A novel culture system for adult porcine intestinal crypts.
A novel culture system for adult porcine intestinal crypts.
复制标题
DOI：
10.1007/s00441-016-2367-0
复制
发表时间：
2016-07
影响因子：
3.6
通讯作者：
Stelzner MG
中科院分区：
文献类型：
--
作者：
Khalil HA;Lei NY;Brinkley G;Scott A;Wang J;Kar UK;Jabaji ZB;Lewis M;Martín MG;Dunn JC;Stelzner MG
猪模型可用于研究短肠综合征的治疗方法，并可能用于肠道干细胞(ISC)移植。虽然小鼠和人类的干细胞培养和遗传操作技术已经很成熟，但猪干细胞的类似方法还没有报道。从小鼠、人、幼年和成年猪的小肠中分离出空肠隐窝，悬浮在Matrigel中，与同种肠道上皮下肌成纤维细胞(ISEMF)共培养或在不同的培养液中不加饲养细胞进行培养。在含有表皮生长因子、noggin和R-pondin 1的培养液(ENR)中添加不同组合的Wnt3a或ISEMF条件培养液(CM)和糖原合成酶激酶3抑制剂(GSK3i)，并在培养的隐窝上进行研究。免疫组织化学(IHC)和定量聚合酶链式反应(QPCR)检测细胞系分化。用慢病毒载体对培养的猪细胞进行连续传代和转导。而ENR培养液支持小鼠肠样生长，但不能维持猪隐窝超过5天。添加Wnt3a-CM和GSK3i可形成具有萌芽延伸的复合猪肠样体。这些肠样组织含有干细胞和分化细胞的混合物，并在GSK3i存在下成功传代。在添加了猪ISEMF-CM的培养液中生长的隐窝形成的球体比肠样体分化得更差。用慢病毒高效地转染肠和球体。我们描述了一种在培养中长期保持幼猪和成年猪隐窝细胞的方法。猪肠球体和球体可以通过慢病毒载体成功地传代和转导。
Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. While techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM), and glycogen synthase kinase 3 inhibitor (GSK3i) and studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry (IHC) and quantitative PCR (qPCR). Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth; it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. We describe a method to maintain juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced using lentiviral vectors.