Methionine and its hydroxy analogue (MHA) have been shown to benefit mouse intestinal regeneration. The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development. Here, we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development. The results showed that 125-μm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting. We found that transforming growth factor-β inhibitor (A8301) supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells (ISC). The mitogen-activated protein kinase p38 inhibitor (SB202190) promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells, goblet cells and Paneth cells. However, the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301. The glycogen synthase kinase 3 inhibitor (CHIR99021), valproic acid (VPA) alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells. Chicken serum significantly improved organoid formation, especially in the presence of A8301, SB202190, CHIR99021, and VPA, but inhibited the differentiation of Paneth cells and enteroendocrine cells. Chicken serum at a concentration of 0.25% meets the requirement of chicken intestinal organoid development, and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1. Moreover, commercial mouse organoid culture medium supplemented with A8301, SB202190, CHIR99021, VPA, and chicken serum promotes chicken organoid budding. Based on the chicken intestinal organoid model, we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size, and this effect was reinforced with increased cycloleucine concentrations. Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine. In conclusion, our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.

甲硫氨酸及其羟基类似物 (MHA) 已被证明有益于小鼠肠道再生。肠道类器官是一个很好的模型，可以直接反映某些营养素或化学物质对肠道发育的影响。在这里，我们旨在首先建立鸡肠道类器官培养方法，然后利用该模型探索蛋氨酸缺乏和 MHA 对肠道类器官发育的影响。结果表明，125-μm 细胞过滤器在鸡胚隐窝收获方面表现出最高的效率。我们发现补充转化生长因子-β 抑制剂 (A8301) 可促进肠细胞分化，但会损害肠干细胞 (ISC) 的增殖。丝裂原活化蛋白激酶 p38 抑制剂 (SB202190) 促进肠道类器官形成和肠细胞分化，但抑制肠内分泌细胞、杯状细胞和 Paneth 细胞的分化。然而，SB202190 对肠内分泌细胞和潘氏细胞分化的抑制在 A8301 的存在下得到缓解。糖原合酶激酶 3 抑制剂 (CHIR99021)、丙戊酸 (VPA) 单独及其组合促进鸡肠道类器官形成和肠上皮细胞分化，但牺牲了 Paneth 细胞和杯状细胞的表达。鸡血清显着改善了类器官的形成，尤其是在 A8301、SB202190、CHIR99021 和 VPA 的存在下，但抑制了潘氏细胞和肠内分泌细胞的分化。浓度为 0 的鸡血清。25%满足鸡肠道类器官发育的需要，鸡血清对鸡肠道类器官培养的有益作用是胎牛血清和胰岛素样生长因子-1所不能替代的。此外，补充有 A8301、SB202190、CHIR99021、VPA 和鸡血清的商业小鼠类器官培养基可促进鸡类器官的出芽。基于鸡肠道类器官模型，我们发现由环亮氨酸模拟的甲硫氨酸缺乏抑制了类器官的形成和类器官的大小，并且这种效果随着环亮氨酸浓度的增加而得到加强。与使用 L-甲硫氨酸获得的结果相比，甲硫氨酸羟基类似物促进了 ISC 的再生，但降低了细胞分化。综上所述，