Establishment of an automated process based on FastLane technology for screening of chemically modified siRNAs

Optimizing the conditions for transfecting siRNA is a laborious and time-consuming process that involves testing many different parameters. The combination of FastLane technology and a Beckman Coulter robotic system enabled automated screening of transfection conditions, significantly shortening the time to establish protocols for automated “high-content” siRNA screening. This novel method is of great benefit to applications such as identification of potent chemically modified siRNAs for therapeutic purposes

Mass Cytometry of CSF Identifies an MS-Associated B-cell Population

OBJECTIVE To identify an MS-specific immune cell population by deep immune phenotyping and relate it to soluble signaling molecules in CSF. METHODS We analyzed surface expression of 22 markers in paired blood/CSF samples from 39 patients using mass cytometry (cytometry by time of flight). We also measured the concentrations of 296 signaling molecules in CSF using proximity extension assay. Results were analyzed using highly automated unsupervised algorithmic informatics. RESULTS Mass cytometry objectively identified a B-cell population characterized by the expression of CD49d, CD69, CD27, CXCR3, and human leukocyte antigen (HLA)-DR as clearly associated with MS. Concentrations of the B cell-related factors, notably FCRL2, were increased in MS CSF, especially in early stages of the disease. The B-cell trophic factor B cell activating factor (BAFF) was decreased in MS. Proteins involved in neural plasticity were also reduced in MS. CONCLUSION When analyzed without a priori assumptions, both the soluble and the cellular compartments of the CSF in MS were characterized by markers related to B cells, and the strongest candidate for an MS-specific cell type has a B-cell phenotype

Single-cell and bulk transcriptomics of the liver reveals potential targets of NASH with fibrosis.

Fibrosis is characterized by the excessive production of collagen and other extracellular matrix (ECM) components and represents a leading cause of morbidity and mortality worldwide. Previous studies of nonalcoholic steatohepatitis (NASH) with fibrosis were largely restricted to bulk transcriptome profiles. Thus, our understanding of this disease is limited by an incomplete characterization of liver cell types in general and hepatic stellate cells (HSCs) in particular, given that activated HSCs are the major hepatic fibrogenic cell population. To help fill this gap, we profiled 17,810 non-parenchymal cells derived from six healthy human livers. In conjunction with public single-cell data of fibrotic/cirrhotic human livers, these profiles enable the identification of potential intercellular signaling axes (e.g., ITGAV-LAMC1, TNFRSF11B-VWF and NOTCH2-DLL4) and master regulators (e.g., RUNX1 and CREB3L1) responsible for the activation of HSCs during fibrogenesis. Bulk RNA-seq data of NASH patient livers and rodent models for liver fibrosis of diverse etiologies allowed us to evaluate the translatability of candidate therapeutic targets for NASH-related fibrosis. We identified 61 liver fibrosis-associated genes (e.g., AEBP1, PRRX1 and LARP6) that may serve as a repertoire of translatable drug target candidates. Consistent with the above regulon results, gene regulatory network analysis allowed the identification of CREB3L1 as a master regulator of many of the 61 genes. Together, this study highlights potential cell-cell interactions and master regulators that underlie HSC activation and reveals genes that may represent prospective hallmark signatures for liver fibrosis

Immune cell landscaping reveals a protective role for Tregs during kidney injury and fibrosis

Acute kidney injury (AKI) and chronic kidney diseases (CKD) are associated with high mortality and morbidity in hospitalized patients. Although the underlying mechanisms determining the transition from acute to irreversible chronic injury are not well understood, immune mediated inflammatory processes are critical in renal injury. We have performed a comparison of two mouse models leading to either kidney regeneration or fibrosis, to better characterize the cellular and molecular events leading to both outcomes. Global gene expression profiling and histological analysis revealed a major upregulation of immune system related pathways during fibrosis. Further unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed that tissue resident macrophages and T cells were major altered populations. In fibrotic kidneys, there was a marked increase in tissue resident IL33R+ and IL2Ra+ regulatory T cells (Tregs). Indeed, prophylactic expansion of this population resulted in protection from kidney injury and fibrosis. Transcriptional profiling of Tregs showed a differential up-regulation of regenerative and pro-angiogenic set of genes in the regeneration model, whereas they expressed markers of hyper activation and fibrosis in the fibrosis model. This suggests that Tregs could exert different functions in the same tissue, dictated by environmental cues. Overall, we have provided a detailed cellular and molecular characterization of murine kidneys after injury and identified key changes in immune cell populations during fibrosis development

CellSIUS provides sensitive and specific detection of rare cell populations from complex single cell RNA-seq data

We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease

Genome-wide screening in human kidney organoids identifies novel aspects of nephrogenesis

Human organoids allow studying proliferation, lineage specification, and three-dimensional tissue development. Here, we present the first genome-wide CRISPR screen in iPSC-derived kidney organoids. The combination of inducible genome editing, longitudinal sampling and endpoint sorting of tubular and stromal cells generated a complex, high quality dataset uncovering a broad spectrum of novel biology from early development to ‘adult’ epithelial morphogenesis. Our functional dataset allows improving mesoderm induction by ROCK inhibition, contains monogenetic and complex trait kidney disease genes, confirms two novel CAKUT genes (CCDC170 and MYH7B), and provides a large candidate list of ciliopathy-related genes. Finally, the identification of a cis-inhibitory effect of Jagged1 controlling epithelial proliferation shows how mosaic knockouts in pooled CRISPR screening can discover novel ways of communication between heterogeneous cell populations in complex tissues. Collectively, these data serve both as a rich resource for the kidney community and as a benchmark for future iPSC-derived organoid CRISPR screens

Immune cell landscaping reveals a protective role for regulatory T cells during kidney injury and fibrosis

Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis

Sustained Trem2 stabilization accelerates microglia heterogeneity and Aβ pathology in a mouse model of Alzheimer’s disease

TREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer’s disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. Here, we generate a transgenic mouse model of reduced Trem2 shedding (Trem2-Ile-Pro-Asp [IPD]) through amino-acid substitution of an ADAM-protease recognition site. We show that Trem2-IPD mice display increased Trem2 cell-surface-receptor load, survival, and function in myeloid cells. Using single-cell transcriptomic profiling of mouse cortex, we show that sustained Trem2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aβ pathology in a mouse model of Alzheimer’s disease. Our data indicate that reduction of Trem2 proteolytic cleavage aggravates neuroinflammation during the course of Alzheimer’s disease pathology, suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states

Single-cell and bulk transcriptomics of the liver reveals potential targets of NASH with fibrosis

Fibrosis is characterized by the excessive production of collagen and other extracellular matrix (ECM) components and represents a leading cause of morbidity and mortality worldwide. Previous studies of nonalcoholic steatohepatitis (NASH) with fibrosis were largely restricted to bulk transcriptome profiles. Thus, our understanding of this disease is limited by an incomplete characterization of liver cell types in general and hepatic stellate cells (HSCs) in particular, given that activated HSCs are the major fibrogenic population during liver fibrosis development. To help fill this gap, we profiled 17,810 non-parenchymal cells derived from six healthy human liver tissues. In conjunction with public single-cell data of fibrotic/cirrhotic human liver, it enables the assessment and identification of potential intercellular communications (e.g., ITGAV–LAMC1, TNFRSF11B–VWF and NOTCH2–DLL4 signaling axes) and regulons (e.g., RUNX1 and CREB3L1) responsible for the activation of HSCs during fibrogenesis. Bulk RNA-seq data of NASH patient livers and rodent models for liver fibrosis of diverse etiologies allowed us to evaluate the translatability of candidate therapeutic targets for NASH with fibrosis. We identified 61 liver fibrosis-associated genes (e.g., AEBP1, PRRX1 and LARP6) that may serve as a repertoire of translatable drug target candidates. Consistent with the above regulon results, gene regulatory network analysis allowed the identification of CREB3L1 as a master regulator of many of the 61 genes. Together, this study sheds light on potential cell-cell interactions and regulons that underlie HSC activation and reveals genes that may represent prospective hallmark signatures for liver fibrosis

Systematic dissection of transcriptional regulatory networks by genome-scale and singlecell CRISPR screens

No Abstrac