Single-cell transcriptomic comparison of the developing human fetal stomach and pluripotent stem cell-derived gastric organoids

The goal of engineering increasingly complex in vitro human organoid models is to more accurately model human organogenesis and disease. Recently, human antral gastric organoids (hAGOs) were engineered to contain splanchnic mesenchyme (SM) and enteric neural crest cells (NCCs), resulting in the formation of functional three germ layer gastric organoids. It is unclear how three-germ layer hAGOs compare with the human fetal stomach and how the added germ layers alter gastric differentiation in vitro. To determine the robustness of hAGOs and how additional germ layers impact development, we benchmarked hAGO with and without added SM and NCC to a single cell atlas of the developing human stomach. A comparison of hAGOs in vitro to fetal organs confirmed that they were gastric and most similar to 7 week stomach with the epithelium comprised primarily of gastric precursors and surface mucous cells. The SM added to hAGOs formed fetal gastrointestinal-like mesenchyme whereas the NCCs in hAGOs differentiated into neuronal precursors and glial cells similar to those found in developing endodermal organs. The addition of SM and NCCs did not drastically alter the cellular diversity of the epithelium in vitro but maintained more uncommitted precursors similar to 7 week stomach. Following transplantation and growth for 10 weeks, hAGOs with SM grew and matured into tissue more similar to the 14 week fetal stomach. Bioinformatic inference of cell-cell communication confirmed known signaling crosstalk between germ layers but also identified new signaling candidates that may regulate tissue assembly. Together, these data show that three-germ layer hAGOs faithfully modeled the multilayer complexity of the fetal stomach at a single cell transcriptomics level and provided insight into human stomach development. Overall design: Day 37 in vitro human antral gastric organoids with added splanchnic mesenchyme and/or neural crest cells (one to two biological replicates), day 35 HIO (one replicate), or HAGO with mesenchyme following 10 wk in vivo grown under mouse kidney capsule were dissociated to single cells were subjected to 10X single cell RNA sequencing.