Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt.
Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt.
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肠上皮细胞的荧光标记揭示了隐窝中独立的长寿命肠干细胞。
DOI：
10.1016/j.bbrc.2014.10.091
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
M. Watanabe
中科院分区：
文献类型：
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作者：
N. Horita;K. Tsuchiya;R. Hayashi;K. Fukushima;S. Hibiya;M. Fukuda;Y. Kano;T. Mizutani;Y. Nemoto;S. Yui;R. Okamoto;T. Nakamura;M. Watanabe
背景和目的肠道干细胞的动态对于肠道功能的调节和维持至关重要。尽管已经通过基因标记方法在肠道中鉴定了隐窝干细胞，但尚未实现对多个隐窝干细胞的鉴定，因为它们以相同的颜色可视化。方法将肠道类器官转移到与编码mCherry的慢病毒混合的Matrigel®中。使用延时成像分析 mCherry 阳性细胞的动态，并使用 3D 免疫荧光分析 mCherry 阳性细胞的定位。 结果我们建立了一种原创方法，将转基因引入小鼠小肠生成的类器官中，导致部分类器官细胞中 mCherry 蛋白出现连续荧光。使用共聚焦显微镜进行的三维分析显示，在培养超过 1 年的类器官隐窝中存在单个 mCherry 阳性细胞，这表明同一隐窝中存在长寿的 mCherry 阳性和阴性干细胞。此外，隐窝中的单个 mCherry 阳性干细胞产生隐窝基底柱状细胞和转运扩增细胞。每个mCherry阳性和阴性细胞都有助于类器官的生成。结论使用我们原创的慢病毒转基因系统来标记单个类器官隐窝干细胞表明，长寿的多个隐窝干细胞可能独立充当肠上皮细胞，从而形成功能完整的绒毛。
Background and aimsThe dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.MethodsIntestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.ResultsWe established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.ConclusionsThe use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.