Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California 94305, USA.
Gastroenterology. 2012 May;142(5):1195-1205.e6. doi: 10.1053/j.gastro.2012.02.006. Epub 2012 Feb 11.
BACKGROUND & AIMS: Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells.
We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.
Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased.
cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.
潘氏细胞有助于小肠 Lgr5(+)干细胞的小生境。尽管结肠也含有 Lgr5(+)干细胞，但它不含潘氏细胞。我们研究了是否存在具有独特转录特征并支持 Lgr5(+)干细胞的结肠潘氏样细胞。
我们使用多色荧光激活细胞分选，根据已知标记物，从分离的小鼠结肠上皮中分离出结肠隐窝的不同亚区。我们进行了多重单细胞基因表达分析，使用定量逆转录聚合酶链反应，随后进行层次聚类分析，以表征不同的细胞类型。我们使用免疫染色和荧光激活细胞分选分析，以及体内给予 Notch 抑制剂和体外类器官培养，来表征不同的细胞类型。
多色荧光激活细胞分选可以分离出不同的结肠隐窝区域。通过对选定的单细胞群体进行基因表达分析，揭示了四种主要的上皮亚型或转录状态。其中一种，杯状细胞，含有一个独特的 cKit/CD117(+)隐窝基底亚群，表达 Dll1、Dll4 和表皮生长因子，类似于也被 cKit 标记的潘氏细胞。在结肠中，cKit(+)杯状细胞与 Lgr5(+)干细胞交织在一起。在体内，这种结肠 cKit(+)细胞群受 Notch 信号调节；给小鼠施用 γ-分泌酶抑制剂会增加 cKit(+)细胞的数量。当从小鼠结肠中分离出来时，cKit(+)细胞促进了 Lgr5(+)干细胞的类器官形成，Lgr5(+)干细胞表达 Kitl/干细胞因子，是 cKit 的配体。当使用毒素结合抗体从类器官中耗尽 cKit(+)细胞时，类器官形成减少。
cKit 标记小肠潘氏细胞和结肠杯状细胞的一个子集，受 Notch 信号调节，并支持 Lgr5(+)干细胞。
