Single-Cell Transcriptomics Uncovers Zonation of Function in the Mesenchyme during Liver Fibrosis.

Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.

Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity.

Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies.

TGFß-Responsive HMOX1 Expression Is Associated with Stemness and Invasion in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell-surface markers HMOX1, SLC16A1, CADM1, SCAMP3, and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non-GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N = 8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFß. siRNA-mediated silencing of HMOX1 impaired GBM invasion-a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18% ± 5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276-2289.

Neural stem cell differentiation is dictated by distinct actions of nuclear receptor corepressors and histone deacetylases.

Signaling factors including retinoic acid (RA) and thyroid hormone (T3) promote neuronal, oligodendrocyte, and astrocyte differentiation of cortical neural stem cells (NSCs). However, the functional specificity of transcriptional repressor checkpoints controlling these differentiation programs remains unclear. Here, we show by genome-wide analysis that histone deacetylase (HDAC)2 and HDAC3 show overlapping and distinct promoter occupancy at neuronal and oligodendrocyte-related genes in NSCs. The absence of HDAC3, but not HDAC2, initiated a neuronal differentiation pathway in NSCs. The ablation of the corepressor NCOR or HDAC2, in conjunction with T3 treatment, resulted in increased expression of oligodendrocyte genes, revealing a direct HDAC2-mediated repression of Sox8 and Sox10 expression. Interestingly, Sox10 was required also for maintaining the more differentiated state by repression of stem cell programming factors such as Sox2 and Sox9. Distinct and nonredundant actions of NCORs and HDACs are thus critical for control of lineage progression and differentiation programs in neural progenitors.

Novel alterations in the epigenetic signature of MeCP2-targeted promoters in lymphocytes of Rett syndrome patients.

Rett syndrome (RTT) is a neurodevelopmental disorder with neurological symptoms, such as motor disorders and mental retardation. In most cases, RTT is caused by mutations in the DNA binding protein MeCP2. In mice, MeCP2 gene deletion has been reported to result in genome-wide increased histone acetylation. Transcriptional regulation of neurotrophic factor BDNF and transcription factor DLX5, essential for proper neurogenesis, is further altered in MeCP2-deleted animals. We therefore investigated the chromatin environment of MeCP2 target genes BDNF and DLX5 in lymphocytes from RTT patients and human controls, and analyzed the density of histones H3, H2B and H1, as well as the levels of methylation and acetylation on selected lysines of histone H3. Notably, we found a general increase in the density of histone H3 in RTT patients' lymphocytes compared with controls, and decreased levels of trimethylation of lysine 4 on histone H3 (H3K4me3), a modification associated with transcriptional activation. The levels of acetylation of lysine 9 (H3K9ac) and 27 (H3K27ac) did not show any statistically significant changes when normalized to the decreased histone H3 levels; nevertheless, an average decrease in acetylation was noted. Our results reveal an unexpected alteration of the chromatin state of established MeCP2 target genes in lymphocytes of human subjects with RTT.

Red wine triggers cell death and thioredoxin reductase inhibition: effects beyond resveratrol and SIRT1.

Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5-3 microM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (> or =10 microM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1-5% v/v)--but not white wine--induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.