Isolation, identification and serum-free culture of mouse intestinal stem cells

• Main Authors: Mahmoud Shaaban Mohamed Abdelrasoul, 穆罕默德
• Other Authors: Chao-Ling Yao
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/sv824s

碩士 === 元智大學 === 生物科技與工程研究所 === 102 === Stem cells are located in many mammalian tissues with the ability of self-renewal and differentiation. One of these tissues is the intestinal tissue. Intestinal stem cells (ISCs) are located at the bottom of the intestinal crypts. Additionally, intestinal stem cells have the ability of differentiation into transit amplifying cells which in turn will give rise to all the mature epithelial cells. Recently, intestinal stem cells have taken a great attention as a promising stem cell therapy for many intestinal diseases such as short bowel syndrome. However, many Challenges were facing the study of ISCs because of the lack of definitive markers and definitive isolation and culture methods. ISCs were identified by a number of biomarkers such as Msi-1, Bmi-1, DCAMKL1 and Lgr5. Thus, developing a definitive culture system and serum-free medium of intestinal stem cells will be promising in the clinical applications and may help in the small intestine tissue engineering. In the present study, we are showing ISCs isolation from mouse small intestine. Furthermore, we have determined the effect of a group of essential growth factors on ISC growth in vitro. Thus, we have selected five significant growth factors; ethanolamine, ascorbic acid phosphate, transferrin, glutathione and sodium selenite which could enhance ISC proliferation in vitro and replace the serum components. Moreover, our serum-free medium could maintain the ISC growth in 3D culture and could enhance the enteroid formation ability of the intestinal crypts in matrigel. The results of gene expression analysis of ISC markers including Lgr5, Bmi1, Msi1 and PTEN have confirmed that our optimum and serum-free media could maintain the stem cell state in this culture system. Additionally, flow cytometric analysis showed that Lgr5+ cells were highly increased after growing for one week in our optimum and serum-free conditions. Furthermore, CD24+ and CD44+ cells increased slightly. Our results suggested that our optimum and serum free growth media can improve the ability of ISCs to form enteroid structures in vitro. Besides, our media can maintain ISC self-renewal and stemmness properties. In a conclusion, these results may help in the enhancement of ISC expansion and understanding the major signaling pathways which maintain the ISC self-renewal and differentiation. Moreover, this serum-free medium will be a good tool in the clinical applications.