A single-cell survey of the small intestinal epithelium

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Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.

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Download references


We thank L. Gaffney for help with figure preparation, the Broad Flow Cytometry Facility (P. Rogers, S. Saldi and C. Otis), C. Hafemeister and R. Satija for use of the ‘How Many Cells’ tool, S. Riesenfeld and A. Dixit for statistical advice, and T. Tickle for help with the Single Cell Portal. This study was supported by the Klarman Cell Observatory at the Broad Institute, NIH RC2DK114784 (A.R. and R.J.X.), HHMI (A.R.), Food Allergy Science Initiative (FASI) at the Broad Institute (A.R. and R.J.X.), and a Broadnext10 award (A.R. and R.J.X.). M.B. is supported by a postdoctoral fellowship from the Human Frontiers Science Program (HFSP). R.J.X. is supported by NIH DK43351, DK097485 and the Helmsley Charitable Trust.

Author information

Author notes

    • Adam L. Haber
    • , Moshe Biton
    •  & Noga Rogel

    These authors contributed equally to this work.

    • Moshe Biton
    • , Ramnik J. Xavier
    •  & Aviv Regev

    These authors jointly supervised this work.


  1. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA

    • Adam L. Haber
    • , Moshe Biton
    • , Noga Rogel
    • , Rebecca H. Herbst
    • , Karthik Shekhar
    • , Christopher Smillie
    • , Grace Burgin
    • , Toni M. Delorey
    • , Itay Tirosh
    • , Danielle Dionne
    • , Raktima Raychowdhury
    • , Wendy S. Garrett
    • , Orit Rozenblatt-Rosen
    • , Omer Yilmaz
    • , Ramnik J. Xavier
    •  & Aviv Regev
  2. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Moshe Biton
    •  & Ramnik J. Xavier
  3. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Rebecca H. Herbst
    •  & Yarden Katz
  4. Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA

    • Toni M. Delorey
  5. Departments of Immunology and Infectious Diseases and Genetics and Complex Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts 02115, USA

    • Michael R. Howitt
    •  & Wendy S. Garrett
  6. The David H. Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Semir Beyaz
    •  & Omer Yilmaz
  7. Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Semir Beyaz
  8. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Semir Beyaz
  9. Mucosal Immunology and Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA

    • Mei Zhang
    •  & Hai Ning Shi
  10. Departments of Pathology, Gastroenterology, and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA

    • Omer Yilmaz
  11. Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Ramnik J. Xavier
  12. Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02140, USA

    • Aviv Regev


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A.L.H., M.B. and N.R. contributed equally to this study; M.B., R.J.X. and A.R. co-conceived the study; M.B., N.R., A.L.H., R.J.X. and A.R. designed experiments and interpreted the results; N.R. and M.B. carried out all experiments; G.B., T.M.D., M.R.H., S.B., D.D., M.Z. and R.R. assisted with experiments; A.L.H. designed and performed computational analysis with assistance from R.H.H., K.S., C.S., Y.K., I.T. and A.R.; M.R.H. and W.S.G. assisted with tuft and follicle-associated epithelium experiments; M.Z. and H.N.S. assisted with pathogen infections; S.B. and O.Y. assisted with epithelial cell sorting; D.D. and O.R.-R. assisted with scRNA-seq; and A.L.H., M.B., N.R., R.J.X. and A.R. wrote the manuscript, with input from all authors.

Competing interests

A.R. is a member of the scientific advisory board of ThermoFisher, Syros Pharmaceuticals and Driver Group. R.J.X. is a consultant at Novartis, Janssen and Celgene. A.H., M.B., N.R., R.H., K.S., C.S., O.R., R.X. and A.R. are co-inventors on a provisional patent application filed by the Broad Institute relating to this manuscript.

Corresponding authors

Correspondence to Moshe Biton or Ramnik J. Xavier or Aviv Regev.

Reviewer Information Nature thanks L. Vermeulen 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

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1 - Summary of single-cell RNAseq experiments.

    This table provides the number (after quality filtering, see Methods) of individual intestinal epithelial cells profiled in each of the in this study.

  2. 2.

    Supplementary Table 2 - Cell-type specific signature genes – droplet-based dataset.

    This table provides the lists of genes specific to each of the identified clusters of intestinal epithelial cells, identified using 3’ droplet-based scRNA-seq data (Figure 1b).

  3. 3.

    Supplementary Table 3 - Cell-type specific signature genes – plate-based dataset.

    This table provides the lists of genes specific to each of the identified clusters of intestinal epithelial cells, identified using full-length scRNA-seq data (Extended Data Fig. 2a).

  4. 4.

    Supplementary Table 4 - Consensus cell-type specific signature genes – both datasets.

    This table provides high-confidence lists of genes specific to each subtype of intestinal epithelial cells in both 3’ droplet-based and full-length scRNA-seq datasets.

  5. 5.

    Supplementary Table 5 - Cell-type specific TFs and receptors.

    This table provides lists of genes annotated as either transcription factors (TFs), G protein-coupled receptors (GPCRs), or leucine-rich repeat (LRR) proteins, enriched in each subtype of intestinal epithelial cells in full-length plate-based scRNA-seq data.

  6. 6.

    Supplementary Table 6 - Enteroendocrine cell subset signature genes.

    This table provides the lists of genes specific to each of the identified subsets of tuft cells, identified using both 3’ droplet-based and full-length scRNA-seq data.

  7. 7.

    Supplementary Table 7 - Consensus tuft cell subset signature genes.

    This table provides the lists of genes specific to each of the identified subsets of tuft cells, identified using both 3’ droplet-based and full-length scRNA-seq data.

  8. 8.

    Supplementary Table 8 - In vitro and in vivo M cell signature genes.

    This table provides the lists of genes specific to intestinal microfold (M) cells, using 3’ droplet-based scRNA-seq data from in vitro cells derived from RANKL-treated organoids, and in vivo cells derived from the mouse follicle associated epithelia (FAE).

  9. 9.

    Supplementary Table 9 - Intestinal epithelial response to pathogenic infection.

    This table provides estimates of differential gene expression in response to infection with H. polygyrus and Salmonella enterica, for each epithelial cell type, using both full-length and 3’ droplet-based scRNA-seq data.

  10. 10.

    Supplementary Table 10 - Markers of proximal and distal Paneth cells.

    This table provides estimates of differential gene expression between two subsets of Paneth cells identified by clustering and interpreted (post-hoc) as derived from proximal and distal small intestine (Extended Data Fig. 3).