metacell 2d projection  (MetaCell Inc)

Journal: Nature

Article Title: Tuft cells act as regenerative stem cells in the human intestine

doi: 10.1038/s41586-024-07952-6

Figure Lengend Snippet: a , Log-normalized expression of KIT , projected on a Metacell 2D representation of scRNA-seq data of primary human adult small intestine . n = 15,184 single epithelial cells. b , Co-staining of KIT and Phalloidin in human colon tissue. Two independent experiments were performed on one donor with similar results. c - d , Representative image ( c ) and quantification ( d ) of KIT staining in AVIL-Clover organoids. n = 303 cells of 17 organoids from 2 donors. e , Representative flow cytometry (left) and quantification (right) of KIT (phycoerythrin, PE) labeled AVIL-Clover organoids differentiated in tuft cell medium with IL-4/IL-13. Each dot is one well, n = 24 wells pooled from 3 donors (Supplementary Fig. ). f - g , Representative flow cytometry ( f ) and quantification ( g ) of the KIT + and AVIL + cells in AVIL-Clover organoids triggered with the depicted cytokines for 48 h, followed by 6 days culturing in tuft cell differentiation medium with IL-4/IL-13. Each dot is a well. n = 2 wells per condition. Results are representative of 2 donors (Supplementary Fig. ). h , KIT expression across epithelial cell types in healthy, IBD non-inflamed, and IBD inflamed human colon tissue . Dot color relates to mean expression values and dot size to fraction of expressing cells. n = 4,428 cells. i , Gating strategy of sorting KIT + cells from human primary intestine tissue. Adult colon is shown. j , Distribution of tuft cell gene expression signature across different epithelial populations from human adult intestine tissue , as well as within sorted populations of KIT + cells from primary adult ileum and colon. Horizontal line indicates an optimal separation based on the unenriched intestine dataset. Percentage of classified tuft cells in KIT + populations is indicated. The tuft cell signature is based on 222 core tuft cell genes shown in Fig. . n = 311 ileal KIT + cells, and 271 colon KIT + cells. k , Representative flow cytometric analysis (left) and quantification (right) of the S/G2/M phase in KIT + cells in FUCCI reporter organoids differentiated for 3 days in the indicated media. Each dot is one well. n = 8 (Diff) or 6 (Diff+IL-4/13) wells from 6 or 4 independent experiments. l - m , Snapshots of dividing AVIL + cells by live-cell imaging in AVIL-Clover reporter organoids differentiated in tuft cell medium. l , Each dividing AVIL + cell and its progeny are colored, 3 independent experiments were performed on 2 donors with similar results (Supplementary Video ). m , Shown one tuft cell dividing twice within 24 h, 2 donors were examined with similar results (Supplementary Videos - ). n , Experimental design (left) and quantification (right) of the KI67 + AVIL + cells percentage at indicated time points., n = 800 (day 0), 631 (day 4, Base), 776 (day 4, Base+IL-4/13), 806 (day 7, Base+IL-4/13) AVIL + cells. Results are pooled from 3 independent experiments on two organoid lines (Supplementary Fig. ). o , Representative flow cytometric analysis (left) and relative quantification (right) of KIT + cells differentiated for 3 days in different regimens. Each dot is one well. n = 3 wells per condition, pooled from two experiments on FUCCI line (Supplementary Fig. ). p , Representative flow cytometric analysis (left) and quantification (right) of AVIL + cell frequency in organoids differentiated for 2 days in EGF Noggin medium (without Wnt, R-spondin1 and DAPT), followed by 4 days culturing in indicated media. Each dot is a well, n = 7 wells per condition, pooled from 3 independent experiments on two lines from one donor. q , Experimental design (top) and qPCR quantification (bottom) of tuft cell and stem cell genes in sorted single cells from organoids cultured in human expansion medium. Each dot is a donor, n = 2 donors. b , c , l , m , Scale bar, 50 µm. d , e , g , k , n - q , Data are presented as mean values +/− standard error ( d , e , k , n - p ) or as mean values ( g , q ). k , n , o , P values are derived from two-tailed t-test. TA cells: Transit-Amplifying Cells; EEC: Enteroendocrine cells; Diff: tuft cell differentiation medium.

Article Snippet: Fig. 3 Single tuft cells give rise to organoids containing all epithelial lineages. a , Organoid outgrowth of single S/G2/M-phase KIT − and KIT + cells sorted from ileum FUCCI reporter organoids, cultured in standard human intestinal expansion medium. n = 3 independent experiments. b , c , MetaCell 2D projection of 10,311 single cells isolated from passage 1 KIT − or KIT + cell-derived organoids, differentiated with or without IL-4/IL-13.

Techniques: Expressing, Staining, Flow Cytometry, Labeling, Cell Differentiation, Gene Expression, Live Cell Imaging, Quantitative Proteomics, Cell Culture, Derivative Assay, Two Tailed Test

Journal: Nature

Article Title: Tuft cells act as regenerative stem cells in the human intestine

doi: 10.1038/s41586-024-07952-6

Figure Lengend Snippet: a , Schematic overview of the scRNA-seq experiment. b , Tuft cell frequency in different medium and gating combinations, as determined by scRNA-seq analysis. c , scRNA-seq statistics, depicting number of reads per cell (top) and number of unique molecular identifiers (UMI) per cell (bottom). Each dot is a cell, colored by its gating and medium conditions. Quality threshold of 500 UMI per cell is indicated by a horizontal line. d , Gene expression profiles of stem cell and goblet cells. n = 373 single non-tuft cells. e , Expression of tuft cell marker genes projected on the metacell 2D layout as in Fig. . Dot color indicate log normalized expression. n = 953 single cells. f , Summary of number of up-regulated differentially expressed genes (DEG) across the tuft-1-4 states, when compared to non-tuft epithelium. g , Top 10 Gene ontology terms enriched in DEG of tuft-1-4, when compared to the non-tuft cells as in ( f ). Values represent -log 10 P value. h , Staining of tuft-1-4 specific markers (red, as indicated) in human ileum AVIL-reporter organoids cultured in tuft cell differentiation medium (tuft 1-2) or supplemented with IL-4/IL-13 (tuft 3-4). n = 2 independent experiments on one donor with similar results. Scale bar, 5 µm. i , Gene expression profiles of primary KIT + tuft cells from human ileum and colon tissue. Shown are tuft-3 genes, as well as KIT , POU2F3 and AVIL . Cells are colored by their tissue origin, and by classification into cycling cells (cc) or non-cycling cells (no-cc). n = 271 single colon and 311 single ileum KIT + cells. j , Differential expression of all tuft-3 genes between 311 primary ileal cycling and non-cycling KIT + cells as in i . Genes with significant differential expression (c2 test; FDR-adjusted P value < 10 −3 ) are colored. k , Estimation of the fraction of proliferating tuft cells across human primary intestine tissues from two published scRNA-seq datasets , . Each dot is a donor, n = 1,240 single tuft cells from 14 human donor samples. Data are presented as mean values +/− standard error. l , Gene expression profiles of 541 primary KIT + non-cycling (no-cc) tuft cells as in i . Shown are tuft-4 genes, as well as KIT , POU2F3 and AVIL . Cells are ordered by their expression of the aggregated tuft-4 program (top panel), and colored by their tissue origin. m , Expression patterns of six genes along the tuft-4 activation gradient in 213 KIT + no-cc ileal cells. Shadings indicate 95% confidence in binomial estimation of the mean. Data was down-sampled to 1,000 UMI per cell. n , Aggregate expression of the tuft-4 program in cycling and non-cycling cells in colon and ileum. n = 582 single cells. o , Aggregate expression of the tuft-4 program across tuft cell substates in organoids as in Fig. . n = 573 single tuft cells from organoids. n - o , Boxplots show data from the 25th–75th percentile and whiskers extending to the minimum and maximum within 1.5 × inter-quartile range, with dots marking outliers. P values are derived from two tailed Mann-Whitney test. Diff: tuft cell differentiation medium; cc: cell cycle. Schematic in a was created using BioRender (J. van Es BioRender.com/k22v672 ; 2024).

Article Snippet: Fig. 3 Single tuft cells give rise to organoids containing all epithelial lineages. a , Organoid outgrowth of single S/G2/M-phase KIT − and KIT + cells sorted from ileum FUCCI reporter organoids, cultured in standard human intestinal expansion medium. n = 3 independent experiments. b , c , MetaCell 2D projection of 10,311 single cells isolated from passage 1 KIT − or KIT + cell-derived organoids, differentiated with or without IL-4/IL-13.

Techniques: Gene Expression, Expressing, Marker, Staining, Cell Culture, Cell Differentiation, Quantitative Proteomics, Activation Assay, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Tuft cells act as regenerative stem cells in the human intestine

doi: 10.1038/s41586-024-07952-6

Figure Lengend Snippet: a , b , MetaCell 2D projection of 953 ileum organoid-derived single cells. Cells are coloured by their gating and medium condition ( a ) or by annotation to cell subsets ( b ). c , Gene-expression profiles of tuft cell-specific core genes shared by all tuft cell clusters across epithelial subsets. a – c , n = 953 single cells. d , Distribution of the four tuft cell states in ileum-derived organoids with or without IL-4/IL-13. e , Gene-expression profiles of tuft cell state-specific genes. d , e , n = 573 single tuft cells. f , Flow cytometry quantification of AVIL + cell frequency in organoid mutant lines of selected transcription factors (homozygous knock-outs). Organoids were differentiated for 11 days (or 6 days with IL-4/IL-13). Each dot is one well. n = 11 (WT Diff), 6 ( SOX9 −/− Diff), 4 ( SPIB −/− / HOXB8 −/− / POU2F3 −/− Diff + IL-4/13) and 3 (rest) wells. Results are representative of three independent experiments (Supplementary Fig. ). P values are derived from FDR-adjusted two-tailed Student’s t -test against the WT levels. g , h , Representative fluorescence image ( g ) and quantification ( h ) of AVIL-Clover organoids costained for POU2F3. g , n = 3 donors (Supplementary Fig. and Supplementary Videos – ). Scale bars, 40 µm. h , n = 993 positive cells from 17 organoids pooled from three donors. d , f , h , Data are presented as binomial estimation of the mean ± 95% confidence intervals ( d ) or as mean values ± standard error ( f , h ).

Article Snippet: Fig. 3 Single tuft cells give rise to organoids containing all epithelial lineages. a , Organoid outgrowth of single S/G2/M-phase KIT − and KIT + cells sorted from ileum FUCCI reporter organoids, cultured in standard human intestinal expansion medium. n = 3 independent experiments. b , c , MetaCell 2D projection of 10,311 single cells isolated from passage 1 KIT − or KIT + cell-derived organoids, differentiated with or without IL-4/IL-13.

Techniques: Derivative Assay, Gene Expression, Flow Cytometry, Mutagenesis, Two Tailed Test, Fluorescence

Journal: Nature

Article Title: Tuft cells act as regenerative stem cells in the human intestine

doi: 10.1038/s41586-024-07952-6

Figure Lengend Snippet: a , Organoid outgrowth of single S/G2/M-phase KIT − and KIT + cells sorted from ileum FUCCI reporter organoids, cultured in standard human intestinal expansion medium. n = 3 independent experiments. b , c , MetaCell 2D projection of 10,311 single cells isolated from passage 1 KIT − or KIT + cell-derived organoids, differentiated with or without IL-4/IL-13. Cells are coloured by cell-type annotation ( b ) or by medium condition and organoid founder cell ( c ). d , AVIL lineage-tracing strategy in human organoids. e , Images of AVIL lineage-tracing organoids, derived from sorted AVIL-tdT + iRFP + cells (as in Extended Data Fig. , day 0). f , Representative images of intestinal epithelial lineages markers in differentiated traced organoids. e , f , n = 3 independent experiments with similar results. Scale bars, 100 µm ( a , e ), 20 µm ( f ). tdTom, tdTomato.

Article Snippet: Fig. 3 Single tuft cells give rise to organoids containing all epithelial lineages. a , Organoid outgrowth of single S/G2/M-phase KIT − and KIT + cells sorted from ileum FUCCI reporter organoids, cultured in standard human intestinal expansion medium. n = 3 independent experiments. b , c , MetaCell 2D projection of 10,311 single cells isolated from passage 1 KIT − or KIT + cell-derived organoids, differentiated with or without IL-4/IL-13.

Techniques: Cell Culture, Isolation, Derivative Assay

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: Meteor transcriptional elongation promotes Eomes expression in naive mESCs (A) Perturbations used to analyze the Meteor locus in this study. Shown are the locations of gRNAs used for deleting or inverting the Meteor promoter (pKO) or gene body (fKO) or for inhibiting transcription (dCas9-KD); the locations of insertion of ribozyme (Rz) or polyadenylation (pAS) sequences; and the scheme for knocking in the PGK promoter downstream of the Meteor promoter (pEX). The fKO and Rz-KD mESC lines are the same as in Tuck et al. (B) RNA-seq tracks showing Meteor expression in the various pKO and pInv lines (grown in serum-free 2i/LIF conditions) and dCas9-KD lines (serum/LIF conditions). All tracks are normalized to the same scale. Orange denotes transcription on the plus strand, and blue denotes transcription on the minus strand. (C) RNA-seq quantifications of Meteor and Eomes in Meteor pKO and pInv cell lines grown in serum/LIF (left) or serum-free 2i/LIF (right) conditions. Amounts normalized to WT1. Bars represent standard errors; n = 3. ∗ p < 0.05, ∗∗ p < 0.005. (D) qRT-PCR quantifications of Meteor and Eomes levels in Meteor fKO and Rz-KD mESCs grown in serum-free 2i/LIF conditions. Levels were normalized to WT4 and Ppib for internal control. Bars represent standard errors; n = 8. ∗ p < 0.05, ∗∗ p < 0.005. (E) Same as (D), for Meteor pKO and pInv mESCs grown in primed conditions, normalized to WT1. n = 4. (F) Same as (D), for pAS clones grown in serum-free 2i/LIF conditions. Levels were normalized to WT7. n = 3. (G) Same as (C), for Meteor dCas9-KD lines grown in serum/LIF conditions. Amounts normalized to Ctrl. n = 3. The dCas9-KD efficiencies of Meteor were 85% and 94% for KD1 and KD2, respectively. (H) Same as (D), for pEX clones grown in serum-free 2i/LIF conditions. Levels were normalized to WT9. n = 3. See also Figure S1 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Control, Clone Assay

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: Meteor depletion induces chromatin changes in mESCs (A) (Top) Genome browser image of the region surrounding the Meteor locus. Shown are representative transcript models; RNA-seq tracks where orange denotes transcription on the plus strand and blue denotes transcription on the minus strand; and ENCODE mESC ChIP-seq tracks. (Middle) 4C analysis in the indicated mESC lines using the Meteor or Eomes promoters as viewpoints. Domainograms showing mean contact per fragment end for a series of window sizes are placed below smoothed trend lines and raw counts of the contact profiles. (Bottom) ChIP-seq tracks of CTCF in the indicated mESC lines. All tracks are normalized to the same scale. (B) (Top) Genome browser image of the region surrounding the Meteor locus. RNA-seq track is the same as shown in (A); Cut&Run analysis of H3K27me3 levels in the indicated mESC lines grown in serum-free 2i/LIF conditions. (Bottom) Bar plot shows quantification of signal in the highlighted region, normalized to WT1 and to a H3K27me3-rich region near the Ppib gene (see ). Bars represent standard errors; n = 3. ∗ p < 0.05, one-sided t test. (C) Distribution of reads per kilobase per million (RPKM) of all transcripts identified in an EZH2 RNA immunoprecipitation (RIP) dataset taken from Zhao et al. RPKM of Meteor indicated by a red line. See also Figure S2 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: RNA Sequencing, ChIP-sequencing, RNA Immunoprecipitation

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph. Cells are shown as small dots, whose location indicates similarity to the adjacent cells and metacells (MCs). Color was assigned to each MC according to the ratio of WT and pKO cells that comprise it, with darker shades representing MCs comprised mostly of WT cells and lighter shades representing MCs comprised mostly of pKO cells. (B) Correlation between the log of the fold enrichment values (expression enrichment over the median MC) for the indicated gene pairs, separated by MCs. (C) Same as (A), separately for WT1 and pKO1 cells. (D) Scatterplot comparing gene expression between the indicated groups of MCs. Highlighted are the 20 most differentially expressed genes in each group. (E) Same as (D), for MCs 2 and 4. See also Figure S4 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Expressing, Gene Expression

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: A distinct functional feature of the Meteor locus represses Eomes throughout neuronal differentiation (A) (Top) Genome browser image of the region surrounding the Meteor locus. Shown are representative transcript models, and RNA-seq tracks taken from Hezroni et al. where orange denotes transcription on the plus strand and blue denotes transcription on the minus strand. All tracks are normalized to the same scale. (Bottom) 4C analysis in the indicated cells using the Meteor or Eomes promoters as viewpoints. Domainograms showing mean contact per fragment end for a series of window sizes are placed below smoothed trend lines and raw counts of the contact profiles. (B) RSEM quantifications of Meteor and Eomes expression levels in the indicated cell types; RNA-seq data are the same as shown in (A). (C) ENCODE ChIP-seq tracks in mESCs (top), and H3K27ac ChIP-seq and H3K4me3 Cut&Run tracks in NPCs (bottom). Genomic coordinates are aligned to (A). (D) DESeq2 quantifications of Meteor and Eomes in NPCs derived from the indicated cell lines. Amounts normalized to WT1/Ctrl. Bars represent standard errors; n = 3. ∗∗ p adj < 0.005. (E) qRT-PCR quantifications of Meteor and Eomes in NPCs derived from the indicated cell lines. Levels were normalized to WT9 and Ppib for internal control. Bars represent standard errors; n = 3. (F) Model of Meteor function. In mESCs, the Meteor locus activates Eomes expression. Perturbing elongation through the locus is associated with increased H3K27me3 deposition and decreased Eomes expression, likely through decreasing the Eomes -expressing mESC subpopulation. As a function of the growth conditions of the cells, this might result in reduced efficiency of cardiac mesoderm formation. Following neuronal differentiation, the Meteor locus now represses Eomes levels, with the DNA element or transcription initiation serving as the functional feature. See also Figure S5 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Functional Assay, RNA Sequencing, Expressing, ChIP-sequencing, Derivative Assay, Quantitative RT-PCR, Control

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet:

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Transfection, Gene Expression, Control, Sequencing, Stable Transfection, Expressing, Recombinant, Plasmid Preparation, Software