Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro

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Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro1, 2. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells3, 4, 5, 6 that have therapeutic efficacy in animal models of liver disease7, 8 and diabetes9, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development10. This involved activin-induced definitive endoderm formation11, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis12, 13, 14, and a pro-intestinal culture system15, 16 to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal ‘organoids’ consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers17. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis18, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.

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  1. FGF4 and WNT3A act synergistically in a temporal and dose-dependent manner to specify stable posterior endoderm fate.
    Figure 1: FGF4 and WNT3A act synergistically in a temporal and dose-dependent manner to specify stable posterior endoderm fate.

    ad, Activin A (100ngml−1) was used to differentiate H9 human ES cells into definitive endoderm. Definitive endoderm was treated with the posteriorizing factors FGF4 (50 or 500ng), WNT3A (50 or 500ng), or both for 6, 48 or 96h. Cells were placed in permissive media for 7 days and expression of foregut markers (ALB, PDX1) and the hindgut marker (CDX2) were analysed by RT–qPCR (a) and immunofluorescence (bd). The definitive endoderm of controls was grown for identical lengths of time in the absence of FGF4 or WNT3A. High levels of FGF4+WNT3A for 96h resulted in stable CDX2 expression and lack of foregut marker expression. Scale bars, 50μm. Error bars are s.e.m. (n = 3). *P<0.05, **P<0.001, ***P<0.0001.

  2. Morphogenesis of posterior endoderm into three-dimensional, hindgut-like spheroids.
    Figure 2: Morphogenesis of posterior endoderm into three-dimensional, hindgut-like spheroids.

    a, Bright-field images of definitive endoderm cultured for 96h in media, FGF4, WNT3A or FGF4+WNT3A. FGF4+WNT3A cultures contained three-dimensional epithelial tubes and free-floating spheres (black arrows) b, CDX2 immunostaining (green) and nuclear stain (DRAQ5, blue) on cultures shown in a. Insets show CDX2 staining alone. c, Bright-field image of hindgut-like spheroids. ac, Scale bars, 50μm. df, Analysis of CDX2, basal-lateral laminin and E-cadherin expression demonstrates an inner layer of polarized, cuboidal, CDX2+ epithelium surrounded by non-polarized mesenchymal CDX2+ cells. Scale bar in e is 20μm.g, CDX2 expression in an e8.5 mouse embryo (sagittal section). Inset is a magnified view showing that both hindgut endoderm (E; outlined with a red dashed line) and adjacent mesenchyme (M) are CDX2 positive (green). FG, foregut; HG, hindgut. Scale bar, 100μm.

  3. Human ES cells and iPSCs form three-dimensional intestine-like organoids.
    Figure 3: Human ES cells and iPSCs form three-dimensional intestine-like organoids.

    a, A time course shows that intestinal organoids formed highly convoluted epithelial structures surrounded by mesenchyme after 13 days (d). be, Intestinal transcription factor expression (KLF5, CDX2, SOX9) and cell proliferation on serial sections of organoids after 14 and 28 days (serial sections are b and c, d and e). Ki67, nuclear proliferation antigen. Nuc, nuclei. f, g, Expression of KLF5, CDX2, and SOX9 in mouse fetal intestine at e14.5 (f) and e16.5 (g) is similar to developing intestinal organoids. The right panels show separate colour channels for d, e and g (bracket highlights the region shown in the panels on the right). hj, Whole mount in situ hybridization of 56-day-old organoids showing epithelial expression of SOX9 (h) and restricted ‘crypt-like’ expression of the stem cell markers LGR5 (i) and ASCL2 (j). Insets show sense controls for each probe. Scale bars, 20μm.

  4. Formation and function of intestinal cell types and regulation of enteroendocrine differentiation by NEUROG3.
    Figure 4: Formation and function of intestinal cell types and regulation of enteroendocrine differentiation by NEUROG3.

    ac, Twenty-eight-day iPSC-derived organoids were analysed for villin (VIL) and the goblet cell marker mucin (MUC2) (a), the Paneth cell marker lysozyme (LYZ) (b), or the endocrine cell marker chromogranin A (CHGA) (c). Nuc, nuclei. d, Electron micrograph showing an enterocyte cell with a characteristic brush border with microvilli (inset). e, Epithelial uptake of the fluorescently labelled dipeptide d-Ala-Lys-AMCA (arrowheads) indicating a functional peptide transport system. fh, Adenoviral expression of NEUROG3 (Ad-NEUROG3) causes a fivefold increase in CGA+ cells compared to a GFP control (Ad-GFP). n = 4 biological samples;*P = 0.005. ik, Organoids were generated from human ES cells that were stably transduced with shRNA-expressing lentiviral vectors. Compared to control shRNA organoids, NEUROG3 shRNA organoids had a 95% reduction in the number of CHGA+ cells. n = 3 for shRNA controls and n = 5 for NEUROG3-shRNA; *P = 0.018. Scale bar in a is 10μm; all others are 20μm.Error bars are s.e.m.

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Author information


  1. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA

    • Jason R. Spence,
    • Christopher N. Mayhew,
    • Scott A. Rankin,
    • Matthew F. Kuhar,
    • Kathryn Tolle,
    • Vladimir V. Kalinichenko,
    • Aaron M. Zorn,
    • Noah F. Shroyer &
    • James M. Wells
  2. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA

    • Jefferson E. Vallance &
    • Noah F. Shroyer
  3. Division of Hematology and Oncology Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA

    • Elizabeth E. Hoskins &
    • Susanne I. Wells
  4. Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA

    • Vladimir V. Kalinichenko


J.M.W. and J.R.S. conceived the study and experimental design, performed and analysed experiments and co-wrote the manuscript. S.A.R., M.F.K. and J.E.V. performed experiments. C.N.M., M.F.K., K.T., V.V.K., J.E.V., E.E.H. and S.I.W. provided reagents, conceptual and/or technical support in generating and characterizing iPSC lines and intestinal organoids. N.F.S. and A.M.Z. provided additional conceptual and experimental support and co-funded the project. All authors read and approved the final manuscript.

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The authors declare no competing financial interests.

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Data have been deposited at NCBI under accession number GSE25557.

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