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MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche

Abstract

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3,4,5,6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)–MAP3K2–ERK5–KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.

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Fig. 1: Map3k2 protects mice from DSS-induced colitis by maintaining intestinal stem cell number.
Fig. 2: Intestinal stromal MAP3K2 protects mice from DSS-induced colitis by inducing Rspo1 expression.
Fig. 3: CD90medCD81+CD34+ MRISCs protect intestinal stem cells via augmented R-spondin 1 production.
Fig. 4: MRISCs possess distinct epigenomic features and promote ISC growth via a ROS–MAP3K2–EKR5–KLF2–R-spondin 1 signalling axis.

Data availability

All raw sequencing data that support the findings of this study have been deposited in the Sequence Read Archive (SRA) with the accession code PRJNA595166. All processed data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession GSE156245Source data are provided with this paper.

Code availability

All code used for data visualization of the scRNA-seq and ATAC–seq data can be found at https://github.com/sun758426-china/intestinal-mesenchymal-stromal-cell.

References

  1. 1.

    Kinchen, J. et al. Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease. Cell 175, 372–386.e17 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    McCarthy, N., Kraiczy, J. & Shivdasani, R. A. Cellular and molecular architecture of the intestinal stem cell niche. Nat. Cell Biol. 22, 1033–1041 (2020).

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Roulis, M. et al. Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche. Nature 580, 524–529 (2020).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Degirmenci, B., Valenta, T., Dimitrieva, S., Hausmann, G. & Basler, K. GLI1-expressing mesenchymal cells form the essential WNT-secreting niche for colon stem cells. Nature 558, 449–453 (2018).

    ADS  CAS  PubMed  Article  Google Scholar 

  5. 5.

    Shoshkes-Carmel, M. et al. Subepithelial telocytes are an important source of WNTs that supports intestinal crypts. Nature 557, 242–246 (2018).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    McCarthy, N. et al. Distinct mesenchymal cell populations generate the essential intestinal BMP signaling gradient. Cell Stem Cell 26, 391–402.e5 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Gehart, H. & Clevers, H. Tales from the crypt: new insights into intestinal stem cells. Nat. Rev. Gastroenterol. Hepatol. 16, 19–34 (2019).

    PubMed  Article  Google Scholar 

  8. 8.

    Stzepourginski, I. et al. CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury. Proc. Natl Acad. Sci. USA 114, E506–E513 (2017).

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    ADS  CAS  PubMed  Article  Google Scholar 

  10. 10.

    Yan, K. S. et al. Non-equivalence of WNT and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal. Nature 545, 238–242 (2017).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Zhang, D. et al. Identification of MEKK2/3 serine phosphorylation site targeted by the Toll-like receptor and stress pathways. EMBO J. 25, 97–107 (2006).

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Grün, D. et al. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature 525, 251–255 (2015).

    ADS  PubMed  Article  CAS  Google Scholar 

  13. 13.

    Schuijers, J. et al. Ascl2 acts as an R-spondin/WNT-responsive switch to control stemness in intestinal crypts. Cell Stem Cell 16, 158–170 (2015).

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Takeda, N. et al. Interconversion between intestinal stem cell populations in distinct niches. Science 334, 1420–1424 (2011).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Wu, N. et al. MAP3K2 augments Th1 cell differentiation via IL-18 to promote T cell-mediated colitis. Sci. China Life Sci. (2020).

  16. 16.

    Kim, K. A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005).

    ADS  CAS  PubMed  Article  Google Scholar 

  17. 17.

    Karpus, O. N. et al. Colonic CD90+ crypt fibroblasts secrete semaphorins to support epithelial growth. Cell Rep. 26, 3698–3708.e5 (2019).

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Tian, X. et al. Generation of a self-cleaved inducible Cre recombinase for efficient temporal genetic manipulation. EMBO J. 39, e102675 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Rinkevich, Y. et al. Identification and prospective isolation of a mesothelial precursor lineage giving rise to smooth muscle cells and fibroblasts for mammalian internal organs, and their vasculature. Nat. Cell Biol. 14, 1251–1260 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Guo, Z. et al. Disruption of Mekk2 in mice reveals an unexpected role for MEKK2 in modulating T-cell receptor signal transduction. Mol. Cell. Biol. 22, 5761–5768 (2002).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).

    ADS  CAS  Article  Google Scholar 

  22. 22.

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Madison, B. B. et al. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J. Biol. Chem. 277, 33275–33283 (2002).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. 27.

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  28. 28.

    Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Fornes, O. et al. JASPAR 2020: update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 48, D87–D92 (2020).

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Aibar, S. et al. SCENIC: single-cell regulatory network inference and clustering. Nat. Methods 14, 1083–1086 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Van de Sande, B. et al. A scalable SCENIC workflow for single-cell gene regulatory network analysis. Nat. Protocols 15, 2247–2276 (2020).

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Wei, Z., Zhang, W., Fang, H., Li, Y. & Wang, X. esATAC: an easy-to-use systematic pipeline for ATAC-seq data analysis. Bioinformatics 34, 2664–2665 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. 38.

    Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013).

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Khan, A. & Mathelier, A. Intervene: a tool for intersection and visualization of multiple gene or genomic region sets. BMC Bioinformatics 18, 287 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  41. 41.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Janky, R. et al. iRegulon: from a gene list to a gene regulatory network using large motif and track collections. PLoS Comput. Biol. 10, e1003731 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Y. Zhuang, G. Su, Z. Yang, and Q. Wang for reading the manuscript and making suggestions; C. Gao, X. Liu, and Y. Han for their initial help and suggestions in scRNA-seq, organoid culture, and the generation of Map3k2 flox mice, respectively; all the members of the Su laboratory for discussion and suggestions; and the sequencing and flow cytometry core facilities at Shanghai Institute of Immunology for their support. This work was supported in part by grants from the National Natural Science Foundation of China (3201101152, 91942311 and 31930035), Shanghai Science and Technology Commission (20410714000).

Author information

Affiliations

Authors

Contributions

N.W. and H.S. contributed equally to this study. N.W., H.S. and B.S. planned the study and experimental design. N.W. performed all major experiments with help from D.L., Y.Q., Q.W. and X.N. including mouse work, organoid culture, flow cytometry, immunoblotting, qRT–PCR and so on. J.T. and N.W. performed orthotopic surgical delivery procedure. M.R., J.C., D.Z., R.A.F. and H.-B.L. provided expert advice on intestinal biology. L.C. provided help for RNA-seq. H.S. analysed bulk and single cell RNA-seq data, and performed FACS sorting and ATAC–seq. Zhaoyuan Liu and F.G. provided expert advice about scRNA-seq data analysis. M.N., L.G.N. and Y.Y. provided expert advice in ATAC–seq data analysis. X.Z. and Y.Z. performed RNAScope and immunostaining with help from Zhiduo Liu and Y.A.Z. The Col1a2-creERT2 mouse line was provided by L.H. and B.Z. All authors discussed the results and commented on the manuscript. H.S., N.W. and B.S. wrote the manuscript with help from all authors. B.S. conceived, supervised, and directed the study.

Corresponding author

Correspondence to Bing Su.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Dominic Grun, Toshiro Sato and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Map3k2 is required for maintaining the number of multiple intestinal epithelial cell subsets upon DSS-induced colitis.

a, Average stool scores of DSS-treated wild-type mice (n = 6) and Map3k2−/− co-housed littermates (n = 6) or untreated (control) wild-type mice (n = 4) and Map3k2−/− littermates (n = 3). b, Left, haematoxylin and eosin (H&E)-stained colons from mice in a. Right, quantification of corresponding histology scores. Arrows, infiltrating leukocytes. Scale bars, 50 μm. c, Alcian Blue/Periodic Acid Schiff (AB/PAS)-stained colons from wild-type and Map3k2−/− mice treated with DSS for 0, 3, 5, or 7 days followed by water administration for 2 days (to day 9) (n = 3–4 mice per time-point). Scale bars, 50 μm. d, Quantification of goblet cells per crypt in the colons of mice in c (20 crypts were counted in total from n = 3–4 mice per time-point). e, qRT–PCR of Muc2 expression in colons as in c (n = 6–8 colon segments from 3–4 independent mice). f, qRT–PCR of Reg4 expression in colons from untreated (control) and DSS-exposed wild-type and Map3k2−/− mice (n = 3). g, qRT–PCR of Lgr5, Ascl2 and Hopx expression in colons from untreated (control) or DSS-exposed wild-type and Map3k2−/− mice (n = 6). h, Representative flow cytometry plots show the percentages of intestinal stem cells (ISCs) (circled CD45CD326+CD44+CD24low population in bottom panels) in colon epithelial cells from untreated (control) and DSS-exposed wild-type and Map3k2−/− mice (n = 4 per group). i, Percentage of intestinal stem cells among total colon epithelial cells in mice as in h. j, Ki67 immunostaining of colons from untreated (control) and DSS-exposed wild-type and Map3k2−/− mice. Results are representative of three independent experiments. Scale bars, 50 μm. k, Numbers of Ki67+ cells per crypt in colons as in j (n = 20 crypts were counted over three independent experiments). exp, expression. Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 2 Haematopoietic or epithelial Map3k2 is not required for protection against DSS-induced colitis.

a, Changes in body weight of DSS-treated wild-type recipient mice transplanted with wild-type or Map3k2−/− bone marrow cells (n = 8 mice per group). b, c, Average stool scores (b) and colon lengths (c) of mice in a. d, Left, H&E-stained colons from mice in a. Right, quantification of corresponding histology scores. Scale bars, 50 μm. e, qRT–PCR of Lgr5 and Gob5 expression in colons from mice in a. f, Changes in body weight of DSS-treated wild-type or Map3k2−/− recipient mice transplanted with wild-type bone marrow cells (n = 6 mice per group). g, h, Average stool scores (g) and colon lengths (h) of mice in f. i, Left, H&E-stained colons from mice in f. Right, quantification of corresponding histology scores. Scale bars, 50 μm. j, qRT–PCR of Lgr5 and Gob5 expression in colons from mice in f. k, Immunoblots of MAP3K2 in IECs and intestinal mesenchymal stromal cells (IMSCs) from the colons of Map3k2fl/fl and Vil1-CreMap3k2fl/fl mice. GAPDH is loading control. Results are representative of two independent experiments. For gel source data, see Supplementary Fig. 1. l, Changes in body weight of DSS-treated Map3k2flfl and Vil1-cre:Map3k2fl/fl co-housed littermate mice (n = 4 mice per group). m, n, Average stool scores (m) and colon lengths (n) of mice in l (n = 4 mice per group). o, Left, H&E-stained colons from mice in l. Right, quantification of corresponding histology scores (n = 4 mice per group). Scale bars, 100 μm. p, qRT–PCR of Lgr5 and Il6 expression in colons (n = 4) from mice in l. Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 3 RNA sequencing revealed that Map3k2 regulates WNT signal in tissue repair by inducing Rspo1 upon DSS-induced colitis.

ac, GSEA enrichment plots of the chemokine and MAPK signalling pathways (a), TCF4 target genes (b) and the Notch pathway and EGF response target genes (c) in colons from DSS-exposed wild-type and Map3k2−/− mice (n = 3 mice per group). d, qRT–PCR of Axin2 and Fzd8 expression in colons from untreated (control) or DSS-treated wild-type and Map3k2−/− mice (n = 6 colon segments from 3 independent mice). e, GSEA enrichment plots of WNT ligands, WNT receptors, and WNT signal mediators in colons as in a. f, Average stool scores of DSS-treated Map3k2−/− mice (n = 5 mice per group) treated with recombinant R-spondin 1 (Rspo1) or saline daily for 9 days. g, qRT–PCR of Il6 and Tnf expression in colons from mice (n = 5 mice per group) as in f. h, Left, AB/PAS-stained colons from mice in f. Right, number of goblet cells per crypt (30 crypts were counted over 5 mice per group). Scale bars, 100 μm. i, j, Changes in body weight (i) and average stool scores (j) for DSS-treated wild-type mice (n = 5 mice per group) injected with recombinant R-spondin 1 or saline daily for 9 days. k, A step-by-step illustration of abdominal orthotopic surgery for local delivery of substances to the colon. The top row shows the preparation for a sterile open incision through the skin and the peritoneum to expose the colon (right). The middle row shows the exposed colon with the injection area (yellow box), which is about 2 cm from the caecum, where the substance is delivered into the submucosa layer using a U-40 insulin syringe (31G gauge needle; right middle). The bottom row shows the colon being returned into the abdomen and the opening having being sutured by stitching abdominal wall and clipping skin. Scale bar, 2 mm. l, The proximal location of colon samples taken for further analysis after orthotopic injection. Asterisk, injection site; −1, 0, +1 indicate the colon segments (0.5 cm) to be taken for RNA extraction and imaging analysis. m, n, qRT–PCR of Il6, Axin2, and Ascl2 expression in the colon described in l of wild-type mice orthotopically treated with PBS or LPS (m), or PBS or recombinant R-spondin 1 (n) (n = 4 colon segments over two independent experiments). Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 4 Map3k2 in intestinal stromal cells protects mice from DSS-induced colitis in vivo.

a, Representative flow cytometry plots showing the percentages of CD326+ intestinal epithelial cells (IECs), CD45+ leukocytes, GP38+CD90 intestinal mesenchymal stromal cells, and GP38+CD90+ intestinal mesenchymal stromal cells in the colon of wild-type mice (n = 4 mice). b, c, qRT–PCR of Rspo1 (b) and Map3k2 expression (c) in the indicated cell types described in a. d, Representative flow cytometry plots and histograms showing percentages of tdTomato+ cells in IECs, CD45+ leukocytes, lymphatic endothelial cells (LECs), blood endothelial cells (BECs), GP38+ cells, and GP38 cells from the colonic lamina propria of Col1a2-creERT2Rosa26-loxP-stop-loxP-tdTomato mice as indicated. e, qRT–PCR of Map3k2 expression in CD45+ leukocytes, IECs, CD31+ endothelial cells, and GP38+ intestinal mesenchymal stromal cells from the colons of tamoxifen-treated Map3k2fl/fl and Col1a2-creERT2Map3k2fl/fl mice (n = 3 mice per group). f, g, Average stool scores (f) and colon lengths (g) of DSS-treated Map3k2fl/fl and Col1a2-creERT2Map3k2fl/fl mice (n = 3 mice per group) that had been previously induced with tamoxifen. h, Left, H&E-stained colons from mice in f. Right, quantification of the corresponding histology scores. Scale bars, 100 μm. i, qRT–PCR of Il6 expression in the colons (n = 3) of mice in f. Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 5 CD90 is highly expressed in LECs and moderately expressed in stromal cells.

a, Lgr5-EGFP-creERT2 colon immunostained for CD90 (cyan), GP38 (red) and DAPI (blue). Box in left image (sub-crypt) area is expanded in the second image, and shown on cyan and red single channels, respectively, in the third and fourth images. Results are representative of three independent experiments. Scale bars, 30 μm. b, Volumetric reconstruction of whole-mount imaging of wild-type mouse colon stained with anti-CD90 (cyan) and anti-CD326 (red) antibodies. Results are representative of two independent experiments. c, Wild-type mouse colon immunostained for CD90 (green) and LYVE1 (red). Left, merged image. Results are representative of three independent experiments. Scale bars, 30 μm. d, Representative flow cytometry plots of IECs, CD45+ leukocytes, LECs, BECs, GP38+ stromal cells, and GP38 cells from wild-type colonic lamina propria. The numbers show the percentages of the indicated cell populations from the respectively gated areas. e, Histograms of CD90 expression in the indicated cell types described in d. Results are representative of three independent experiments.

Extended Data Fig. 6 scRNA-seq reveals two distinctive Rspo1-expressing intestinal mesenchymal stromal cells.

a, Violin plots of the expression of the indicated genes in clusters (C) 1, 2, 3, and 5. b, GO terms enriched in genes that are differentially expressed between cluster 2 and cluster 5 stromal cells. FDR, false discovery rate. c, Violin plots of the expression of non-transcription factor genes (non-TF) and transcriptional regulator genes (TF) in clusters 1, 2, 3, and 5. d, Representative flow cytometry plots of cluster 5 and cluster 2 stromal cells in colonic lamina propria isolated from wild-type mice. The numbers show the percentages of the indicated cell populations. e, f, Quantification of cluster 5 cells (e) and cluster 2 cells (f) as percentages of GP38+ stromal cells as assessed by flow cytometry in colons from untreated (control) or DSS-exposed (2 day) wild-type and Map3k2−/− mice (n = 3). Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 7 RSPO1+CD81+ CD34+ CD31CD90med mesenchymal stromal cells are located near the intestinal crypt.

a, Representative flow cytometry plots show the percentages of CD34+CD81+ populations (numbers in the upper right quadrants in the bottom panels) in IECs, CD45+ leukocytes, GP38 cells, LECs, BECs, GP38+CD90 stromal cells, and GP38+CD90med stromal cells from wild-type colon. b, Wild-type colon immunostained for CD81 (red), CD34 (green), and CD31 (blue). Top left, three-colour merged image; top middle, a magnified image of boxed area. Yellow in top right shows CD81+CD34+ double-positive cells. Bottom row, CD81+CD34+ cells (yellow) superimposed on three separate channels as indicated. Results are representative of three independent experiments. Scale bars, 30 μm. c, Diagram of the Rspo1 gene locus (wild-type allele) containing translation starting codon ATG, the targeting vector that contains a tdTomato-WPRE-polyA cassette inserted in-frame with the ATG codon of the Rspo1 gene in exon 2, and the targeted allele (Rspo1-tdTomato) in which the tdTomato-WPRE-polyA cassette is inserted in exon 2 by Cas9-mediated incision-induced homologous recombination. P1 and P2 are primers used for PCR to distinguish the wild-type and Rspo1-tdTomato alleles. d, Genotyping of Rspo1-tdTomato mice with P1 and P2 primers. DNA was prepared from toes of wild-type and Rspo1-tdTomato mice. Results are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. e, Representative flow cytometry plots for tdTomato+ and tdTomato intestinal mesenchymal stromal cells from Rspo1-tdTomato mice. f, qRT–PCR of tdTomato, Rspo1, and Rspo3 expression in tdTomato+ and tdTomato intestinal mesenchymal stromal cells from Rspo1-tdTomato mice (n = 3 mice). g, Representative flow cytometry plots of tdTomato+ cells in IECs, CD45+ leukocytes, LECs, BECs, GP38+ and GP38 stromal cells in colonic lamina propria from Rspo1-tdTomato mice. The numbers show the percentages of the indicated cell populations. h, qRT–PCR of Rspo1 and Tomato expression in tdTomato+ cells in cluster 5 stromal cells from untreated (control) or DSS-treated Rspo1-tdTomato mice (n = 3 mice). Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 8 MRISCs require MAP3K2 to promote intestinal organoid growth via R-spondin 1.

a, Bright-field images of organoids cultured for 0, 3, or 5 days alone (no stromal cells), or co-cultured with MRISCs or with non-MRISC stromal cells in the absence (−) or presence (+) of R-spondin 1 or anti-R-spondin 1 antibody (Ab). Results are representative of three independent experiments. Scale bars, 100 μm. b, Average circumferences of colon organoids in a at day 5. Each symbol represents a single organoid (n = 15 organoids over three independent experiments). c, Dark-field images of organoids co-cultured with MRISCs or CD138+ cluster 2 stromal cells with an anti-R-spondin 1 antibody (Ab) or a control antibody (isotype). Bottom panels show magnified images of boxed areas. Results are representative of three independent experiments. Scale bars, 500 μm (top); 200 μm (bottom). d, Average circumferences of colon organoids in c at day 4. Each symbol represents a single organoid (n = 15 organoids over three independent experiments). e, Images of tdTomato+ cells in the colons of wild-type mice orthotopically injected with PBS alone or with tdTomato+ colon stromal cells in PBS (Extended Data Fig. 3k). White dashed lines indicated baseline of colon crypt. Results are representative of two independent experiments. Scale bars, 200 μm. f, qRT–PCR of tdTomato expression in the colons of wild-type mice orthotopically injected with PBS or tdTomato+ colon stromal cells (n = 4 colon segments over two independent experiments) (Extended Data Fig. 3k). g, qRT–PCR of Axin2, Lgr5, Rspo1, and tdTomato expression in colons from DSS-exposed wild-type mice (n = 4 colon segments over two independent experiments) orthotopically injected with PBS or with wild-type or Map3k2−/− MRISCs from colons of wild-type or Map3k2−/−R26-mTmG mice (Extended Data Fig. 3k). Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 9 ATAC–seq of different colonic cell populations.

a, Representative flow cytometry plots show IECs, myofibroblasts, CD138+ cluster 2 stromal cells, MRISCs, and CD34SP stromal cells that were sorted for ATAC–seq. b, Pearson correlation heat map of MRISCs and IECs based on all ATAC–seq peaks. c, Chromatin accessibility of Col1a2, Pdpn, Cdh1, Ptprc, and Cdh5 in the indicated cell types normalized to Hprt loci. d, Pearson correlation heat map of the indicated cell types based on all ATAC–seq peaks. e, Venn diagram indicating differentially regulated ATAC–seq peaks among the indicated cell types. f, Top six motifs enriched within ±100 bp of the summits described in Fig. 4d. g, Chromatin accessibility of Fez1, Pcolce2, Il33, Hoxb2, Stmn2, Ch25h, Agt, Actg2, and Gapdh in the indicated cell types. h, Transcriptional regulatory networks for the indicated stromal cell types generated using iRegulon. Target genes were determined by intersecting genes whose nearby enhancers (±20 kb from transcription start site (TSS) were cell-type-specific accessible regions identified from the ATAC–seq dataset with the cell-type-specific marker genes identified from the scRNA-seq dataset.

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Extended Data Fig. 10 ROS and Klf2 are highly active in MRISCs.

a, t-SNE plots show the KLF2 and HOXB2 regulons, respectively, identified using pySCENIC based on scRNA-seq data. b, Sequence alignments of the evolutionarily conserved human, rat, and mouse (chr4: 124985668–124985674) KLF binding motifs (core binding sequence shown in red) in the Rspo1 promoter region. c, Chromatin accessibility of Rspo1 locus in the indicated cell types. Highlighted KLF motif was identified using JASPAR database. d, t-SNE plots show Klf2, Klf3, Klf4, Klf6, Klf7, Klf9, Klf10, and Klf13 expression at the single-cell level. e, qRT–PCR showing expression of Klf family transcription factors in MRISCs (n = 3 biologically independent cells). f, qRT–PCR of Klf3, Klf4, Klf6, and Klf9 expression in MRISCs from the colons of DSS-treated or untreated (control) wild-type and Map3k2−/− mice (n = 3 mice per group). g, qRT–PCR of Klf2 and Rspo1 expression in Klf2-silenced (shKlf2) or control empty vector (EV) stromal cells (n = 4 biologically independent cells). h, qRT–PCR of Rspo1 expression in colons of untreated (control) or DSS-exposed wild-type mice in the absence (PBS) or presence of NAC (n = 3 mice per group). i, Histograms of ROS levels in MRISCs from the colons of untreated (control) or DSS-exposed wild-type and Map3k2−/− mice. Numbers in the histogram show the MFI of indicated cell populations. Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

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Supplementary information

Supplementary Figures

This file contains Supplementary Figs 1-3, including the uncropped scans of the blots and gel and gating strategies for FACs analysis.

Reporting Summary

Supplementary Table 1

Primer sequences for qPT-PCR, mice genotyping, and Chip-q-PCR analysis, and shRNA sequences.

Video 1

: Whole mount 3D imaging of WT mouse colon A video showing the whole mount 3D imaging (related to Extended Data Fig. 5b) of WT mouse colon after clearing and stained with anti-CD90 (cyan) and CD326 (red) for analysis using a Leica SP8 microscope with 20x objective lens. Data are representative of two independent experiments.

Video 2

: 3D imaging of WT mouse colon A video showing the 3D imaging (related to Extended Data Fig. 7b) of WT mouse colon section stained with anti-CD81 (red), anti-CD34 (green), anti-CD31 (blue) visualized using a Leica SP8 microscope with 63x oil objective lens. The CD81 and CD34 double positive staining is marked with a pseudo-yellow color. Data are representative of three independent experiments.

Video 3

: 3D imaging of Rspo1-tdTomato mouse colon A video showing the 3D imaging (related to Fig. 3i) of Rspo1-tdTomato mouse colon section stained with anti-CD81 (cyan), anti-CD34 (blue), anti-CD31 (green) and then visualized using a Leica SP8 microscope with 63x oil objective lens. The CD81, CD34, and tdTomato triple positive cells marked with a pseudo-white color. Data are representative of three independent experiments.

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Wu, N., Sun, H., Zhao, X. et al. MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche. Nature 592, 606–610 (2021). https://doi.org/10.1038/s41586-021-03283-y

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