Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.
Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.
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作者：
肠上皮细胞的体外分析由于缺乏合适的培养系统而受到阻碍。在这里，我们描述了强大的长期小肠和大肠文化的方法，将气液界面和底层基质元素。这些培养物显示出延长的肠上皮扩张为具有增殖和多谱系分化的球形类器官。Wnt生长因子家族在体内积极调节肠上皮细胞的增殖。因此，培养物生长被Wnt拮抗剂Dickkopf-1（Dkk 1）抑制，并被Wnt激动剂R-spondin-1和免疫球蛋白Fc（RSpo 1-Fc）之间的融合蛋白显著刺激。此外，用γ-分泌酶抑制剂二苯并氮杂卓和神经生成素-3过表达处理分别诱导杯状细胞和肠内分泌细胞分化，这与内源性Notch信号传导和谱系可塑性一致。来自富含亮氨酸重复序列的G蛋白偶联受体5阳性（Lgr 5+）和B淋巴瘤莫洛尼鼠白血病病毒插入区同源物1阳性（Bmi 1+）谱系的上皮细胞，代表推定的肠干细胞（ISC）群体，在体外存在并通过用RSo 1-Fc处理扩增;随后在体内证实了RSpo 1-Fc处理后Lgr 5+细胞数量的增加。我们的研究结果表明，成功的长期肠道培养的微环境准确重现Wnt和Notch依赖的ISC生态位。
The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the γ-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein–coupled receptor-5–positive (Lgr5+) and B lymphoma moloney murine leukemia virus insertion region homolog-1–positive (Bmi1+) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5+ cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.
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