长期可再生的单层人肠上皮干细胞：临床应用潜力
Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use.
作者信息
Scott Andrew, Rouch Joshua D, Jabaji Ziyad, Khalil Hassan A, Solorzano Sergio, Lewis Michael, Martín Martín G, Stelzner Matthias G, Dunn James C Y
机构信息
Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA.
出版信息
J Pediatr Surg. 2016 Jun;51(6):995-1000. doi: 10.1016/j.jpedsurg.2016.02.074. Epub 2016 Mar 2.
PURPOSE
Current culture schema for human intestinal stem cells (hISCs) frequently rely on a 3D culture system using Matrigel™, a laminin-rich matrix derived from murine sarcoma that is not suitable for clinical use. We have developed a novel 2D culture system for the in vitro expansion of hISCs as an intestinal epithelial monolayer without the use of Matrigel.
METHODS
Cadaveric duodenal samples were processed to isolate intestinal crypts from the mucosa. Crypts were cultured on a thin coat of type I collagen or laminin. Intestinal epithelial monolayers were supported with growth factors to promote self-renewal or differentiation of the hISCs. Proliferating monolayers were sub-cultured every 4-5days.
RESULTS
Intestinal epithelial monolayers were capable of long-term cell renewal. Less differentiated monolayers expressed high levels of gene marker LGR5, while more differentiated monolayers had higher expressions of CDX2, MUC2, LYZ, DEF5, and CHGA. Furthermore, monolayers were capable of passaging into a 3D culture system to generate spheroids and enteroids.
CONCLUSION
This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation.
LEVEL OF EVIDENCE
1 Experimental.
目的
目前人类肠道干细胞（hISCs）的培养方案通常依赖于使用基质胶（Matrigel™）的三维培养系统，基质胶是一种富含层粘连蛋白的基质，源自鼠肉瘤，不适合临床应用。我们开发了一种新型二维培养系统，用于在不使用基质胶的情况下将hISCs作为肠上皮单层进行体外扩增。
方法
对尸体十二指肠样本进行处理，从黏膜中分离出肠隐窝。将隐窝培养在I型胶原蛋白或层粘连蛋白的薄涂层上。用生长因子支持肠上皮单层，以促进hISCs的自我更新或分化。每4-5天对增殖的单层进行传代培养。
结果
肠上皮单层能够长期进行细胞更新。分化程度较低的单层表达高水平的基因标志物LGR5，而分化程度较高的单层CDX2、MUC2、LYZ、DEF5和CHGA的表达较高。此外，单层能够传代到三维培养系统中以生成球体和类器官。
结论
该二维系统是扩大hISCs用于进一步实验研究和临床细胞移植的重要一步。
证据水平
1 实验性。
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