In Vitro Isolation of Intestinal Side Population Cells from Newborn Mice

Abstract:

To isolate side population cells from newborn mice small intestinal mucosa, and to investigate the feasibility of constructing the murine intestinal stem cell population by fluorescence activated cell sorting. Small intestine mucosa organoids of mice were isolated and dissociated into single cells. The side population cells were stained with Hoechst 33342 and propidium iodide, then sorted using fluorescence activated cell sorting. Total RNA and protein were purified from sorted fractions to detect Musashi-1 expressions by RT-PCR and Western-blotting. Single cell suspension from mouse small intestine mucosa contained a viable population of cells, which showed the side population phenotype and were sensitive to verapamil. These cells were enriched for Musashi-1 mRNA and MSI-1 protein expression. Results demonstrated that the side population fraction separated from mice intestinal mucosa is enriched for intestinal stem cells, the murine intestinal stem cell population can be successfully constructed with fluorescence activated cell sorting.

• Periodical: Applied Mechanics and Materials (Volume 140)
• Pages: 157-161
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.140.157
• Online since: November 2011
• Authors: Yong Feng Zhang, Jin Yang, Yong Gang Tan, Ying Hong Li, Min Yang
• Keywords: Fluorescence Activated Cell Sorting, Intestinal Stem Cells, Mice, Musashi-1, Side Population Cells

References

[1] Wang L, Liu J. Isolation, culture, induction, differentiation and application of hepatic stem cells. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2008;12(12):2355-2358.

[2] Wang Y, Liu H, Chen Y, et al. Progress in intestinal epithelial stem cells and colorectal cancer stem cells. Xiandai Linchuang Yixue. 2009;35(3):165-167.

[3] Wang R, Jiang JT, Yang YL, et al. Isolation and purification of tumor stem cells. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2009;13(1):161-164.

[4] Yan H. Gastrointestinal stem cells and stem cell transplantation for gastrointestinal diseases. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2007; 11(20): 4009-4012.

[5] Han Q, He DN, Liu YN, et al. Repair of intestinal irradiation injury by Flk-1~+ MSCs and SP cells. Jichu Yixue yu Linchuang.2008;28(6):563-569.

[6] The Ministry of Science and Technology of the People's Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.

[7] He DN, Qin H, Liao L, et al. Small intestinal organoid-derived SP cells contribute to repair of irradiation-induced skin injury.Stem Cells Dev. 2005;14(3):285-291.

DOI: 10.1089/scd.2005.14.285

[8] Wang ZL, Han Q, Zhao CH, et al. Characterization of intestine side population cells from fetal rats. Yixue Yanjiusheng Xuebao.2007;20(7):693-696.

[9] Ye ZQ, Liu GL, Chen ZQ, et al. Construction of Murine Intestinal Stem Cell Population. Huazhong Keji Daxue Xuebao:Yixueban.2006;35(6):828-830.

[10] Zhu LB, Liu ZJ, Li ZR, et al. A primary report on the culture in vitro of enteric neural stem cells in rats. Wenzhou Yixueyuan Xuebao. 2007;37(1):5-8.

[11] Li WD, Zhang XG, Chang J, et al. Isolation and culture of murine embryonic stem cells of Kunming species. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu.2008;12(21):4060-4064.

[12] Zhu YL, Zhong X, Zheng S. Conditionally immortalized human colorectal crypt cell line. Zhejiang Daxue Xuebao:Yixueban. 2004;33(5):379-384.

[13] Luo YQ, Wen Y, Wu CT. The advances on Isolation and culture of intestinal epithelial stem cells. Guoji Waike Zazhi. 2007;34(2):101-103.

[14] Xu YY, Zhu HY, Ai J, et al. Research Progress of SP cells.Shengming de Huaxue. 2008;28(5):536-538.

[15] Li YT, Hu WH, Dou J. SP. Research progress of side population cells. Shengwu Jishu Tongxun. 2008;19(3):430-432.

[16] He QZ, Lu HJ, Ma AG. The relationships and advances on stem cells、tumor stem cells and side population cells. Xiandai Zhongliu Yixue. 2008;16(10): 1803-1806.

[17] Hao YP, Hu SM, Zhang SD, et al. Expression and significance of stem cell marker LGR5 proteins in the development and progression of human colorectal carcinoma. Shandong Daxue Xuebao: Yixueban. 2009;47(8):85-88.

[18] Asai R, Okano H, Yasugi S. Correlation between Musashi-1 and chairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse.Dev Growth Differ. 2005;47(8):501-510.

DOI: 10.1111/j.1440-169x.2005.00825.x

[19] Wang YX, Wu CT. Research progress of intestinal stem cell markers. Zhongguo Putong Waike Zazhi.2008;17(10):1013-1015.

[20] Zhao Y, Li JS, Ma CL. Identification of human gastrointestinal stem cell markers. Weichangbingxue he Ganbingxue Zazhi.2006;15(2):199-202.

[21] Gong JF, Zhu WM, Gao X, et al. Cloning and functional analysis of the promoter region of an intestinal stem cell specific expressed gene,Musashi-1. Changnei yu Changwai Yingyang. 2006;13(5):257-260.

[22] Gu EY, Ha F, Li LF, et al. Identification method of neural stem cells. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu.2009;13(10):1947-1950.