two-dimensional (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