Skip to main content

Generation of Gastrointestinal Organoids from Human Pluripotent Stem Cells

Part of the Methods in Molecular Biology book series (MIMB,volume 1597)


Over the past several decades, developmental biologists have discovered fundamental mechanisms by which organs form in developing embryos. With this information it is now possible to generate human “organoids” by the stepwise differentiation of human pluripotent stem cells using a process that recapitulates organ development. For the gastrointestinal tract, one of the first key steps is the formation of definitive endoderm and mesoderm, a process that relies on the TGFb molecule Nodal. Endoderm is then patterned along the anterior-posterior axis, with anterior endoderm forming the foregut and posterior endoderm forming the mid and hindgut. A-P patterning of the endoderm is accomplished by the combined activities of Wnt, BMP, and FGF. High Wnt and BMP promote a posterior fate, whereas repressing these pathways promotes an anterior endoderm fate. The stomach derives from the posterior foregut and retinoic acid signaling is required for promoting a posterior foregut fate. The small and large intestine derive from the mid and hindgut, respectively.

These stages of gastrointestinal development can be precisely manipulated through the temporal activation and repression of the pathways mentioned above. For example, stimulation of the Nodal pathway with the mimetic Activin A, another TGF-β superfamily member, can trigger the differentiation of pluripotent stem cells into definitive endoderm (D’Amour et al., Nat Biotechnol 23:1534–1541, 2005). Exposure of definitive endoderm to high levels of Wnt and FGF promotes the formation of posterior endoderm and mid/hindgut tissue that expresses CDX2. Mid-hindgut spheroids that are cultured in a three-dimensional matrix form human intestinal organoids (HIOs) that are small intestinal in nature Spence et al., Nature 2011. In contrast, activation of FGF and Wnt in the presence of the BMP inhibitor Noggin promotes the formation of anterior endoderm and foregut tissues that express SOX2. These SOX2-expressing foregut spheroids can be further patterned into posterior foregut by addition of retinoic acid. Once formed, these posterior foregut spheroids can be grown in three-dimensional human gastric organoids (HGOs) that have all of the cell types of antral part of the stomach (Mc Cracken et al. 2014).

Here, we describe the detailed methods for generating stomach/human gastric organoids (HGOs) and human intestinal organoids (HIOs) from human pluripotent stem cells. We first present a method for generating definitive endoderm from pluripotent stem cells followed by differentiation of definitive endoderm into either posterior foregut spheroids or mid-hindgut spheroids. We then describe how three-dimensional culturing of these spheroids results in the formation of HGOs and HIOs, respectively.

Key words

  • Definitive endoderm
  • Foregut
  • Hindgut
  • Human pluripotent stem cells
  • Gastric organoids
  • Intestinal organoids

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-6949-4_12
  • Chapter length: 11 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
USD   129.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-6949-4
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   169.99
Price excludes VAT (USA)
Hardcover Book
USD   169.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. McCracken KW, Cata EM, Crawford CM et al (2014) Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516:400–404

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  2. McCracken KW, Howell JC, Wells JM et al (2011) Generating human intestinal tissue from pluripotent stem cells in vitro. Nat Protoc 6:1920–1928

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  3. Spence JR, Mayhew CN, Rankin SA et al (2011) Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470:105–109

    CrossRef  PubMed  Google Scholar 

  4. Wells JM, Spence JR (2014) How to make an intestine. Development 141:752–760

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. D’Amour KA et al (2005) Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23:1534–1541. doi:10.1038/nbt1163

    CrossRef  PubMed  Google Scholar 

  6. Sato T et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265. doi:10.1038/nature07935

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to James M. Wells .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Múnera, J.O., Wells, J.M. (2017). Generation of Gastrointestinal Organoids from Human Pluripotent Stem Cells. In: Tsuji, T. (eds) Organ Regeneration. Methods in Molecular Biology, vol 1597. Humana Press, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6947-0

  • Online ISBN: 978-1-4939-6949-4

  • eBook Packages: Springer Protocols