Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP^−/−Vim^−/− mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP^−/−Vim^−/− than in wild-type (WT) mice; GFAP^−/−, Vim^−/− and WT mice had the same infarct volume. Endothelin B receptor (ET_BR) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP^−/−Vim^−/− astrocytes. In WT astrocytes, ET_BR colocalized extensively with bundles of IFs. GFAP^−/−Vim^−/− astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP^−/−Vim^−/− than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET_BR-mediated control of gap junctions, and PAI-1 expression.

Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP^−/−Vim^−/−). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell–cell contact. GFAP^−/−Vim^−/− astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP^−/−Vim^−/− astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP^−/−Vim^−/− mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin. STEM Cells2012;30:2320–2329

The identification of breast cancer cell subpopulations featuring truly malignant stem cell qualities is a challenge due to the complexity of the disease and lack of general markers. By combining extensive single-cell gene expression profiling with three functional strategies for cancer stem cell enrichment including anchorage-independent culture, hypoxia, and analyses of low-proliferative, label-retaining cells derived from mammospheres, we identified distinct stem cell clusters in breast cancer. Estrogen receptor (ER)α+ tumors featured a clear hierarchical organization with switch-like and gradual transitions between different clusters, illustrating how breast cancer cells transfer between discrete differentiation states in a sequential manner. ERα- breast cancer showed less prominent clustering but shared a quiescent cancer stem cell pool with ERα+ cancer. The cellular organization model was supported by single-cell data from primary tumors. The findings allow us to understand the organization of breast cancers at the single-cell level, thereby permitting better identification and targeting of cancer stem cells.

Single-cell gene expression levels show substantial variations among cells in seemingly homogenous populations. Astrocytes perform many control and regulatory functions in the central nervous system. In contrast to neurons, we have limited knowledge about functional diversity of astrocytes and its molecular basis. To study astrocyte heterogeneity and stem/progenitor cell properties of astrocytes, we used single-cell gene expression profiling in primary mouse astrocytes and dissociated mouse neurosphere cells. The transcript number variability for astrocytes showed lognormal features and revealed that cells in primary cultures to a large extent co-express markers of astrocytes and neural stem/progenitor cells. We show how subpopulations of cells can be identified at single-cell level using unsupervised algorithms and that gene correlations can be used to identify differences in activity of important transcriptional pathways. We identified two subpopulations of astrocytes with distinct gene expression profiles. One had an expression profile very similar to that of neurosphere cells, whereas the other showed characteristics of activated astrocytes in vivo.

Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.