胃肠道干细胞在球状体培养中的体外扩增与基因修饰
In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.
作者信息
Miyoshi Hiroyuki, Stappenbeck Thaddeus S
机构信息
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
出版信息
Nat Protoc. 2013 Dec;8(12):2471-82. doi: 10.1038/nprot.2013.153. Epub 2013 Nov 14.
Abstract
It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell-enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell-enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.
摘要
能够在体外培养富集的干细胞群体是很有用的。将干细胞培养成组织球体是允许成年上皮细胞进行可持续培养的关键方法。胃肠道干细胞可以通过使用来自支持性细胞系（L-WRN）的条件培养基来增殖。本方案描述了如何制备条件培养基以及如何从小鼠胃肠道培养富含干细胞的上皮球体。这些球体也适合用重组慢病毒进行基因改造。该系统使许多已用永生化细胞系进行的细胞生物学检测能够应用于球体。从小鼠分离上皮细胞单位最多需要2小时，3天内可获得富含干细胞的胃肠道球体。用慢病毒进行基因改造的球体可在2周内获得。
相似文献
1
In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.胃肠道干细胞在球状体培养中的体外扩增与基因修饰
Nat Protoc. 2013 Dec;8(12):2471-82. doi: 10.1038/nprot.2013.153. Epub 2013 Nov 14.
2
L-WRN conditioned medium for gastrointestinal epithelial stem cell culture shows replicable batch-to-batch activity levels across multiple research teams.用于胃肠道上皮干细胞培养的L-WRN条件培养基在多个研究团队中显示出批次间可重复的活性水平。
Stem Cell Res. 2019 May;37:101430. doi: 10.1016/j.scr.2019.101430. Epub 2019 Mar 27.
3
Isolation and In Vitro Culture of Human Gut Progenitor Cells.人肠道祖细胞的分离与体外培养
Methods Mol Biol. 2019;2029:49-62. doi: 10.1007/978-1-4939-9631-5_5.
4
Lotus seedpod-inspired hydrogels as an all-in-one platform for culture and delivery of stem cell spheroids.莲房启发的水凝胶作为干细胞球体培养和递送的一体化平台。
Biomaterials. 2019 Dec;225:119534. doi: 10.1016/j.biomaterials.2019.119534. Epub 2019 Sep 30.
5
A novel three-dimensional culture system for isolation and clonal propagation of neural stem cells using a thermo-reversible gelation polymer.一种使用热可逆凝胶聚合物分离和克隆增殖神经干细胞的新型三维培养系统。
Tissue Eng Part C Methods. 2009 Dec;15(4):615-23. doi: 10.1089/ten.TEC.2008.0516.
6
Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells.球体培养增强牙周膜间充质干细胞的成骨潜能。
J Periodontal Res. 2018 Oct;53(5):870-882. doi: 10.1111/jre.12577. Epub 2018 Jun 14.
7
Multi-Spheroid-Loaded Human Acellular Dermal Matrix Carrier Preserves Its Spheroid Shape and Improves In Vivo Adipose-Derived Stem Cell Delivery and Engraftment.多球体负载人去细胞真皮基质载体保持其球体形状，并改善体内脂肪来源干细胞的递送和植入。
Tissue Eng Regen Med. 2020 Jun;17(3):271-283. doi: 10.1007/s13770-020-00252-w. Epub 2020 Apr 20.
8
Spheroid model for functional osteogenic evaluation of human adipose derived stem cells.用于人脂肪来源干细胞功能性成骨评估的球体模型
J Biomed Mater Res A. 2017 Apr;105(4):1230-1236. doi: 10.1002/jbm.a.35974. Epub 2017 Feb 2.
9
Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.源自人诱导多能干细胞的心肌细胞球体改善了小鼠心肌损伤的恢复。
Am J Physiol Heart Circ Physiol. 2018 Aug 1;315(2):H327-H339. doi: 10.1152/ajpheart.00688.2017. Epub 2018 Apr 6.
10
Lungosphere Assay: 3D Culture of Lung Epithelial Stem/Progenitor Cells.肺球测定法：肺上皮干细胞/祖细胞的三维培养
Methods Mol Biol. 2017;1612:149-165. doi: 10.1007/978-1-4939-7021-6_11.
引用本文的文献
1
Three-dimensional cell sheet model improves prediction accuracy of osteogenic potential for biodegradable magnesium-based metals.三维细胞片层模型提高了可降解镁基金属成骨潜力的预测准确性。
Bioact Mater. 2025 Aug 23;54:291-310. doi: 10.1016/j.bioactmat.2025.08.013. eCollection 2025 Dec.
2
Helicobacter hepaticus promotes hepatic steatosis through CdtB-induced mitochondrial stress and lipid metabolism reprogramming.肝螺杆菌通过CdtB诱导的线粒体应激和脂质代谢重编程促进肝脂肪变性。
Nat Commun. 2025 Aug 26;16(1):7954. doi: 10.1038/s41467-025-63351-z.
3
Clinical applications of circulating tumor cells in metastasis and therapy.循环肿瘤细胞在转移和治疗中的临床应用
J Hematol Oncol. 2025 Aug 22;18(1):80. doi: 10.1186/s13045-025-01733-y.
4
Fructose enhances intestinal development and barrier function while driving hepatic lipogenesis and inflammation in broilers.果糖可促进肉鸡肠道发育和屏障功能，同时引发肝脏脂肪生成和炎症。
Poult Sci. 2025 Aug 6;104(11):105645. doi: 10.1016/j.psj.2025.105645.
5
Gastrointestinal tract, its pathophysiology and models: A "quick" reference guide to translational studies.胃肠道、其病理生理学及模型：转化研究的“快速”参考指南。
World J Gastroenterol. 2025 Jul 28;31(28):108297. doi: 10.3748/wjg.v31.i28.108297.
6
Metabolomics and proteomics reveal blocking argininosuccinate synthetase 1 alleviates colitis in mice.代谢组学和蛋白质组学研究表明，抑制精氨酸琥珀酸合成酶1可缓解小鼠结肠炎。
Nat Commun. 2025 Jul 30;16(1):6983. doi: 10.1038/s41467-025-62217-8.
7
Gasdermin C cleavage by Cathepsin S modulates Rab7 vesicles in intestinal epithelial cells to amplify anti-helminth immunity.组织蛋白酶S对Gasdermin C的切割作用调节肠道上皮细胞中的Rab7囊泡，以增强抗蠕虫免疫。
Immunity. 2025 Jul 16. doi: 10.1016/j.immuni.2025.06.018.
8
A multicellular star-shaped actin network underpins epithelial organization and connectivity.一个多细胞的星形肌动蛋白网络支撑着上皮组织的结构和连接性。
Nat Commun. 2025 Jul 4;16(1):6201. doi: 10.1038/s41467-025-61438-1.
9
A CXCR4 partial agonist improves immunotherapy by targeting immunosuppressive neutrophils and cancer-driven granulopoiesis.一种CXCR4部分激动剂通过靶向免疫抑制性中性粒细胞和癌症驱动的粒细胞生成来改善免疫治疗。
Cancer Cell. 2025 Jun 21. doi: 10.1016/j.ccell.2025.06.006.
10
Phosphocreatine Rescues Intestinal Epithelial Metabolic Dysfunction Related to Creatine Kinase Loss and Is Protective in Murine Colitis.磷酸肌酸可挽救与肌酸激酶缺失相关的肠上皮代谢功能障碍，并对小鼠结肠炎具有保护作用。
Cell Mol Gastroenterol Hepatol. 2025 Jun 23;19(11):101557. doi: 10.1016/j.jcmgh.2025.101557.
本文引用的文献
1
Wnt5a potentiates TGF-β signaling to promote colonic crypt regeneration after tissue injury.Wnt5a 增强 TGF-β 信号以促进组织损伤后的结肠隐窝再生。
Science. 2012 Oct 5;338(6103):108-13. doi: 10.1126/science.1223821. Epub 2012 Sep 6.
2
Controlled gene expression in primary Lgr5 organoid cultures.在原代 Lgr5 类器官培养物中进行基因表达的控制。
Nat Methods. 2011 Dec 4;9(1):81-3. doi: 10.1038/nmeth.1802.
3
Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.从人结肠、腺瘤、腺癌和 Barrett 食管中扩增上皮类器官。
Gastroenterology. 2011 Nov;141(5):1762-72. doi: 10.1053/j.gastro.2011.07.050. Epub 2011 Sep 2.
4
Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.肠隐窝稳态源自对称分裂的 Lgr5 干细胞之间的中性竞争。
Cell. 2010 Oct 1;143(1):134-44. doi: 10.1016/j.cell.2010.09.016.
5
Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.Lgr5(+) 干细胞驱动胃的自我更新，并在体外构建长寿的胃单位。
Cell Stem Cell. 2010 Jan 8;6(1):25-36. doi: 10.1016/j.stem.2009.11.013.
6
Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.单个Lgr5干细胞在体外无需间充质微环境即可构建隐窝-绒毛结构。
Nature. 2009 May 14;459(7244):262-5. doi: 10.1038/nature07935. Epub 2009 Mar 29.
7
Wnt signaling, lgr5, and stem cells in the intestine and skin.肠道和皮肤中的Wnt信号通路、Lgr5与干细胞
Am J Pathol. 2009 Mar;174(3):715-21. doi: 10.2353/ajpath.2009.080758. Epub 2009 Feb 5.
8
Wnt signaling and stem cell control.Wnt信号传导与干细胞调控。
Cold Spring Harb Symp Quant Biol. 2008;73:59-66. doi: 10.1101/sqb.2008.73.035. Epub 2008 Nov 21.
9
Identification of stem cells in small intestine and colon by marker gene Lgr5.通过标记基因Lgr5鉴定小肠和结肠中的干细胞。
Nature. 2007 Oct 25;449(7165):1003-7. doi: 10.1038/nature06196. Epub 2007 Oct 14.
10
A ROCK inhibitor permits survival of dissociated human embryonic stem cells.一种ROCK抑制剂可使解离的人胚胎干细胞存活。
Nat Biotechnol. 2007 Jun;25(6):681-6. doi: 10.1038/nbt1310. Epub 2007 May 27.
Zotero 插件