An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5–expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3β, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.

能否安全地利用成体干细胞强大的再生潜力进行临床应用，关键取决于对调节其活性的潜在机制的全面了解。上皮类器官培养准确地概括了体内干细胞驱动的上皮更新的许多特征，为研究关键调控机制提供了一个极好的离体平台。在这里，我们使用小鼠胃上皮类器官采用基因组规模聚集、定期间隔、短回文重复 (CRISPR) 敲除 (KO) 筛选试验来鉴定胃黏膜中 Wnt 驱动的干细胞依赖性上皮更新的调节剂。除了已知的 Wnt 通路调节剂，如Apc，我们发现Alk的 KO ，Bclaf3或Prkra支持离体胃上皮细胞的 Wnt 独立自我更新。在成年小鼠中，这些因子的表达主要局限于腺体基底上方不表达Lgr5的干细胞区域，暗示这些因子在抑制自我更新或促进胃上皮细胞分化中的关键作用。值得注意的是，我们发现 Alk 通过磷酸化 Gsk3β 的酪氨酸来抑制 Wnt 信号传导，而 Bclaf3 和 Prkra通过调节上皮白细胞介素的表达来抑制再生胰岛衍生( Reg ) 基因。因此，Alk、Bclaf3 和 Prkra 可以抑制干性/增殖并作为胃上皮分化的新调节剂发挥作用。