Department of Microbiology and CHA Organoid Research Center, School of Medicine, CHA University, South Korea.
Department of Radiation Oncology, Bundang CHA Medical Center, South Korea.
Biochem Biophys Res Commun. 2019 Jan 8;508(2):430-439. doi: 10.1016/j.bbrc.2018.11.003. Epub 2018 Nov 28.
An organoid is a complex, multi-cell three-dimensional (3D) structure that contains tissue-specific cells. Epithelial stem cells, which are marked by leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), have the potential for self-renewal and expansion as organoids. However, in the case of intestinal organoids from Lgr5-EGFP-IRES-CreERT2 transgenic mice, in vitro expansion of the Lgr5 expression is limited in a culture condition supplemented with essential proteins, such as epidermal growth factor (E), noggin (N), and R-spondin 1 (R). In this study, we hypothesized that self-renewal of Lgr5+ stem cells in a 3D culture system can be stimulated by defined compounds (CHIR99021, Valproic acid, Y-27632, and A83-01). Our results demonstrated that dissociated single cells from organoids were organized into a 3D structure in the four compounds containing the ENR culture medium in a 3D and two-dimensional (2D) culture system. Moreover, the Lgr5 expression level of organoids from the ENR- and compound-containing media increased. Furthermore, the conversion of cultured Lgr5+ stem cells from 2D to 3D was confirmed. Therefore, defined compounds promote the expansion of Lgr5+ stem cells in organoids.
类器官是一种复杂的、多细胞的三维（3D）结构，其中包含组织特异性细胞。富含亮氨酸重复的 G 蛋白偶联受体 5（Lgr5）标记的上皮干细胞具有自我更新和扩增为类器官的潜力。然而，在 Lgr5-EGFP-IRES-CreERT2 转基因小鼠的肠类器官中，在补充表皮生长因子（E）、诺金（N）和 R 螺旋蛋白 1（R）等必需蛋白的培养条件下，Lgr5 表达的体外扩增是有限的。在这项研究中，我们假设在 3D 培养系统中，Lgr5+干细胞的自我更新可以通过定义的化合物（CHIR99021、丙戊酸、Y-27632 和 A83-01）来刺激。我们的结果表明，在含有 ENR 培养基的四种化合物中，类器官的分离单细胞在 3D 和二维（2D）培养系统中组织成 3D 结构。此外，ENR 和含化合物培养基中的类器官的 Lgr5 表达水平增加。此外，还证实了培养的 Lgr5+干细胞从 2D 向 3D 的转化。因此，定义的化合物促进了类器官中 Lgr5+干细胞的扩增。
