Human gastric (stomach) organoids can now be assembled in vitro from three pluripotent-stem-cell-derived germ-layer progenitors — epithelium, splanchnic mesenchyme, and enteric neural crest cells — yielding tissue with patterned glands, layered smooth muscle, and functional enteric nerves (Eicher et al., Cell Stem Cell 2022). That work established that all three lineages must communicate: enteric neural crest cells promote mesenchymal growth and glandular morphogenesis but, through BMP signaling, posteriorize the epithelium toward a non-gastric, Brunner's-gland-like fate unless a robust mesenchymal population is present. The molecular identities of most of the underlying signals and the causal logic of this three-way crosstalk remain unresolved: which enteric-nervous-system-derived signals drive mesenchymal proliferation and gastric gland morphogenesis, and which mesenchyme-derived signals maintain gastric epithelial identity against posteriorization. A 2025 Nature 'gastroids' study provided a first partial entry by showing that neural populations instruct fundic–antral patterning through WNT-mediated crosstalk with NR2F2 (a nuclear receptor transcription factor) as a key mediator, while 2025–2026 work materially advanced the sibling protocol-engineering directions (scalability, maturation, transplantation).

Background

Human pluripotent stem cells (hPSCs, comprising both embryonic and induced pluripotent stem cells) can be directed to differentiate into the founder tissues of the embryo's three primary germ layers — endoderm, mesoderm, and ectoderm — from which all organs arise. Gastrointestinal (GI) organs are assembled from all three: the lining epithelium from endoderm, the connective tissue and smooth muscle from splanchnic mesenchyme (the visceral mesoderm that envelops the gut tube), and the enteric nervous system (ENS, the intrinsic neuronal network of the gut wall that controls motility and secretion) from enteric neural crest cells (ENCCs), an ectodermal derivative that migrates into the gut during embryogenesis. Organoids — self-organizing three-dimensional cultured tissues — had previously been grown for the human stomach and intestine from hPSCs, but these contained essentially only the epithelial layer; ENCCs had been incorporated into human intestinal organoids by Workman et al. (Nature Medicine, 2017), demonstrating functional neurons and neurally evoked contractions, yet a system containing epithelium, mesenchyme, and ENS together did not exist for the stomach. Eicher et al. (Cell Stem Cell, 2022; bioRxiv preprint 2021) filled this gap by separately differentiating the three progenitor populations from hPSCs and recombining them: foregut spheroids were coated with splanchnic mesenchyme and ENCCs, generating three-germ-layer human antral gastric organoids (antral, from the antrum, the distal lower region of the stomach) that, after growth under the kidney capsule of immunocompromised mice (a standard in vivo 'incubator' that vascularizes the graft and promotes maturation), formed glandular gastric tissue with all antral cell types, organized smooth muscle layers, a neuroglial plexus, and neuron-controlled rhythmic contractions; the assembly paradigm was extended to fundic (upper-stomach) and esophageal tissue. Mechanistically, this system revealed a three-way dependency: ENCCs alone posteriorize the gastric epithelium — shifting it toward a Brunner's-gland-like identity rather than an intestinal one (the glandular epithelium of these grafts lacked the gastric markers CLDN18, SOX2 and MUC5AC and the intestinal markers CDX2 and CDH17; Brunner's glands are mucus-secreting duodenal glands whose embryonic development is poorly understood in any species) — through BMP (bone morphogenetic protein) signaling, which the authors blocked with a BMP inhibitor; mesenchyme alone gives poorly organized tissue; only the combination of robust splanchnic mesenchyme and ENCCs yields well-organized smooth muscle, a neuroglial plexus, and properly patterned gastric glands. WNT signaling, a master regulator of intestinal-versus-gastric identity and anterior–posterior patterning of the gut, is the other principal pathway implicated in the region's patterning. The authors framed the central unresolved need as follows: to understand congenital GI disorders, 'we need to better understand the processes of epithelial-mesenchyme-ENS communication during normal organ development', with the new system being 'ideally suited to interrogate such signaling pathways'. What the paper established is phenomenology — which lineage combinations are required and that BMP is one involved signal — not the molecular mechanism: the identity of the ENS-derived signals that drive mesenchymal proliferation and glandular morphogenesis, and of the mesenchyme-derived signals that maintain gastric epithelial identity against posteriorization, remain unknown.

Problem Statement

Identify the molecular mechanisms of inter-germ-layer communication — between the epithelium, the splanchnic mesenchyme, and the enteric nervous system — that govern assembly, gastric epithelial identity, and glandular morphogenesis of the developing human stomach, as accessed with human pluripotent stem cell-derived three-germ-layer organoid systems. In particular: (i) which ENS-derived signals drive mesenchymal proliferation and gastric glandular morphogenesis, given that enteric neural crest cells promote both but the responsible molecules are unknown; and (ii) which mesenchyme-derived signals maintain gastric epithelial identity and prevent the BMP-mediated posteriorization of the epithelium toward the non-gastric, Brunner's-gland-like fate that occurs when ENCCs are added without robust mesenchyme. The source work established the phenomenology (the joint requirement for mesenchyme and ENCCs; BMP involvement in posteriorization) and later work added neural instruction of fundic–antral patterning through WNT-mediated crosstalk, with the nuclear receptor transcription factor NR2F2 as a key mediator, in self-organized co-developing 'gastroids' — but the specific ligand–receptor pathways, their cellular sources and targets, and the causal logic explaining why both germ layers are jointly required for glandular morphogenesis remain unidentified, and the division of labor among the three lineages in building patterned gastric tissue remains unexplained. The two numbered sub-questions (i) and (ii) are the outcomes to be resolved, scoped to the stomach, where the phenotypes to be explained were observed (antral three-germ-layer organoids, with the assembly paradigm also applied to fundic tissue); the overarching process of assembly and the division of labor among the three lineages frame the problem rather than adding required outcomes, and differences among antrum, fundus and esophagus are outside this question.

References

  1. Eicher AK, Kechele DO, Sundaram N, Berns HM, Poling HM, Haines LE, Sanchez JG, Kishimoto K, Krishnamurthy M, Han L, Zorn AM, Helmrath MA, Wells JM. 'Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells.' Cell Stem Cell 29(1):36–51.e6 (2022). DOI: 10.1016/j.stem.2021.10.010, https://doi.org/10.1016/j.stem.2021.10.010. (An earlier bioRxiv preprint of the same work was posted under the different title 'Engineering functional human gastrointestinal organoid tissues using the three primary germ layers separately derived from pluripotent stem cells': DOI 10.1101/2021.07.15.452523, https://doi.org/10.1101/2021.07.15.452523.)
  2. Workman MJ, Mahe MM, Trisno S, Poling HM, Watson CL, Sundaram N, Chang CF, Schiesser J, Aubert P, Stanley EG, Elefanty AG, Miyaoka Y, Mandegar MA, Conklin BR, Neunlist M, Brugmann SA, Helmrath MA, Wells JM. 'Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.' Nature Medicine 23(1):49–59 (2017). DOI: 10.1038/nm.4233, https://doi.org/10.1038/nm.4233
  3. Lin M, Hartl K, Heuberger J, Beccaceci G, Berger H, Li H, Liu L, Müllerke S, Conrad T, Heymann F, Wöhler A, Tacke F, Rajewsky N, Sigal M. 'Establishment of gastrointestinal assembloids to study the interplay between epithelial crypts and their mesenchymal niche.' Nature Communications 14:3025 (2023). DOI: 10.1038/s41467-023-38780-3, https://doi.org/10.1038/s41467-023-38780-3
  4. Childs CJ, Poling HM, Chen K, Tsai YH, Wu A, Vallie A, Eiken MK, Huang S, Sweet CW, Schreiner R, Xiao Z, Spencer RC, Paris SA, Conchola AS, Villanueva JW, Anderman MF, Holloway EM, Singh A, Giger RJ, Mahe MM, Loebel C, Helmrath MA, Walton KD, Rafii S, Spence JR. 'Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature.' Cell Stem Cell 32(4):640–651.e9 (2025). DOI: 10.1016/j.stem.2025.02.007, https://doi.org/10.1016/j.stem.2025.02.007
  5. Li X, Lin F, Cui Q, Sun S, Li S, Wang Y, Wang Y, Bai J, Liu S, Guo J, Han Y, Zhang M, Chang T, Zheng Y, Liu J, Liu L, Wang L, Fu J, Liu X, Bai B, Shao Y. 'Human gastroids to model regional patterning in early stomach development.' Nature 646(8086):893–902 (2025). DOI: 10.1038/s41586-025-09508-8, https://doi.org/10.1038/s41586-025-09508-8
  6. Poling HM, Noël T, Singh A, Fisher GW, Thorner K, Chaturvedi P, Nattamai K, Srivastava K, Batie MR, Hausfeld T, Pitstick AL, Brown NE, Ménaret S, Anegon I, Barrile R, Mayhew CN, Takebe T, Wells JM, Helmrath MA, Mahe MM. 'Large-scale and innervated functional human gut tissues for transplantation via transient spheroid confinement.' Nature Biomedical Engineering (2026). DOI: 10.1038/s41551-026-01688-6, https://doi.org/10.1038/s41551-026-01688-6
  7. Kechele DO, Enriquez JR, Eicher AK, Thorner K, Chaturvedi P, Kishimoto K, Adair B, Sanchez JG, Tominaga K, Zorn AM, Wells JM. 'Single-cell transcriptomic comparison of the developing human fetal stomach and pluripotent stem cell-derived gastric organoids.' Development 153(4) (2026). DOI: 10.1242/dev.205196, https://doi.org/10.1242/dev.205196