Hubrecht Institute, KNAW and University Medical Centre Utrecht, Utrecht, The Netherlands.
Gastroenterology. 2011 Nov;141(5):1762-72. doi: 10.1053/j.gastro.2011.07.050. Epub 2011 Sep 2.
BACKGROUND & AIMS: We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon.
Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems.
Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus.
We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.
我们之前建立了长期培养条件，使从小鼠小肠中分离得到的单个隐窝或干细胞能够长时间扩增。扩增的隐窝经历多次隐窝分裂事件，同时产生类绒毛的上皮区域，其中包含所有分化类型的细胞。我们已经适应了这些培养条件，能够从小鼠结肠和人小肠及结肠中培养出类似的上皮类器官。
基于从小鼠小肠的培养体系，我们对小鼠和人结肠的培养体系进行了优化。
在应用于小鼠结肠隐窝的生长因子组合中添加 Wnt3A ，使其能够无限期扩增。在长期培养人小肠和结肠组织时，需要添加烟酰胺，以及一种 Alk 的小分子抑制剂和一种 p38 的抑制剂。该培养系统还允许生长小鼠 Apc 缺陷型腺瘤、人结直肠癌细胞以及 Barrett 食管区域的人化生上皮。
我们开发了一种可用于研究人类胃肠道感染、炎症或肿瘤组织的技术。这些工具可能通过体外扩增肠上皮在再生生物学中有应用。对这些培养物的研究表明，在体外，成人干细胞（或海弗利克极限）不存在固有复制潜能限制。
