Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, small cycling cells located at crypt bottoms1, 2. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells, that are known to produce bactericidal products such as lysozyme and cryptdins/defensins3. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells4. Here, we note a close physical association of Lgr5 stem cells with Paneth cells in vivo and in vitro. CD24+ Paneth cells express EGF, TGFalpha, Wnt3 and the Notch-ligand Dll4, all essential signals for stem cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell. We used intestinal cell fractions from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations: stem cells were sorted based on high level of GFP expression (GFPhi) and Paneth cells were sorted based on high level of CD24 expression (CD24hi) and high side-scatter (SSChi). Differentially labelled cRNA from GFPhi and CD24hi/SSChi cells from two different sorts (each combining ten individual mice) were hybridized on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays.小肠隐窝的自我更新稳态，源自隐窝底部的小型增殖细胞——Lgr5干细胞（Lgr5 stem cells）之间的中性竞争1,2。Lgr5干细胞与终末分化的潘氏细胞（Paneth cells）交错分布，后者已知可产生溶菌酶（lysozyme）、隐窝素/防御素（cryptdins/defensins）等杀菌物质3。单个表达Lgr5的干细胞可在非上皮性龛细胞缺失的条件下体外培养，形成可长期存活、自我组织的隐窝-绒毛类器官（organoids）4。本研究观察到，在体内与体外环境中，Lgr5干细胞均与潘氏细胞存在紧密的物理关联。表达CD24的潘氏细胞可表达EGF、TGFα、Wnt3以及Notch配体Dll4，上述分子均为体外培养中维持干细胞存活所必需的信号分子。将分选得到的干细胞与潘氏细胞共培养，可显著提升类器官形成效率。该潘氏细胞的支持作用可通过单次施加外源性Wnt予以替代。在体内通过遗传学手段清除潘氏细胞，会导致Lgr5干细胞随之丢失。在结肠隐窝中，位于Lgr5干细胞之间的CD24阳性细胞或为潘氏细胞的同源细胞类型。综上，我们得出结论：Lgr5干细胞需要竞争由特化子代细胞——潘氏细胞所提供的必需龛信号。本研究使用了来自Lgr5-EGFP-ires-CreERT2小鼠的肠道细胞组分，该小鼠的Lgr5启动子可调控GFP的表达。RNA从两类经荧光激活细胞分选（FACS）得到的细胞群中提取：干细胞基于高水平GFP表达（GFPhi）进行分选，潘氏细胞则基于高水平CD24表达（CD24hi）与高侧向散射光（SSChi）特征进行分选。将来自两次独立分选（每次分选均混合10只小鼠）的GFPhi细胞与CD24hi/SSChi细胞的cRNA进行差异化标记后，在4X44K安捷伦（Agilent）全小鼠基因组双色微阵列（G4122F）上进行杂交，共完成两次染料交换（dye swap）实验，最终得到四张独立芯片数据。