Isolation and characterization of a putative intestinal stem cell fraction from mouse jejunum
Isolation and characterization of a putative intestinal stem cell fraction from mouse jejunum
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DOI：
10.1053/j.gastro.2005.08.011
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发表时间：
2005-11-01
期刊：
影响因子：
29.4
通讯作者：
Henning, SJ
中科院分区：
文献类型：
--
作者：
Dekaney, CM;Rodriguez, JM;Henning, SJ
背景与目的：尽管近年来在肠道干细胞生物学方面取得了许多进展，但由于缺乏分离这些细胞的方法，这一领域受到了很大的阻碍。因此，本研究的目的是探索一种假设，即活的肠道干细胞可以通过荧光激活的细胞分选法分离出来作为一个侧群(SP)。方法：取小鼠空肠全粘膜或上皮细胞，经Hoechst 33342和碘化丙啶染色后，用荧光激活细胞分选法进行分选。用荧光标记的细胞表面标志物、细胞内标志物和膜联蛋白V检测早期细胞凋亡。提取总RNA，进行实时定量逆转录聚合酶链式反应，检测细胞系标志物和肠道干细胞标志物Musashi-1的表达。结果：成人和新生儿空肠均含有SP表型的活细胞群，对维拉帕米敏感。这组细胞(来自粘膜和上皮)包括CD45阴性的部分，对应于非造血细胞，这表明通常在其他组织的干细胞上发现的表面标记和在间充质细胞中发现的细胞内标记的表达很少。此外，这些细胞富含Musashi-1和PI-整合素，细胞角蛋白阳性，在培养中存活长达14天。结论：CD45阴性的SP组分虽然不是纯的，但代表着成功分离出显著富含小肠上皮干细胞的活细胞群。
Background & Aims: Although there have been many recent advances regarding the biology of intestinal stem cells, the field has been hampered significantly by the lack of a method to isolate these cells. Therefore, the aim of this study was to explore the hypothesis that viable intestinal stem cells can be isolated as a side population (SP) by fluorescence-activated cell sorting after staining with the DNA-binding dye Hoechst 33342.Methods: Preparations of individual cells from either whole mucosa or epithelium of mouse jejunum were stained with Hoechst 33342 and propidium iodide and then sorted using fluorescence-activated cell sorting. Cells were characterized using fluorochrome-labeled antibodies to surface markers, intracellular markers, and annexin V to detect early apoptosis. Total RNA was isolated from sorted fractions and used for quantitative real-time reverse-transcription polymerase chain reaction to evaluate the expression of cell lineage markers and the intestinal stem-cell marker, Musashi-1.Results: Adult and neonatal jejunum contain a viable population of cells that shows the SP phenotype and is sensitive to verapamil. This population of cells (from both mucosal and epithelial preparations) includes a CD45-negative fraction corresponding to nonhematopoietic cells, which shows minimal expression of surface markers typically found on stem cells from other tissues and of intracellular markers found in mesenchymal cells. Additionally, these cells were enriched for Musashi-1 and PI-integrin, were cytokeratin positive, and survived in culture for up to 14 days.Conclusions: The CD45-negative SP fraction, although not pure, represents the successful isolation of a viable population significantly enriched in small intestinal epithelial stem cells.