Volume 26, Issue 13, Pages 3698-3708.e5 (March 2019) Colonic CD90+ Crypt Fibroblasts Secrete Semaphorins to Support Epithelial Growth  Olga N. Karpus, B. Florien Westendorp, Jacqueline L.M. Vermeulen, Sander Meisner, Jan Koster, Vanesa Muncan, Manon E. Wildenberg, Gijs R. van den Brink  Cell Reports  Volume 26, Issue 13, Pages 3698-3708.e5 (March 2019) DOI: 10.1016/j.celrep.2019.02.101 Copyright © 2019 Amsterdam UMC Terms and Conditions

Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions

Figure 1 Gli1 Expression Can Be Used to Classify Intestinal Fibroblasts into Specific Subpopulations (A) Colon cell suspensions from Gli1CreERT2;R26-ZsGreen mice were analyzed by using flow cytometry, and cells were gated on Epcam-CD45-CD31-gp38+. Histograms and percentages of fibroblast markers Sca1 and PDGFRα expressed on Gli1ZsGreenneg (light green) and Gli1ZsGreenpos (dark green) are shown. Representative example of at least three independent experiments is shown for each marker. (B) Double immunofluorescent stainings of ZsGreen and fibroblast markers in colons from Gli1CreERT2;R26-ZsGreen mice. Representative image of three independent experiments is shown. Scale bar, 100 μm. (C) qRT-PCR analysis of sorted Gli1ZsGreenneg and Gli1ZsGreenpos fibroblasts, CD45+ hematopoietic cells, and Epcam+ epithelial cells (n = 4 mice). Bars are mean ± SEM. ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001. See also Figure S1. Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions

Figure 2 Gli1ZsGreenpos Fibroblasts Support Organoid Growth and Express Established Stem Cell Niche Factors (A and B) (Left) Perimeter of small intestinal (A) and colon (B) organoids co-cultured for 4 days with sorted fibroblast subpopulations in R-spondin-reduced medium. One of three independent experiments is shown. Every dot represents a single organoid; black line is the mean. (Right) Representative photo of a single organoid (30–40 organoids/well) co-cultured with Gli1ZsGreenneg or Gli1ZsGreenpos fibroblasts. (C–G) Heatmaps of selected genes from Hedgehog (C), regulation of stem cell pluripotency (D), proteoglycans (E), focal adhesion (F) pathways, and selected membrane markers (G) upregulated in flow-sorted Gli1ZsGreenpos in comparison to Gli1ZsGreenneg fibroblasts. ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001. See also Figure S2 and Table S1. Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions

Figure 3 CD90 and Itga8 as Candidate Membrane Markers for Stem Cell Niche Fibroblasts (A) Immunofluorescent stainings of fibroblast markers ITGA9, SDC2, SDC3, CD90, and ITGA8 on frozen sections of mouse colon. Nuclei 4′,6-diamidino-2-phenylindole (DAPI) staining is shown below. Scale bar, 50 μm. (B) Flow cytometry dot plot of CD90 and gp38 for colon cell suspension, gated on Epcam-CD45-CD31-gp38+ cells stained with anti-CD90 (top) or no antibodies (bottom). Representative plot of five independent stainings is shown. (C) qRT-PCR analysis of flow-sorted CD90− and CD90+ subpopulations of fibroblasts (n = 4 mice). One of three independent experiments is shown. Bars are mean ± SEM. (D) (Left) Perimeter of organoids co-cultured for 4 days with CD90- or CD90+ sorted fibroblasts in R-spondin reduced medium. One of four independent experiments is shown. Every dot represents a single organoid; black line is the mean. (Right) Representative photo of a single organoid (20–30 organoids/well) co-cultured with CD90− or CD90+ fibroblasts is depicted. (E) Flow cytometry dot plot of Itga8 and gp38 for colon cell suspension, gated on Epcam-CD45-CD31-gp38+ cells stained with anti-Itga8 (top) or no antibodies (bottom). Representative plot of three independent stainings is shown. (F) qRT-PCR analysis of flow-sorted Itga8− and Itga8+ subpopulations of fibroblasts (n = 4 mice). (G) (Left) Perimeter of organoids co-cultured for 4 days with Itga8− or Itga8+ sorted fibroblasts in R-spondin-reduced medium. One of four independent experiments is shown. Every dot represents single organoid; black line is the mean. (Right) Representative photo of a single organoid (20–30 organoids/well) co-cultured with Itga8− or Itga8+ fibroblasts is shown. ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001. See also Figures S3 and S4. Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions

Figure 4 CD90+ Cells Are Located in Colon Crypt Bottoms and in between Crypts (A–C) In situ hybridization (RNAscope) for CD90 mRNA in combination with immunofluorescent staining for (A) gp38, (B) BrdU, and (C) Lgr5. Single red dots represent expression of a single CD90 mRNA copy. Representative image of three independent experiments is shown. Scale bar, 50 μm. Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions

Figure 5 CD90− and CD90+ Fibroblasts Have Miscellaneous Functions (A) Clustering of upregulated genes (>8 fold) in CD90− (cluster 1) or CD90+ (cluster 2 and cluster 3) fibroblasts. Cluster 2 is zoomed in the right panel. Arrows indicate mesenchymal stem cell niche genes Rspo3, Wnt2b, and Grem1 within the cluster analysis. (B) Gene set enrichment analysis (GSEA) for HALLMARK pathways shows presence of Epithelial-mesenchymal transition gene set in CD90+ fibroblasts and IL2/STAT5 signaling in CD90− fibroblasts. (C) GSEA for GO pathways shows presence of Regulation of inflammatory response gene set in CD90+ fibroblasts and Smooth muscle contraction in CD90− fibroblasts. (B and C) HALLMARK and GO gene sets enriched in CD90+ (left) and in CD90- (right) fibroblasts. Enrichment score (ES), normalized enrichment score (NES), and p values are indicated in each image. Some representative genes from enrichment analysis are shown below. See also Figures S4 and S5 and Table S1. Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions

Figure 6 Class 3 Semaphorins Are Expressed by CD90+ Fibroblasts and Able to Increase Organoid Growth (A) Overview of selected soluble factors upregulated in CD90+ cells. (B) qRT-PCR analysis for semaphorin 3 family members (Sema3a–g) in CD90− and CD90+ subpopulations (n = 4 mice). (C) RNAscope for Sema3a mRNA (red) in combination with immunofluorescent staining for gp38 (green). Single red dot represents expression of single Sema3a mRNA copy. Representative image of three independent experiments is shown. Scale bar, 50 μm. (D and E) Effect of class 3 semaphorin on organoid growth. (Left) Perimeter of colon organoids (D) and small intestinal organoids (E) cultured for respectively 2 and 4 days with 2 μg/ml Sema3a or vehicle control. One of four independent experiments is shown. Every dot represents a single organoid; the red line indicates the mean. (Right) Representative image of a single organoid (20–30 organoids/well) supplemented with 2 μg/ml Sema3a or vehicle control. ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001. See also Figure S6 and Table S3. Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions

Figure 7 Blocking Sema3 Signaling Abrogates CD90+-Driven Organoid Growth (A) Epcam+ epithelial cells, gp38+CD31− fibroblasts, and CD45+ immune cells were flow sorted from mouse colon suspensions (n = 3 mice). Expression of Sema3 receptors by the sorted populations is shown by qRT-PCR analysis. (B) RNAscope for Nrp2 mRNA (red) in combination with immunofluorescent staining for epithelial marker E-cadherin (green). Single red dot represents expression of a single Nrp2 mRNA copy. Scale bar, 50 μm. (C) Perimeter of colon organoids co-cultured for four days with CD90+ fibroblasts in R-spondin-reduced medium with a vehicle control (red dots) or with recombinant Nrp2 (gray dots). One of three independent experiments is shown. Every dot represents a single organoid; red line is the mean (left). Representative photo of a single organoid (30–40 organoids/well) supplemented with vehicle control or 5 μg/ml of recNrp 2 (right). (D) Colon organoids were transfected with lentiviral constructs against the receptor Nrp2. Perimeter of the knockdown versus short hairpin control organoids (ShControl) was measured after two days when co-cultured with CD90− and CD90+ organoids in R-spondin-reduced medium lacking CHIR-99021 (left). One of two independent experiments is shown. Every dot represents a single organoid; blue line indicates the mean. (right) Representative photo of a single organoid is shown. ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001. See also Figure S6. Cell Reports 2019 26, 3698-3708.e5DOI: (10.1016/j.celrep.2019.02.101) Copyright © 2019 Amsterdam UMC Terms and Conditions