Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.
Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.
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DOI：
10.1053/j.gastro.2013.04.050
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发表时间：
2013-08
期刊：
影响因子：
29.4
通讯作者：
Li L
中科院分区：
文献类型：
--
作者：
Wang F;Scoville D;He XC;Mahe MM;Box A;Perry JM;Smith NR;Lei NY;Davies PS;Fuller MK;Haug JS;McClain M;Gracz AD;Ding S;Stelzner M;Dunn JC;Magness ST;Wong MH;Martin MG;Helmrath M;Li L
肠干细胞（ISCs）的鉴定在很大程度上依赖于在小鼠中使用转基因报告基因，但这种方法受到嵌合表达模式的限制，并且难以直接应用于人体组织。我们试图鉴定ISCs的可靠表面标志物，并建立一种稳健的功能测定来表征来自小鼠和人组织的ISCs。我们使用免疫组化，实时逆转录聚合酶链反应，荧光激活细胞分选（FACS）分析肠上皮细胞分离小鼠和人类肠道组织。我们比较了基于Lgr 5-绿色荧光蛋白表达分离的ISCs之间表面标记物的不同组合。我们开发了一种培养方案，以促进从小鼠中鉴定功能性ISCs，然后用人肠隐窝和推定的ISCs测试该测定。通过FACS从小鼠小肠和结肠分离的CD 44 + CD 24 loCD 166+细胞表达高水平的干细胞相关基因。然后基于GRP 78或c-Kit的表达排除过渡扩增细胞和祖细胞。来自小鼠小肠的CD 44 + CD 24 loCD 166 + GRP 78 lo/−推定干细胞包括Lgr 5-GFPhi和Lgr 5-GFP med/lo细胞。将这些细胞与GSK抑制剂CHIR 99021和E-钙粘蛋白稳定剂Thiazovivin孵育，导致25%至30%的单一分选的ISC形成集落。我们开发了一种培养方案，根据细胞表面标记物从小鼠和人组织中鉴定推定的ISCs。CD 44 + CD 24 loCD 166+、GRP 78 lo/−和c-Kit−分别促进了小鼠小肠和结肠中推定干细胞的鉴定。CD 44 + CD 24 −/loCD 166+也鉴定了推定的人ISCs。这些发现将促进小鼠和人类ISCs的功能研究。
Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/− putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/−, and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
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影响因子：
30.8
作者：
Sangiorgi, Eugenio;Capecchi, Mario R.
通讯作者：
Capecchi, Mario R.
影响因子：
21.3
作者：
通讯作者：
--
影响因子：
64.5
作者：
Powell AE;Wang Y;Li Y;Poulin EJ;Means AL;Washington MK;Higginbotham JN;Juchheim A;Prasad N;Levy SE;Guo Y;Shyr Y;Aronow BJ;Haigis KM;Franklin JL;Coffey RJ
通讯作者：
Coffey RJ
DOI：
10.1126/science.1180794
发表时间：
2010-01-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Li L;Clevers H
通讯作者：
Clevers H
DOI：
10.1073/pnas.1013004108
发表时间：
2011-01-04
影响因子：
11.1
作者：
Montgomery, Robert K.;Carlone, Diana L.;Breault, David T.
通讯作者：
Breault, David T.