Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Generating human intestinal tissue from pluripotent stem cells in vitro


Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2+SOX17+ endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2+ mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1–3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Passaging hPSCs and differentiation to DE.
Figure 2: Spheroid formation from hPSC-derived definitive endoderm.
Figure 3: Three-dimensional growth of intestinal organoids in Matrigel.


  1. Spence, J.R., Lauf, R. & Shroyer, N.F. Vertebrate intestinal endoderm development. Dev. Dyn. 240, 501–520 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Wells, J.M. & Melton, D.A. Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development 127, 1563–1572 (2000).

    CAS  PubMed  Google Scholar 

  3. Dessimoz, J., Opoka, R., Kordich, J.J., Grapin-Botton, A. & Wells, J.M. FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo. Mech. Dev. 123, 42–55 (2006).

    CAS  Article  PubMed  Google Scholar 

  4. McLin, V.A., Rankin, S.A. & Zorn, A.M. Repression of Wnt/beta-catenin signaling in the anterior endoderm is essential for liver and pancreas development. Development 134, 2207–2217 (2007).

    CAS  Article  PubMed  Google Scholar 

  5. Spence, J.R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).

    Article  PubMed  Google Scholar 

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

    CAS  Article  Google Scholar 

  7. Leibach, F.H. & Ganapathy, V. Peptide transporters in the intestine and the kidney. Annu. Rev. Nutr. 16, 99–119 (1996).

    CAS  Article  PubMed  Google Scholar 

  8. Adibi, S.A. The oligopeptide transporter (Pept-1) in human intestine: biology and function. Gastroenterology 113, 332–340 (1997).

    CAS  Article  PubMed  Google Scholar 

  9. Yamada, T. et al. In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells 20, 146–154 (2002).

    Article  PubMed  Google Scholar 

  10. Ishikawa, T. et al. Characterization of in vitro gutlike organ formed from mouse embryonic stem cells. Am. J. Physiol. Cell Physiol. 286, C1344–C1352 (2004).

    CAS  Article  PubMed  Google Scholar 

  11. Matsuura, R. et al. Crucial transcription factors in endoderm and embryonic gut development are expressed in gut-like structures from mouse ES cells. Stem Cells 24, 624–630 (2006).

    Article  PubMed  Google Scholar 

  12. Konuma, N. et al. Mouse embryonic stem cells give rise to gut-like morphogenesis, including intestinal stem cells, in the embryoid body model. Stem Cells Dev. 18, 113–126 (2009).

    CAS  Article  PubMed  Google Scholar 

  13. Axelrod, H.R. Embryonic stem cell lines derived from blastocysts by a simplified technique. Dev. Biol. 101, 225–228 (1984).

    CAS  Article  PubMed  Google Scholar 

  14. Cao, L. et al. Intestinal lineage commitment of embryonic stem cells. Differentiation 81, 1–10 (2011).

    CAS  Article  PubMed  Google Scholar 

  15. Hakala, H. et al. Comparison of biomaterials and extracellular matrices as a culture platform for multiple, independently derived human embryonic stem cell lines. Tissue Eng. Part A 15, 1775–1785 (2009).

    CAS  Article  PubMed  Google Scholar 

  16. van der Flier, L.G. & Clevers, H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241–260 (2009).

    CAS  Article  PubMed  Google Scholar 

Download references


This work was supported by the Juvenile Diabetes Research Foundation (JDRF-2-2003-530 to J.M.W.) and the US National Institutes of Health (R01GM072915, R01DK080823A1 and S1 to J.M.W.). This work was also supported in part by PHS grants P30 DK078392 (to J.R.S.) and K01 DK091415 (to J.R.S.). J.C.H. is supported by an Endocrine Fellows Foundation Developmental Research Grant in Diabetes, Obesity and Fat Cell Biology. K.W.M. is partially supported by the Training Program in Organogenesis award 5T32HD046387-05 and by award no. T32-GM063483 from the National Institute of General Medical Sciences. We also acknowledge core support from the Pluripotent Stem Cell Facility of Cincinnati Children's Hospital (supported by grant no. U54 RR025216). We thank J. Whitsett (Cincinnati Children's Hospital Medical Center) for rabbit anti-FOXA2 antibodies and the Pluripotent Stem Cell Facility of Cincinnati Children's Hospital for iPSCs.

Author information

Authors and Affiliations



J.M.W., J.R.S., J.C.H. and K.W.M. conceived the study and experimental design. J.M.W. and J.R.S. analyzed data and co-wrote the manuscript. J.C.H. and K.W.M. performed experiments.

Corresponding author

Correspondence to James M Wells.

Ethics declarations

Competing interests

J.M.W. and J.R.S. are inventors on a patent involving the system described in this protocol.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

McCracken, K., Howell, J., Wells, J. et al. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat Protoc 6, 1920–1928 (2011).

Download citation

  • Published:

  • Issue Date:

  • DOI:

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing