Engineering functional human gastrointestinal organoid tissues using the three primary germ layers separately derived from pluripotent stem cells

Alexandra K. Eicher, Daniel O. Kechele, Nambirajan Sundaram, H. Matthew Berns, Holly M. Poling, Lauren E. Haines, J. Guillermo Sanchez, Keishi Kishimoto, Mansa Krishnamurthy, Lu Han, Aaron M. Zorn, Michael A. Helmrath, James M. Wells

Preprint posted on 16 July 2021

Article now published in Cell Stem Cell at

Germ layer human gastrointestinal organoid tissues: A novel model system to understand stomach development.

Selected by Niveda Udaykumar


The stomach is a major organ of the gastrointestinal (GI) tract, and aids in the mechanical and chemical breakdown of ingested food. Like other organ development, the components of the stomach develop from the three germ layers. The endoderm forms the inner epithelial lining, the smooth muscle layer forms from the mesoderm, and the ectoderm gives rise to the enteric nervous system (ENS) – all three of which are necessary for the proper functioning of the stomach [1-4].

Signalling pathways play important roles in regulating morphogenesis, progenitor differentiation, and establishing regional identity during embryonic development [5]. Evidence from chick embryos indicates the presence of a reciprocal signalling module involving sonic hedgehog (Shh), which is secreted by epithelial cells and regulates BMP signalling in the adjacent mesenchyme [6-8]. This eventually results in proliferation/differentiation of the ENCCs (enteric neural crest cells) 9], and patterning, growth, and maturation of the stomach mesenchyme [10].

Although animal models facilitate the study of stomach development and disease, major structural and developmental differences exist in the stomach between different species [11]. Humans lack the forestomach found in rodents, and the avian gizzard is very different from the human stomach antrum [12]. In addition, mouse and chick animal models have accessibility and genetics difficulties, that limit the study of stomach development.

To overcome these issues, in this preprint, Eicher et. al., have generated a novel organoid system to recapitulate normal gastric development. This system consists of hPSCs (human pluripotent stem cells)-derived splanchnic mesenchyme and the ENCCs incorporated into human antral gastric organoids (hAGOs) and human fundic gastric organoids (hFGOs). This approach resulted in functional organoids with the epithelial glands innervated by smooth muscle. These functional organoids may be valuable in studying various aspects of gut development, including communication between germ layers, the role of human ENCCs during/in embryonic stomach development, and tissue morphogenesis.

Key findings

Generating three germ layer gastric organoids from hPSCs

The stomach tissue consists of glandular units of simple columnar epithelial cells surrounded by many layers of differently oriented smooth muscle layers. To best recapitulate the human stomach morphogenesis in vitro, the authors generated gastric organoids by incorporating enteric neural crest cells (ENCCs) and splanchnic mesenchyme (SM) with the hAGO spheroids [ENCCs+SM+hAGO], with each layer, derived separately from hPSCs. First,the neural crest cells (NCCs) were differentiated from RFP labelled human pluripotent stem cells (hPSCs) and incorporated with GFP labelled SM into hAGOs spheroids. Second, GFP-labelled splanchnic mesenchyme (SM) was generated from an hPSC line with constitutive GFP expression by a two-step process. The hPSCs were differentiated first into lateral plate mesoderm (LPM), as LPM can give rise to SM and cardiac fate. Treatment of LPM to retinoic acid (RA) induces SM fate, detected by the upregulation of the SM marker, FOXF1 followed by the downregulation of cardiac markers.

The spheroids were monitored for proper incorporation of the germ layers in the hAGOs in vitro followed by their transplantation into mice for 10-12 weeks for growth and maturation. Histological analysis of organoids showed well-organized smooth muscle layers with the distinct cellular architecture of the gastric tissue. In addition, the organoids also showed the characteristic glandular structures found in the human stomach.

The appearance of the gastric epithelial marker CLDN18 and the lack of intestinal epithelial marker CDH17, as identified by immunostaining, confirmed the gastric identity of the organoids. Additionally, the hAGO glands contained all expected cell types along with a neural network-like plexus embedded within the smooth muscle layer. Also, the 12-week hAGO was like the 38-week human gastric tissue, suggesting that the engineered three germ layer hAGO recapitulated the morphology and development of the human gastric tissue.  Taken together, this suggests that the gastric organoids derived from the three germ layers separately may be a potential model system for the study of human stomach development.

Figure 1: Schematic demonstrating the procedure to engineer three germ layer gastric organoids. (Adapted from Figure 2, Eicher et al., 2021).

The germ layer hAGOs display functional muscle contractions

The mechanical breakdown of food in the stomach occurs via contraction of the smooth muscles, which is controlled by the ENS. To investigate the ability of the organoid ENS and smooth muscle layer to form a neuromuscular junction, they monitored contractibility in an organ bath chamber. After a brief period of equilibration, the organoids showed spontaneous contractile oscillations. The oscillations demonstrated the presence of intramuscular interstitial cells of Cajal (ICCs), which was further supported by the expression of c-KIT, a marker of ICC, and the neuroglial cell marker TUJ1.

Next, the ENS of the engineered organoids were assessed for their ability to control gastric tissue contractions using electric field stimulation (EFS). EFS helps trigger neuronal firing followed by smooth muscle contractions. Administration of EFS pulses in the organoids resulted in an increase in their contractile activity. Treatment of the organoids with tetrodotoxin (TTX) resulted in a decrease in contractile activity, demonstrating that the ENS can regulate smooth muscle contractions in the engineered hAGO organoids.

Figure 2: A. Contractile activity of isolated tissue from one thAGO+SM(blue) and three thAGO+SM+ ENCC (red) triggered by EFS. B. Loss of contractile activity triggered by EFS in the three germ layer organoids on treatment with TTX. (Adapted from Figure 4, Eicher et al., 2021).

The engineered hAGOs may be used to study the contribution of the three germ layers during gastric tissue development

To study this aspect of gastric tissue development, hPSCs were differentiated to migrating vagal-like ENCCs, recombined with hAGOs [ENCC+hAGO], and assessed for their ability to form the ENS in the absence of the exogenous mesenchyme layer. Although the ENCCs differentiated into different neuron subtypes with appropriate morphology, the spatial orientation and ENS development in these organoids were abnormal, suggesting that the mesenchyme is required for the proper spatial organization and ENS development during gastric tissue development. Transplantation of the ENCC+hAGO organoids in mice demonstrated that the ENCCs supported the growth and glandular morphogenesis of the organoid epithelium in vivo. Further, a time-course analysis of the transplanted organoids showed differentiated smooth muscle and neuroglial cells, demonstrating the contribution of the ENCCs during gastric tissue development. In addition, analysis of the organoid epithelium displayed similarities to Brunner’s glands, both morphologically and molecularly by combinatorial expression profiling. Brunner’s glands secrete sodium bicarbonate to neutralize gastric acids and are found in the submucosa. The absence of the mesenchyme and the added ENCCs induced a posterior fate to the gastric epithelium, possibly through BMP signalling. Culturing the organoids in the presence of noggin, a BMP signalling inhibitor, resulted in the absence of Brunner’s glands in the gastric epithelium. Taken together, the ENCCs are required for proper ENS development, growth, and production of posteriorizing factors such as BMP4 and BMP7.

Why I chose to highlight this preprint

I chose to highlight this preprint as I thought the authors used a clever approach to engineer hAGOs using the three germ layers and performed a comprehensive study to understand gastric tissue development. This preprint also beautifully unpicks the role of the individual germ layers in the development of gastric tissue. Additionally, generating functional organoids using this technique may open new avenues to engineer, study and understand the development of various tissues that are limited by the lack of appropriate animal model systems.

Questions to the authors

  • How did the group come up with the idea of engineering the hAGOs with the three germ layers?
  • What factor(s) in the mesenchyme do you think contributes to the precise spatial organization of the ENS during gastric tissue development?


1.Furness, J. B., Di Natale, M., Hunne, B., Oparija-Rogenmozere, L., Ward, S. M., Sasse, K. C.,

2. Powley, T. L., Stebbing, M. J., Jaffey, D. and Fothergill, L. J. (2020) ‘The identification of neuronal control pathways supplying effector tissues in the stomach’, Cell Tissue Res, 382(3) pp. 433-445.

3.Zhao, C. M., Martinez, V., Piqueras, L., Wang, L., Taché, Y. and Chen, D. (2008) ‘Control of gastric acid secretion in somatostatin receptor 2 deficient mice: shift from endocrine/paracrine to neurocrine pathways’, Endocrinology, 149(2), pp. 498-505.

4.Norlen, P., Ericsson, P., Kitano, M., Ekelund, M. and Hakanson, 1221 (2005) ‘The vagus regulates histamine mobilization from rat stomach ECL cells by controlling their sensitivity to gastrin’, J Physiol, 564(Pt 3), pp. 895-905.

5.Rydning, A., Lyng, O., Falkmer, S. and Grønbech, J. E. (2002) ‘Histamine is involved in gastric vasodilation during acid back diffusion via activation of sensory neurons’, Am J Physiol Gastrointest Liver Physiol, 283(3), pp. G603-11.

6.Le Guen, L., Marchal, S., Faure, S. and de Santa Barbara, P. (2015) ‘Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration, Cell Mol Life Sci, 72(20), pp. 3883-96.

7. Roberts, D. J., Johnson, R. L., Burke, A. C., Nelson, C. E., Morgan, B. A. and Tabin, C. (1995).’Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut’, Development, 121(10), pp. 3163-74.

8.Faure, S., de Santa Barbara, P., Roberts, D. J. and Whitman, M. (2002).Endogenous patterns of BMP signaling during early chick development, Dev Biol, 244(1), pp. 44-65.

9.De Santa Barbara, P., Williams, J., Goldstein, A. M., Doyle, A. M., Nielsen, C., Winfield, S., Faure, S. and Roberts, D. J. (2005) ‘Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development’, Dev Dyn, 234(2), pp. 312-22.

10. Nagy, N., Barad, C., Graham, H. K., Hotta, R., Cheng, L. S., Fejszak, N. and Goldstein, A. M. (2016) ‘Sonic hedgehog controls enteric nervous system development by patterning the extracellular matrix’, Development, 143(2), pp. 264-75.

11. Faure, S., McKey, J., Sagnol, S. and de Santa Barbara, P. (2015) ‘Enteric neural crest cells regulate vertebrate stomach patterning and differentiation, Development, 142(2), pp. 331-42.

12. de Santa Barbara, P., van den Brink, G. R. and Roberts, D. J. (2002) ‘Molecular etiology of gut malformations and diseases’, Am J Med Genet, 115(4), pp. 221-30.

13.Kim, T. H. and Shivdasani, R. A. (2016) ‘Stomach development, stem cells and disease’, Development, 143(4), pp. 554-65.

Tags: developmental biology, model system, organoids

Posted on: 14 August 2021


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Author's response

Alexandra Eicher shared

1) How did the group come up with the idea of engineering the hAGOs with the three germ layers?

Our previous work to engineer human intestinal organoids (HIOs) with a functional enteric nervous system (ENS) (Workman et al. 2017) laid the initial groundwork to consider innervating further human organoids. We focused on the more proximal stomach organoids as there is a significant disparity between the published knowledge of intestinal versus stomach ENS, with intestinal ENS research far surpassing that of the stomach. However, it quickly became apparent that hAGOs, in contrast to HIOs, lacked the mesenchymal compartment necessary to support proper ENS development. So, we utilized a new differentiation protocol for splanchnic mesenchyme that was recently published in Han et al. 2020 to engineer hAGOs with cell types from all three germ layers.

2) What factor(s) in the mesenchyme do you think contributes to the precise spatial organization of the ENS during gastric tissue development?

For gastric, as well as more proximal gastrointestinal (GI) development, we actually think this is the other way around. While there is evidence that distal GI mesenchyme is necessary for intestinal and colonic ENS development (Graham et al. 2017 and Bourret et al. 2017), there is opposite evidence for more proximal GI organs like the stomach. In chick (Faure et al. 2015), ENCCs are shown to regulate the correct patterning and proliferative growth of gastric mesenchyme and we see similar results with our hAGOs engineered with only an ENS and no additional mesenchyme. These discrete ENCCs are capable of surviving, differentiating, and forming a primitive neuroglial network independent of robust mesenchyme. They even pattern and encourage the growth of what little mesenchyme is present within the system. However, they organize quite closely to the hAGO epithelium. It is only with a proper quantity of additional mesenchyme do ENCCs spatially organize away from the epithelium and into characteristic plexi layers among the smooth muscle layers.

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