Journal: NAR Genomics and Bioinformatics

Article Title: stPipe: a flexible and streamlined R/Bioconductor pipeline for preprocessing sequencing-based spatial transcriptomics data

doi: 10.1093/nargab/lqaf167

Figure Lengend Snippet: Interactive R-shiny web app created by the Run_Interactive function and visualization of QC metrics created by the Run_Visualization function for data quality assessment. ( a ) Visualization of Slide-seq mouse brain sample Puck_200115_08. The Run_Interactive function offers flexible options for selecting a ROI through four intuitive buttons. The ‘Add Selection’ button allows users to add spatial coordinates along with corresponding metadata, such as UMI count and spatial barcode sequences, each time an ROI is selected. The ‘Clear Last Selection’ button removes the most recently selected ROI from the current selection list. The ‘Reset All Selections’ button resets both the spatial heatmap and clustering plot, providing a clean slate for a new selection. Finally, the ‘Save All Selected ROI’ button saves the finalized selection as ‘selected_ROI’ object in the user’s R global environment, streamlining data management and export. In this example, the selection of cluster 7, highlighted in purple on the t-SNE plot, is found to mostly correspond to the choroid plexus region in the spatial UMI count plot. ( b ) Barplot showing spatial barcode demultiplexing information between 10× Visium probe-based (left) and polyA-based (right) protocols to assess sequencing accuracy. ( c ) Stacked bar plots showing the mapping rate, separated into reads that map to exons, introns, and those that are ambiguously mapped or map elsewhere in the genome (ordered by exon mapping rate) between 10× Visium probe-based (left) and polyA-based (right) protocols. ( d ) UMI duplication plot between a probe-based sample (left) with a higher UMI duplication number than a polyA-based one (right). A distribution skewed toward lower duplication values indicates higher library complexity and minimal redundancy, suggesting that the sequencing depth is well-matched to the diversity of the transcriptome. In contrast, a pronounced tail toward higher duplication values suggests substantial over-sequencing or PCR amplification biases, as many reads may originate from the same underlying transcript molecule. (e) UMI count distribution between sample 709 with two protocols, the first and last two are plotted as distribution of raw UMI count per spot and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\log _{10}$\end{document} UMI count per gene respectively.

Article Snippet: Spatial transcriptomics technology has developed rapidly in recent years, with various sequencing-based platforms such as 10× Visium, Slide-seq, and Stereo-seq becoming widely used by researchers.

Techniques: Selection, Sequencing, Amplification

Journal: Advanced Science

Article Title: Single Cell and Spatial Transcriptomics Define a Proinflammatory and Profibrotic Niche After Kidney Injury

doi: 10.1002/advs.202503691

Figure Lengend Snippet: Single‐cell and spatial transcriptome landscape of healthy and fibrotic kidneys after unilateral ischemia‐reperfusion injury (UIRI). a) Schematic representation of single‐cell RNA sequencing (scRNA‐seq) and spatial transcriptomics (ST) of kidneys from the sham and 10‐day UIRI mice, graphically designed with Biorender ( https://www.biorender.com/ ). b) t‐SNE plot illustrating the intricate cellular diversity in fibrotic kidneys, demonstrating distinct clusters representing glomerular endothelial cells (GEC), podocytes (Podo), mesangial cells (Mesa), Bowman's capsule epithelium (BC), proximal tubules (PT), descending limbs of Henle (DLOH), ascending limbs of Henle (ALOH), distal tubules (DT), principal cells (PC), intercalated cells (IC), fibroblasts (Fib), smooth muscle cells (SMC), extraglomerular endothelial cells (EGEC), monocytes (Mono), dendritic cells (DC), macrophages (Mϕ), plasmacytoid dendritic cells (pDC), proliferating mononuclear lineage (Prolif mono_L), and neutrophils (Neu), B cells (B), T cells (T), proliferating T cells (prolif T), and natural killer cells (NK). These cell types were further categorized into four major compartments: Glomerular, Renal, Interstitium, and Immune, as indicated by color grouping in the plot. c) Bubble plot illustrating the relative proportions of major kidney cell types in sham and UIRI samples. Each dot represents the proportion of a given cell type in a specific sample group, with dot size corresponding to its relative proportion. d) A comprehensive heatmap depicting the unique marker gene signature of major renal cell types. e) UMAP plot illustrating the inferred renal cell region distribution based on integrated spatial transcriptomics data from normal (Sham) and UIRI 10D mouse kidneys, generated using the 10x Genomics Visium platform. The identified regions include glomerular cells (Glom), distinct segments of the proximal tubule (PTS1, PTS1S2, PTS2), injured proximal tubules (InjPT), ascending limbs of Henle in cortex (ALOH(C)), distal tubules (DT), connecting tubules and collecting ducts (CNT_CD), cells at the corticomedullary junction (CMJ), fibrogenic niche regions (Niche1, Niche2), the inner stripe of the outer medulla (IOM), inner medulla (IM), renal capsule (RC), and perirenal tissue (Perirenal). f) Spatial maps illustrating the anatomical distribution of renal cell regions in Sham and UIRI 10D mouse kidneys. Region colors correspond to the classifications defined in panel (e). g) Bubble plot illustrating the relative proportions of major renal cell regions in spatial transcriptomics data from sham and UIRI 10D mouse kidneys. h) Bubble plot depicting the expression patterns of marker genes across distinct renal cell regions in spatial transcriptomics data. Dot color indicates the average gene expression level within each region, while dot size represents the proportion of spatial spots expressing the gene. i) Schematic diagram of nephron segmentation by cell types. j) Comparison of kidney anatomical regions and spatial transcriptomic clusters, showing clusters in kidney tissue (top) and the corresponding Visium H&E‐stained section (bottom). k) Renal tissue structure alterations at the corticomedullary junction (CMJ) in UIRI samples, showing the formation of two distinct fibrogenic niches, Niche1 and Niche2. l) A heatmap showing the deconvolution scores of cell type compositions across different regions in Visium spatial transcriptomics data, obtained using the RCTD method. m) Spatial FeaturePlots of RCTD‐derived cell type scores in the sham (top) and UIRI (bottom) groups, with paired panels sharing a common legend.

Article Snippet: For the preparation of sections for Visium Spatial Transcriptomics sequencing, samples were equilibrated at −18 °C and a 10 μm thick section was cut onto the active sequencing area (6 mm x 6 mm) of a spatial barcoded slide.

Techniques: RNA Sequencing, Marker, Generated, Expressing, Gene Expression, Comparison, Staining, Derivative Assay

Journal: Advanced Science

Article Title: Single Cell and Spatial Transcriptomics Define a Proinflammatory and Profibrotic Niche After Kidney Injury

doi: 10.1002/advs.202503691

Figure Lengend Snippet: High‐resolution spatial transcriptomics and immunostaining reveal the TNC‐enriched fibroblast‐macrophage niche organization in fibrotic kidneys. a) Schematic diagram of the Visium HD workflow applied to kidney tissues from sham and UIRI model mice. b) UMAP visualization of integrated Visium HD spatial transcriptomics data from control mice (obtained from the 10x Genomics public dataset) and UIRI mice (this study), processed using canonical correlation analysis (CCA). This dimensionality reduction visualization reveals distinct clusters representing various renal parenchymal and stromal cell populations, including: Glomerulus, Vasculature, PTS1, PTS2, PTS1S2, InjPT, ascending limbs of Henle in cortex [ALOH(Cortex)], distal tubule and connecting tubule (DT_CNT), connecting tubule and collecting duct (CNT_CD), collecting duct in cortex [CD(Cortex)], PTS3, injured PTS3 (InjPTS3), Fibrogenic Niche, Vasa recta, loop of Henle in outer medulla [LOH(IOM)], collecting duct in outer medulla [CD(IOM)], collecting duct in inner medulla [CD(IM)], thin ascending limbs of Henle in inner medulla [tALOH(IM)], renal capsule (RC), Perirenal Fibrous tissue, and Perirenal Adipose tissue. c) Bubble plot comparing the regional distribution in Control versus UIRI 10d kidneys (Visium HD). d) Bubble plot depicting the expression patterns of marker genes across distinct renal cell regions in Visium HD data. e) Spatial maps generated using Visium HD illustrate the inferred anatomical distribution of renal cell regions in kidney tissues from Control and UIRI mice. f) Spatial Feature Plots of Visium HD data showing the spatial distribution of selected renal cell types in controls (top) and UIRI mice (bottom), based on cell‐type deconvolution using RCTD. g) A heatmap showing the correlation between NMF factors and cell‐type deconvolution scores in standard Visium spatial transcriptomics data. h) Spatial distribution of gene scores associated with the NMF factors most correlated with the fibrogenic niche, along with the contribution of key genes to each factor. i) Spatial FeaturePlots showing the anatomical distribution of Tnc expression in standard Visium. j) A heatmap showing the correlation between NMF factors and cell type deconvolution scores in Visium HD spatial transcriptomics data. k) Spatial distribution of NMF factors (NMF3 and NMF11) associated with the fibrogenic niche in Visium HD data, along with their corresponding high‐contributing genes. l) Spatial FeaturePlots showing the anatomical distribution of Tnc expression in Visium HD datasets. m) Immunofluorescence staining demonstrates colocalization of TNC with macrophages (F4/80⁺) in the CMJ interstitial region. From top to bottom: an overview merged image (Merge), followed by magnified views of TNC, Vimentin, and F4/80 staining in the same region, and an enlarged merged image (Enlarged Merge) at the bottom.

Article Snippet: For the preparation of sections for Visium Spatial Transcriptomics sequencing, samples were equilibrated at −18 °C and a 10 μm thick section was cut onto the active sequencing area (6 mm x 6 mm) of a spatial barcoded slide.

Techniques: Immunostaining, Control, Expressing, Marker, Generated, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Single Cell and Spatial Transcriptomics Define a Proinflammatory and Profibrotic Niche After Kidney Injury

doi: 10.1002/advs.202503691

Figure Lengend Snippet: TLR4 knockout in macrophages attenuates renal inflammation and renal fibrosis in vivo. a) The diagram shows the experimental protocol. Bone marrow chimera models were established by transplanting the WT bone marrow to WT mice, or TLR4 KO bone marrow to WT mice. Mice were irradiated at a single dose of 1100 Rads and then underwent bone marrow transplantation. After 8 weeks of successful transplantation, a unilateral ischemia‐reperfusion (UIRI) model was established. b) PCR‐based identification of kidney genotypes in the recipient mice of bone marrow transplantation models using TLR4 mutation site primers and wild‐type site primers, respectively. c,d) Graphic presentations show serum creatinine (Scr) (c) and blood urea nitrogen (BUN) (d) levels in different groups as indicated at 11 days after IRI. * p < 0.05 versus WT‐WT (n = 4–6). e,f) Western blot analyses show renal expression of TLR4, p‐P65, and P65 in different groups as indicated. Representative Western blot (e) and quantitative data (f) are shown. * p < 0.05 versus WT‐WT (n = 4–6). g) Representative micrographs show renal expression and co‐localization of TLR4 and F4/80 by immunofluorescence staining in different groups as indicated. The areas between the dashed lines represent the corticomedullary junction of the kidney. h,i) Western blot analyses show renal expression of MR, Arg‐1, iNOS, TNF‐α, and CCL2 in different groups as indicated. Representative Western blot (h) and quantitative data (i) are shown. * p < 0.05 versus WT‐WT (n = 4–6). j,k) Western blot analyses show renal expression of TNC, FN, and α‐SMA in different groups as indicated. Representative Western blot (j) and quantitative data (k) are shown. * p < 0.05 versus WT‐WT (n = 4–6). l) A schematic diagram shows a crucial role of TNC in organizing the proinflammatory and profibrotic niche. By integrating single‐cell RNA sequencing and spatial transcriptomics, we unveil TNC as a central organizer of the proinflammatory and profibrotic niche in kidney fibrosis. TNC promotes macrophage activation through TLR4/NF‐κB signaling, leading to macrophage activation, proliferation, and cytokine production.

Article Snippet: For the preparation of sections for Visium Spatial Transcriptomics sequencing, samples were equilibrated at −18 °C and a 10 μm thick section was cut onto the active sequencing area (6 mm x 6 mm) of a spatial barcoded slide.

Techniques: Knock-Out, In Vivo, Irradiation, Transplantation Assay, Mutagenesis, Western Blot, Expressing, Immunofluorescence, Staining, RNA Sequencing, Activation Assay

Journal: eLife

Article Title: Chronic hyperactivation of midbrain dopamine neurons causes preferential dopamine neuron degeneration

doi: 10.7554/eLife.98775

Figure Lengend Snippet: ( A ) DAT IRES cre animals that received clozapine- N -oxide (CNO) (CNO alone, n=2 mice) or were injected with AAV-hM3Dq-mCherry and received vehicle (GqVeh, n=3 mice) or CNO (GqCNO, n=3 mice) were treated for 1 week before brains were flash-frozen for spatial transcriptomic analysis. ( B ) Image of midbrain and striatal sections stained with TH (green), NeuN (purple), and DAPI (blue) shows discs assigned to regions of interest. Inset shows a disc containing two TH+ cell bodies. ( C ) Expression of dopaminergic and striatal genes is confined to expected spatial regions. Expression of genes involved in DA metabolism decreases with chronic CNO. 2–49 discs were compiled per ventral tegmental area (VTA), and 1–7 discs were compiled per SN. 357–560 capture areas were compiled per CP. The thalamus was selected as a midbrain control region, while white matter tracts (white) and the lateral septal complex (LSX) were used as striatal controls. ( D ) Principal components analysis of midbrain regions (top) and the caudate putamen (bottom) for GqCNO, GqVeh, and CNO alone groups. ( E ) The deep learning NEUROeSTIMator model was used to predict neural activity of GqCNO, GqVEH, and CNO alone within the SN and VTA from Visium spatial transcriptomics. Groups were compared using the Kolmogorov-Smirnov (KS) test (see Methods). ( F ) Hits were used for Enrichr pathway analysis if significant in both GqCNO vs CNO alone and GqCNO vs GqVeh comparisons. Gene rankings for hit analysis were established using fold change score (FCS) and signal-to-noise score (SNS). ( G ) Volcano plots comparing GqCNO vs GqVeh in the SN, VTA, and CP. Genes highlighted are also significantly altered when comparing GqCNO vs CNO alone. Scale bars indicate 500 µm. Data indicate mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA followed by Holm-Sidak post hoc test.

Article Snippet: Spatial transcriptomics were acquired with Visium spatial gene expression kits (10x Genomics).

Techniques: Injection, Staining, Expressing, Control, Activity Assay

Journal: Communications Biology

Article Title: Single-cell sequencing uncovers disrupted stromal-macrophage communication as a driver of intrauterine adhesion progression

doi: 10.1038/s42003-025-08634-3

Figure Lengend Snippet: a Haematoxylin and eosin staining of the slides in the Visium arrays from Alonso et al. Two individuals were selected: proliferative phase A13 and secretory phase A30. b , c Estimated amount of mRNA (color intensity) contributed by each stromal cell population ( b ), macrophage subsets ( c ) to each spot (color) shown over the H&E image of proliferative (A13, 152810 slide and 152806 slide) and secretory (A30, 152811 slide and152807 slide) endometrium.

Article Snippet: Fig. 9 Spatial transcriptomics reveals regional distribution of stromal and macrophage subpopulations in human endometrium. a Haematoxylin and eosin staining of the slides in the Visium arrays from Alonso et al. Two individuals were selected: proliferative phase A13 and secretory phase A30. b , c Estimated amount of mRNA (color intensity) contributed by each stromal cell population ( b ), macrophage subsets ( c ) to each spot (color) shown over the H&E image of proliferative (A13, 152810 slide and 152806 slide) and secretory (A30, 152811 slide and152807 slide) endometrium.

Techniques: Staining