrain fate in mice and C. elegans

In the postnatal forebrain regionalized neural stem cells along the ventricular walls produce olfactory bulb (OB) interneurons with varying neurotransmitter phenotypes and positions. To understand the molecular basis of this region-specific variability we analyzed gene expression in the postnatal dorsal and lateral lineages in mice of both sexes from stem cells to neurons. We show that both lineages maintain transcription factor signatures of their embryonic site of origin, the pallium and subpallium. However, additional factors, including Zic1 and Zic2, are postnatally expressed in the dorsal stem cell compartment and maintained in the lineage that generates calretinin-positive GABAergic neurons for the OB. Functionally, we show that Zic1 and Zic2 induce the generation of calretinin-positive neurons while suppressing dopaminergic fate in the postnatal dorsal lineage. We investigated the evolutionary conservation of the dopaminergic repressor function of Zic proteins and show that it is already present in C. elegans. SIGNIFICANCE STATEMENT The vertebrate brain generates thousands of different neuron types. In this work we investigate the molecular mechanisms underlying this variability. Using a genomics approach we identify the transcription factor signatures of defined neural stem cells and neuron populations. Based thereon we show that two related transcription factors, Zic1 and Zic2, are essential to control the balance between two defined neuron types in the postnatal brain. We show that this mechanism is conserved in evolutionary very distant species.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo. Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. Summary: mRNA electroporation is a powerful new method for functional studies in neonatal and adult neural stem cells as well as postmitotic neurons.