Dissection of gastric homeostasis in vivo facilitates permanent capture of isthmus stem cells in vitro [single-cell RNA-seq]

The glandular stomach is comprised of two anatomically and functionally distinct epithelial tissues termed corpus and antrum. While these compartments continuously regenerate to ensure normal organ function, our understanding of the molecular and cellular determinants that maintain these tissues remains incomplete. Here, we established an unbiased account of all gastric epithelial cells of the mouse corpus and antrum using single cell RNA-sequencing, allowing us to compare these adjacent epithelia and their stem cell compartments. Our data reveals that epithelial cell types with equivalent functional roles in the corpus and antrum utilize similar transcriptional programs, and our results support the existence of two discernible stem cell populations within the gland base and isthmus regions of these tissues. To study isthmus stem cells, typically underrepresented in conventional, gland base-enriched organoid cultures, we developed a novel 2D monolayer culture system to propagate mouse and human gastric epithelia. These 2D cultures expand indefinitely via Lgr5- isthmus-like cells and maintain the ability to differentiate into diverse gastric cell types. While 2D corpus and antrum cultures were transcriptionally similar, they retained a molecular memory and differentiation bias that mirrored their tissue of origin. Furthermore, gastric 2D cultures could be readily and reversibly converted into conventional 3D organoids, highlighting the remarkable plasticity of undifferentiated stomach epithelial cells. Finally, we utilized the 2D culture system to show that rare primary epithelial cells with high levels of Sox2 expression, previously hypothesized to be gastric stem cells, most closely resembled enterochromaffin (EC) cells and Sox2 is both necessary and sufficient to generate EC cells in vivo. Together, our data (i) provide important insights into the basis of gastric epithelial regeneration and differentiation, (ii) establish a tractable 2D culture system to capture and manipulate primary gastric isthmus stem cells in vitro and (iii) uncover a role for the stem cell factor Sox2 during EC cell specification. Primary corpus and antrum samples of mouse glandular stomach; mouse and human 2D cultures for antrum and corpus; mouse antrum/corpus organoids.

腺体胃由两种在解剖学与功能上均存在显著差异的上皮组织构成，分别命名为胃体（corpus）与胃窦（antrum）。尽管这些区域持续进行再生以维持器官的正常功能，但目前我们对维持这些上皮组织的分子与细胞调控决定因素的认知仍不全面。本研究通过单细胞RNA测序（single cell RNA-sequencing），对小鼠胃体与胃窦的全部胃上皮细胞开展了无偏倚的全景分析，得以直接比对这两处相邻上皮组织及其干细胞微区域。研究数据显示，在胃体与胃窦中发挥等价功能的上皮细胞类型，采用了高度相似的转录调控程序；结果同时支持，在这些组织的腺体基底与峡部区域，存在两种可明确区分的干细胞群体。为研究常规腺体基底富集类器官培养中通常占比极低的峡部干细胞，我们开发了一种全新的二维单层培养体系，用于扩增小鼠与人类的胃上皮细胞。该二维培养体系可通过Lgr5阴性的峡部样细胞实现无限增殖，并能够稳定分化为多种胃上皮细胞类型。尽管二维培养的胃体与胃窦上皮细胞在转录组层面表现相似，但它们仍保留了源自起源组织的分子记忆与分化偏好特性。此外，胃上皮二维培养体系可便捷且可逆地转化为常规三维类器官，这凸显了未分化胃上皮细胞显著的发育可塑性。最后，我们利用该二维培养体系证实：此前被推测为胃干细胞的、高表达Sox2的稀有原代上皮细胞，最接近于肠嗜铬（enterochromaffin, EC）细胞；且Sox2在体内生成EC细胞的过程中既是必需的调控因子，也是足够的诱导因素。综上，本研究（i）为胃上皮再生与分化的分子基础提供了重要认知；（ii）建立了一套可操作的二维培养体系，用于体外捕获并精准操控原代胃峡部干细胞；（iii）揭示了干细胞因子Sox2在EC细胞特化过程中的关键调控作用。本研究使用的样本包括：小鼠腺体胃的原代胃体与胃窦样本、用于胃窦与胃体研究的小鼠及人类二维培养体系，以及小鼠胃窦/胃体类器官。