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Insulin is expressed by enteroendocrine cells during human fetal development


Generation of beta cells via transdifferentiation of other cell types is a promising avenue for the treatment of diabetes. Here we reconstruct a single-cell atlas of the human fetal and neonatal small intestine. We identify a subset of fetal enteroendocrine K/L cells that express high levels of insulin and other beta cell genes. Our findings highlight a potential extra-pancreatic source of beta cells and expose its molecular blueprint.

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Fig. 1: Fetal human K/L enteroendocrine cells contain a subset of INS+ cells.
Fig. 2: In situ validation for the expression of INS+ cells in the fetal human small intestine.

Data availability

All data generated in this study are available at the Zenodo repository: Immune cells from fetal samples 1100A and 1102 and the neonatal sample 1127 were presented in Olaloye et al.36. Data from Cao et al.8 are available at Data from Baron et al.9 are available at Data from Elmentaite et al.14 are available at

Code availability

All custom code used in this study is available at the Zenodo repository:


  1. Merrell, A. J. & Stanger, B. Z. Adult cell plasticity in vivo: de-differentiation and transdifferentiation are back in style. Nat. Rev. Mol. Cell Biol. 17, 413–425 (2016).

    Article  CAS  Google Scholar 

  2. Jennings, R. E., Berry, A. A., Strutt, J. P., Gerrard, D. T. & Hanley, N. A. Human pancreas development. Development 142, 3126–3137 (2015).

    Article  CAS  Google Scholar 

  3. Habib, A. M. et al. Overlap of endocrine hormone expression in the mouse intestine revealed by transcriptional profiling and flow cytometry. Endocrinology 153, 3054–3065 (2012).

    Article  CAS  Google Scholar 

  4. Talchai, C., Xuan, S., Kitamura, T., DePinho, R. A. & Accili, D. Generation of functional insulin-producing cells in the gut by Foxo1 ablation. Nat. Genet. 44, 406–412 (2012).

    Article  CAS  Google Scholar 

  5. Bouchi, R. et al. FOXO1 inhibition yields functional insulin-producing cells in human gut organoid cultures. Nat. Commun. 5, 4242 (2014).

    Article  CAS  Google Scholar 

  6. Chen, Y.-J. et al. De novo formation of insulin-producing ‘neo-β cell islets’ from intestinal crypts. Cell Rep. 6, 1046–1058 (2014).

    Article  CAS  Google Scholar 

  7. Suissa, Y. et al. Gastrin: a distinct fate of neurogenin3 positive progenitor cells in the embryonic pancreas. PLoS ONE 8, e70397 (2013).

    Article  CAS  Google Scholar 

  8. Cao, J. et al. A human cell atlas of fetal gene expression. Science 370, eaba7721 (2020).

    Article  CAS  Google Scholar 

  9. Baron, M. et al. A single-cell transcriptomic map of the human and mouse pancreas reveals inter- and intra-cell population structure. Cell Syst. 3, 346–360 (2016).

    Article  CAS  Google Scholar 

  10. Blum, B. et al. Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3. Nat. Biotechnol. 30, 261–264 (2012).

    Article  CAS  Google Scholar 

  11. Matschinsky, F. M. & Wilson, D. F. The central role of glucokinase in glucose homeostasis: a perspective 50 years after demonstrating the presence of the enzyme in islets of Langerhans. Front. Physiol. 10, 148 (2019).

    Article  Google Scholar 

  12. Vivot, K. et al. The regulator of G-protein signaling RGS16 promotes insulin secretion and β-cell proliferation in rodent and human islets. Mol. Metab. 5, 988–996 (2016).

    Article  CAS  Google Scholar 

  13. Gross, S. et al. Lmx1a functions in intestinal serotonin-producing enterochromaffin cells downstream of Nkx2.2. Development 143, 2616–2628 (2016).

  14. Elmentaite, R. et al. Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn’s disease. Dev. Cell 55, 771–783 (2020).

    Article  CAS  Google Scholar 

  15. Farack, L. et al. Transcriptional heterogeneity of beta cells in the intact pancreas. Dev. Cell 48, 115–125 (2019).

    Article  CAS  Google Scholar 

  16. Nowak-Sliwinska, P. et al. Oncofoetal insulin receptor isoform A marks the tumour endothelium; an underestimated pathway during tumour angiogenesis and angiostatic treatment. Br. J. Cancer 120, 218–228 (2019).

    Article  CAS  Google Scholar 

  17. Menon, R. K. & Sperling, M. A. Insulin as a growth factor. Endocrinol. Metab. Clin. North Am. 25, 633–647 (1996).

    Article  CAS  Google Scholar 

  18. Herrera, P. L. Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development 127, 2317–2322 (2000).

    Article  CAS  Google Scholar 

  19. Teitelman, G., Alpert, S., Polak, J. M., Martinez, A. & Hanahan, D. Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development 118, 1031–1039 (1993).

    Article  CAS  Google Scholar 

  20. Kaspi, H., Pasvolsky, R. & Hornstein, E. Could microRNAs contribute to the maintenance of β cell identity? Trends Endocrinol. Metab. 25, 285–292 (2014).

    Article  CAS  Google Scholar 

  21. Stras, S. F. et al. Maturation of the human intestinal immune system occurs early in fetal development. Dev. Cell 51, 357–373 (2019).

    Article  CAS  Google Scholar 

  22. Konnikova, L. et al. High-dimensional immune phenotyping and transcriptional analyses reveal robust recovery of viable human immune and epithelial cells from frozen gastrointestinal tissue. Mucosal Immunol. 11, 1684–1693 (2018).

    Article  CAS  Google Scholar 

  23. Xin, H. et al. GMM-Demux: sample demultiplexing, multiplet detection, experiment planning, and novel cell-type verification in single cell sequencing. Genome Biol. 21, 188 (2020).

    Article  CAS  Google Scholar 

  24. Luo, J., Erb, C. A. & Chen, K. Simultaneous measurement of surface proteins and gene expression from single cells. In: T-Cell Receptor Signaling Vol. 2111 35–46 (ed Liu, C.) (Springer, 2020).

  25. Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).

    Article  CAS  Google Scholar 

  26. Van den Brink, S. C. et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat. Methods 14, 935–936 (2017).

    Article  Google Scholar 

  27. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. 57, 289–300 (1995).

    Google Scholar 

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

    Article  Google Scholar 

  29. Massalha, H. et al. A single cell atlas of the human liver tumor microenvironment. Mol. Syst. Biol. 16, e9682 (2020).

    Article  CAS  Google Scholar 

  30. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  31. Scharfmann, R., Staels, W. & Albagli, O. The supply chain of human pancreatic β cell lines. J. Clin. Invest. 129, 3511–3520 (2019).

    Article  Google Scholar 

  32. Sender, R. & Milo, R. The distribution of cellular turnover in the human body. Nat. Med. 27, 45–48 (2021).

    Article  CAS  Google Scholar 

  33. Salomon, L. J., Bernard, J. P. & Ville, Y. Estimation of fetal weight: reference range at 20–36 weeks’ gestation and comparison with actual birth-weight reference range. Ultrasound Obstet. Gynecol. 29, 550–555 (2007).

    Article  CAS  Google Scholar 

  34. Manco, R. et al. Clump sequencing exposes the spatial expression programs of intestinal secretory cells. Nat. Commun. 12, 3074 (2021).

    Article  CAS  Google Scholar 

  35. Buse, M. G., Roberts, W. J. & Buse, J. The role of the human placenta in the transfer and metabolism of insulin. J. Clin. Invest. 41, 29–41 (1962).

    Article  CAS  Google Scholar 

  36. Olaloye, O. O. et al. CD16+CD163+ monocytes traffic to sites of inflammation during necrotizing enterocolitis in premature infants. J. Exp. Med. 218, e20200344 (2021).

    Article  CAS  Google Scholar 

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We thank Y. Dor and R. Scharfmann for insightful comments. S.I. is supported by the Wolfson Family Charitable Trust, the Edmond de Rothschild Foundations, the Fannie Sherr Fund, the Dr. Beth Rom-Rymer Stem Cell Research Fund, the Minerva Stiftung grant, Israel Science Foundation grant number 1486/16, Broad Institute‐Israel Science Foundation grant number 2615/18, the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program (grant no. 768956), Chan Zuckerberg Initiative grant number CZF2019‐002434, the network of Pancreatic Organ Donors with Diabetes (nPOD), the Bert L. and N. Kuggie Vallee Foundation and the Howard Hughes Medical Institute international research scholar award. L.K. is supported by previous start-up funds from the University of Pittsburgh and current start-up funds from Yale University, Binational Science Foundation award number 2019075 and National Institute of Health (NIH) grants R21TR002639 and R21HD102565. No NIH funds were used for the fetal work of these studies.

Author information

Authors and Affiliations



S.I., L.K. and A.E. conceived the study. D.L. and B.M. were involved in sample collection, processing and preparation for single-cell analysis. X.A., F.W. and K.C. were involved in library preparation. A.E. performed computational analysis, smFISH + immunofluorescence experiments and image analysis. L.F. designed the combined protocol. L.F. and K.B.H. performed smFISH + immunofluorescence experiments. L.K. and S.I. supervised the entirety of the project. All authors approved the manuscript.

Corresponding authors

Correspondence to Liza Konnikova or Shalev Itzkovitz.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Medicine thanks Dominic Grun, Gordon Weir and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anna Maria Ranzoni was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 Analysis of differentially-expressed genes between the human fetal and neonatal small intestines.

a-b, UMAP of small intestinal cells colored by age (a) or subject (b). c, MA plot showing differentially expressed genes between fetal and neonatal cells stratified by cell type. Genes in red have q-values below 0.1 and expression above 5e-5. INS, the most up-regulated gene in fetal enteroendocrine cells, is highlighted with a blue box.

Extended Data Fig. 2 Characterization of the fetal enteroendocrine cell types.

a, MA plot showing differentially expressed genes between fetal and neonatal enteroendocrine cells stratified by cell type. Genes in red have q-values below 0.05 and expression above 1e-4. INS, the most up-regulated gene in fetal K/L cells, is highlighted with a blue box. b, Spearman correlation distances between adult beta cells and the fetal endocrine cell types. Each dot represents a distance obtained from one of 100 bootstrap iterations, in which cells were sampled with replacement from the complete dataset. Distances from beta cells are significantly smaller for FIKL cells compared to all other enteroendocrine cell types (two-sided Wilcoxon rank-sum p = 1e-31 for the differences between FIKL cells and K/L INS- cells, the second closest enteroendocrine cell population). White circles are medians, gray boxes mark the 25–75 percentiles, gray lines extend from 1.5 times the interquartile range (IQR) above the 75 percentile to 1.5 times the IQR below the 25 percentile and truncated at the minimal or maximal measured values. c, RGS16 expression (log10 of the sum-normalized UMI counts).

Extended Data Fig. 3 Analysis of gene regulatory networks.

a, Clustergram of TF activities Z-score of mean AUC for each cell type. The list includes TFs with the most differential activities in the different cell types (Methods). b-g, Selected TFs from (a). In b-g, each dot is a cell, white circles are medians, gray boxes mark the 25–75 percentiles, gray lines extend from 1.5 times the interquartile range (IQR) above the 75 percentile to 1.5 times the IQR below the 25 percentile and truncated at the minimal or maximal measured values. Black lines connect the means.

Extended Data Fig. 4 FIKL cells appear in earlier developmental time points.

a-b, Combined UMAP of 1st trimester14 and 2nd trimester colored by enteroendocrine subtype (a) or INS expression (log10(normalized expression)), panel b. Dots with a black outline in a-b are 1st trimester cells, dots without an outline are 2nd trimester cells. c, Heatmap of INS + cells enrichment in each enteroendocrine subtype. Colors are –log10(hypergeometric p-value for the enrichment of INS + cells within each cell cluster).

Extended Data Fig. 5 In-situ validation of FIKL cells at different developmental stages.

a, Intestinal section with a typical example of an INS + cell marked with INS protein (green) from a 12 weeks GA fetus. Scale bar - 50 µm. b, Blowup of the INS + cell from (a) stained with both INS-mRNA (magenta) and INS-protein (green). Scale bar - 5 µm. c-f, Representatives examples of INS + cells in small intestine tissues from 12 weeks GA (c, 3 cells out of 20 found), 14 weeks GA (d, 2 cells out of 3 found), 18 weeks GA (e, 1 cell out of 1 found) and 21 weeks GA (f, 3 cells out of 23 found) stained with INS-mRNA (magenta), INS-protein (green). Scale bar - 5 µm. In all images nuclei are stained by DAPI (blue).

Extended Data Fig. 6 Insulin is not detected in embryonic mouse small intestine.

a, Mouse embryo intestinal section (E16.5) stained with the pan-enteroendocrine cell marker Chga (gray) and blowups of selected Chga+ cells. Scalebar - 50 µm for big image and 10 µm for blowups. b, Mouse embryo pancreatic tissue stained with Ins2-mRNA (magenta) and INS-protein (green) shown as a positive control for the in-situ detection of mouse cells expressing insulin. Scale bar - 10 µm. In all images nuclei are stained by DAPI (blue). Images are representative of 6 E16.5 embryonic subjects and 5 P0 neonatal subjects.

Extended Data Fig. 7 Expression of INSR in small intestinal cell types.

INSR expression in all cell types of 1st trimester small intestinal data from14 (a) and from 2nd trimester small intestine (b). Cell types are ranked by the mean INSR expression. Expression units are Seurat normalized (Methods).

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Egozi, A., Llivichuzhca-Loja, D., McCourt, B.T. et al. Insulin is expressed by enteroendocrine cells during human fetal development. Nat Med 27, 2104–2107 (2021).

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