These organoids have some nerve: Uniting three germ layers in a human gastric model system

Gastrointestinal organoids provide an accessible model for studying human development and disease. In this issue of Cell Stem Cell, Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar direct human pluripotent stem cells to incorporate three germ layers into gastric organoids, recapitulating the structure and function of human gut tissue in an in vitro model. Gastrointestinal organoids provide an accessible model for studying human development and disease. In this issue of Cell Stem Cell, Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar direct human pluripotent stem cells to incorporate three germ layers into gastric organoids, recapitulating the structure and function of human gut tissue in an in vitro model. Human gastrointestinal organoids are a valuable model system for studying development and disease. Organoids expand from a variety of host stem cells and are cultured in three dimensions, closely modeling the native tissue of origin. Several research groups have begun utilizing gastrointestinal organoids to study in vitro human development and disease due to their improved physiological relevance over animal models (McCracken et al., 2017McCracken K.W. Aihara E. Martin B. Crawford C.M. Broda T. Treguier J. Zhang X. Shannon J.M. Montrose M.H. Wells J.M. Wnt/β-catenin promotes gastric fundus specification in mice and humans.Nature. 2017; 541: 182-187Crossref PubMed Scopus (128) Google Scholar; Fujii et al., 2019Fujii M. Clevers H. Sato T. Modeling human digestive diseases with CRISPR-Cas9–modified Organoids.Gastroenterology. 2019; 156: 562-576Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar; Tsuji et al., 2021Tsuji T. Maeda Y. Kita K. Murakami K. Saya H. Takemura H. Inaki N. Oshima M. Oshima H. FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells.Oncogene. 2021; 40: 3072-3086Crossref PubMed Scopus (5) Google Scholar). Biobanks of patient-derived stem cells and human organoids are also popping up worldwide in clinical research centers and core facilities (Yan et al., 2018Yan H.H.N. Siu H.C. Law S. Ho S.L. Yue S.S.K. Tsui W.Y. Chan D. Chan A.S. Ma S. Lam K.O. et al.A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening.Cell Stem Cell. 2018; 23: 882-897.e11Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). These biobanks allow for personalized medicine opportunities and increase accessibility to human tissue for research. Human pluripotent stem cells (hPSCs) have arisen as an alternative to donor tissue for organoid development. The first use of gastric organoids using hPSCs was published in 2014 by McCracken et al. (McCracken et al., 2014McCracken K.W. Catá E.M. Crawford C.M. Sinagoga K.L. Schumacher M. Rockich B.E. Tsai Y.H. Mayhew C.N. Spence J.R. Zavros Y. Wells J.M. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids.Nature. 2014; 516: 400-404Crossref PubMed Scopus (629) Google Scholar). Great strides in organoid cell specificity using hPSCs have since been made with directed differentiation of organoids for specific regions of the gastrointestinal tract, including the antral and fundic stomach domains (Broda et al., 2019Broda T.R. McCracken K.W. Wells J.M. Generation of human antral and fundic gastric organoids from pluripotent stem cells.Nat. Protoc. 2019; 14: 28-50Crossref PubMed Scopus (40) Google Scholar). In this issue of Cell Stem Cell, Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar developed an organoid system incorporating all three germ layers (endoderm, mesoderm, and ectoderm) compared to traditional methods comprising primarily epithelial cell types from the endoderm. Including all germ layers is an essential step in advancing models that recapitulate human tissue architecture and organogenesis. Organs of the gastrointestinal tract emerge from the three germ layers into different components, including the epithelium, smooth muscle, and enteric nervous system (ENS). These components work synergistically to support gut function. Developmental and other disorders can occur among each of these components, creating a need for model systems inclusive of all gastrointestinal functions and interactions. Realizing this need, Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar developed human antral gastric organoids (hAGOs) with progenitor cells from the mesoderm and ectoderm (Figure 1). A recent publication used single-cell transcriptomics to investigate the diversity of mesenchymal cell subtypes and their interactions with the endoderm during organogenesis (Han et al., 2020Han L. Chaturvedi P. Kishimoto K. Koike H. Nasr T. Iwasawa K. Giesbrecht K. Witcher P.C. Eicher A. Haines L. et al.Single cell transcriptomics identifies a signaling network coordinating endoderm and mesoderm diversification during foregut organogenesis.Nat. Commun. 2020; 11: 4158Crossref PubMed Scopus (59) Google Scholar). Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar built off the findings of this work to differentiate hPSCs into splanchnic mesenchyme (SM). Incorporation of the SM with hAGOs (hAGOs+SM) resulted in over one-third of the population expressing mesenchymal αSMA+, a smooth muscle cell marker. The hAGOs+SM were transplanted into mice over 10 to 12 weeks and then compared to human fetal and adult stomach tissue. Most hAGOs without SM did not survive when compared with the hAGOs+SM, which had an impressive 100% survival rate and some diameters over 1 cm, highlighting the mesenchyme as a critical component when modeling gastric tissue. After successfully mastering the incorporation of SM into the hAGOs, Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar focused on including the ENS. A workflow for developing enteric neural crest cells (ENCCs) was utilized with the unique goal of harvesting migrating ENCCs to more closely match embryonic ENCC colonization (Workman et al., 2017Workman M.J. Mahe M.M. Trisno S. Poling H.M. Watson C.L. Sundaram N. Chang C.F. Schiesser J. Aubert P. Stanley E.G. et al.Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.Nat. Med. 2017; 23: 49-59Crossref PubMed Scopus (332) Google Scholar). After transplantation, the addition of the ENCCs to hAGOs+SM (hAGOs+SM+ENCC) resulted in stereotypic glandular structures matching those of the human stomach, not exhibited in hAGOs+SM. The three-germ layer hAGOs closely matched fetal stomach tissue architecture but were not as similar to adult tissue. Following in vivo incubation, organoids expressed the gastric marker CLDN18 and lacked the intestinal epithelial marker CDH17. Tissue contractions were irregular in hAGOs+SM but highly regular in hAGOs+SM+ENCC, suggesting that the ENS may control muscle contractions and interact with intramuscular interstitial cells of Cajal (ICCs). The methods used for three-germ layer hAGOs were applied to human fundic gastric organoids (hFGOs) to model additional stomach regions. Fundic identity of the hFGOs was verified, showing expression of ATP4B+ parietal cells, expression of fundic-specific PGA3+ chief cells (absent in hAGOs), and higher ghrelin expression. The authors noted that glandular morphogenesis of transplanted hFGOs was less than that of the hAGOs. However, in vivo transplantation showed more mature parietal cells and did not “require additional factors for robust parietal cell differentiation, demonstrating that the signaling processes that control gastric cell type specification occur normally in engineered tissue” (Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The authors demonstrated their organoid model functionality to study germ layer interactions by comparing hAGOs+SM+ENCC, hAGOs+ENCC, and hAGOs+SM. ENCCs differentiated into the diverse neuron and glial subtypes (interneuron, sensory, and dopaminergic) in both models. However, the lack of SM showed abnormal and disorganized ENS plexus, highlighting the mesenchyme as a critical player in ENS development. Transplanted hAGOs+ENCC also showed increased amounts of epithelial cells and pronounced glandular epithelial morphogenesis compared to hAGOs alone. The authors concluded that ENCCs promote hAGOs survival, but outcomes do not wholly recapitulate human tissue. The glands observed were not gastric or intestinal, lacking key gastric epithelial markers CLDN18 or SOX2, gastric cell types like MUC5AC-expressing mucosal cells, and intestinal epithelial markers CDX2 and CDH17. Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar concluded that the glandular properties were most similar to Brunner’s glands based on a combinatorial expression profile of 9 different markers. This implies that ENCCs may direct gastric epithelium to a more posterior identity without the endogenous mesenchyme. This work showcases the importance and impact of all three germ components on gastric patterning and gland morphogenesis. Eicher et al., 2022Eicher A.K. Kechele D.O. Sundaram N. Berns M.H. Poling H.M. Haines L.E. Sanchez J.G. Kishimoto K. Krishnamurthy M. Han L. et al.Functional human gastrointestinal organoids 1 can be engineered from three primary germ layers separately derived from pluripotent system cells.Cell Stem Cell. 2022; 29 (Published online December 1, 2021): 36-51https://doi.org/10.1016/j.stem.2021.10.010Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar expanded beyond the stomach and successfully applied the three-germ layer concept to human esophageal organoids (HEOs), supporting the idea that their methods can be applied to diverse applications. Their methods may extend beyond the gastrointestinal tract to organ systems, including the lungs, liver, and bladder. Advancements of these more complex in vitro systems will be invaluable for expanding holistic research of human development and disease. Persisting limitations still exist in organoid models regarding the need for animal-derived extracellular matrix (ECM) hydrogels. Matrigel is commonly used for organoid propagation and is mouse-tumor derived. The composition is not well characterized and variable, hindering the ability to use organoids for clinical applications. Many synthesized, crosslinked hydrogels have limited success with organoids due to elastic forces restricting the fast expansion needed of organoids. Current research has started synthesizing stress-relaxing hydrogels that more closely model natural ECM and support organoid morphogenesis (Chrisnandy et al., 2021Chrisnandy A. Blondel D. Rezakhani S. Broguiere N. Lutolf M.P. Synthetic dynamic hydrogels promote degradation-independent in vitro organogenesis.Nat. Mater. 2021; (Published online November 15, 2021)https://doi.org/10.1038/s41563-021-01136-7Crossref PubMed Scopus (26) Google Scholar). Another remaining limitation is the need for in vivo transplantation to promote differentiation closer to that of human tissue. And still, transplanted organoids more closely match embryonic structures compared to adult. Ideally, future work will identify methods, factors, and master regulators to promote organoid maturation without xenogenic input for well-defined in vitro systems recapitulating human organs. Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cellsEicher et al.Cell Stem CellDecember 1, 2021In BriefEicher et al. have generated complex 3-germ-layer human gastric organoids from pluripotent stem cells. These 3-germ-layer organoids contain glandular epithelium surrounded by multiple organized layers of functionally innervated smooth muscle. Manipulating this system revealed key developmental roles of neural crest cells on gastric epithelium and mesenchyme. Full-Text PDF