Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use.
Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use.
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DOI：
10.1016/j.jpedsurg.2016.02.074
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发表时间：
2016-06
影响因子：
2.4
通讯作者：
Dunn JC
中科院分区：
文献类型：
--
作者：
Scott A;Rouch JD;Jabaji Z;Khalil HA;Solorzano S;Lewis M;Martín MG;Stelzner MG;Dunn JC
目前人类肠道干细胞 (hISC) 的培养方案通常依赖于使用 Matrigel™ 的 3D 培养系统，Matrigel™ 是一种源自鼠肉瘤的富含层粘连蛋白的基质，不适合临床使用。我们开发了一种新型 2D 培养系统，用于在不使用基质胶的情况下以肠上皮单层形式体外扩增 hISC。对尸体十二指肠样本进行处理，以将肠隐窝与粘膜分离。隐窝培养在 I 型胶原蛋白或层粘连蛋白的薄层上。单层肠上皮细胞受到生长因子的支持，以促进 hISC 的自我更新或分化。增殖的单层细胞每 4-5 天进行一次传代培养。肠上皮单层能够长期细胞更新。分化程度较低的单层细胞表达高水平的基因标记物 LGR5，而分化程度较高的单层细胞则具有较高的 CDX2、MUC2、LYZ、DEF5 和 CHGA 表达。此外，单层细胞能够传代到 3D 培养系统中以生成球状体和肠状体。该 2D 系统是扩展 hISC 以进行进一步实验研究和临床细胞移植的重要一步。
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. 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 4–5 days. 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. This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation.