Adult stem cells in the small intestine are intrinsically programmed with their location-specific function

Differentiation and specialisation of epithelial cells in the small intestine is regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine or M-cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signalling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine. By using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signalling. These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals. In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine. Overall design: RNA sequencing of intestinal crypts, villi and cultured organoids derived from mouse duodenum, jejunum and ileum

小肠上皮细胞的分化与特化受两种方式调控。其一，肠道干细胞沿隐窝-绒毛轴（crypt-villus axis）分化为吸收性肠上皮细胞、潘氏细胞（Paneth cell）、杯状细胞、簇细胞、肠内分泌细胞或M细胞，该过程主要受WNT信号通路调控。其二，沿头尾轴（cephalocaudal axis）存在功能特化：十二指肠、空肠与回肠具备不同的吸收与消化功能，这一特化过程受GATA4等多种转录因子调控。然而截至目前，学界仍不清楚位置特异性的功能特性是在干细胞内部固有编程而来，还是需要间质细胞（mesenchymal cell）持续传递信号以维持小肠的位置特异性身份。本研究采用纯上皮类器官（organoid）培养技术，证实小鼠与人类小肠干细胞的区域特异性基因表达谱在长期培养过程中均得以保留，且与Gata4的差异表达呈显著相关。此外，人类类器官培养系统实验表明，仅在WNT信号通路缺失的条件下，Gata4调控的基因表达才会被激活。上述数据证明，位置特异性的功能在成体小肠干细胞中是固有编程的，其分化命运不受位置特异性细胞外信号的调控。鉴于小肠来源的类器官在未来临床应用中的巨大潜力，本研究数据提示，为重建小肠的全部核心生理功能，构建GATA4阳性与GATA4阴性的培养体系至关重要。实验整体设计：对来源于小鼠十二指肠、空肠、回肠的肠道隐窝、绒毛及体外培养的类器官进行RNA测序（RNA sequencing）。