Real-time imaging analysis of proliferation/differentiation of colonic epithelial cells in primary culture

原代培养结肠上皮细胞增殖/分化的实时成像分析

• 批准号: 21590803
• 负责人: NAKAMURA Tetsuya
• 金额: $ 3万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21590803/
• 关键词: 消化管上皮幹細胞; ライブセルイメージング; 初代培養技術; 細胞移植治療; 大腸上皮幹細胞; ライブイメージング; 細胞増殖; 細胞分化; 初代培養; 幹細胞移植; 再生治療; 炎症性腸疾患

We have developed a novel culture method for colonic epithelial cells that allows long-term maintenance and efficient expansion of stem cells in vitro. Colonic crypts from normal adult mice were isolated and three-dimensionally cultured in serum-free medium supplemented with a combination of growth factors. The crypt cells formed a round cystic organoid consisting of epithelial monolayer of multi-lineage cells, continuously grew with increasing size of organoid structure, and could be propagated for months without losing their properties. Importantly, Lgr5+colonic stem cells were constantly maintained for a long time period in our culture. In order to test transplantability of these cultured cells, GFP+colonic cells were re-introduced into the lumen of mouse colon superficially damaged by DSS treatment. As observed 4 weeks post-transplantation, the infused organoids readily integrated into the epithelium to form self-renewing GFP+epithelial patches that were histologically normal. Similar results were obtained with colon organoids that were derived from a single Lgr5+colon stem cell after extensive in vitro expansion.The techniques that we have developed would be of significant help to build a basis to develop a novel colon stem cell therapy based on in vitro expansion of a single adult colonic stem cell.

我们开发了一种新的结肠上皮细胞培养方法，可以在体外长期维持和有效扩增干细胞。从正常成年小鼠的结肠隐窝中分离和三维培养的无血清培养基中补充的生长因子的组合。隐窝细胞形成由多谱系细胞的上皮单层组成的圆形囊状类器官，随着类器官结构的大小的增加而连续生长，并且可以繁殖数月而不丧失其特性。重要的是，Lgr5+结肠干细胞在我们的培养物中持续维持很长一段时间。为了测试这些培养的细胞的可移植性，将GFP+结肠细胞重新引入通过DSS处理表面损伤的小鼠结肠的内腔中。如移植后4周所观察到的，输注的类器官容易整合到上皮中以形成组织学正常的自我更新GFP+上皮斑块。类似的结果与结肠类器官，衍生自一个单一的Lgr5+结肠干细胞后，广泛的体外expansion.The技术，我们已经开发的将是显着的帮助建立一个基础，开发一种新的结肠干细胞治疗的基础上，在体外扩增的一个单一的成年结肠干细胞。

项目成果

大腸上皮幹細胞の単離・培養技術とこれを用いた大腸上皮移植技術

结肠上皮干细胞分离培养技术及使用该技术的结肠上皮移植技术

• 发表时间: 2011

HES1 via notch signaling directly suppresses ATOH1/HATH1 gene transcription, resulting in the undifferentiated state of intestinal epithelial cells.

HES1通过Notch信号直接抑制ATOH1/HATH1基因转录，导致肠上皮细胞处于未分化状态。

• 发表时间: 2010
• 作者: Zheng X;Tsuchiya K;Okamoto R;Okamoto E;Iwasaki M;Kano Yoshihito;Nakamura T;Watanabe M
• 通讯作者: Watanabe M

A long-term, fully-deffined culture system for colonic epithelial cells that alows efficient expansion of stem cell compartment

一种长期、完全明确的结肠上皮细胞培养系统，可有效扩展干细胞区室

• 发表时间: 2010
• 作者: Tetsuya Nakamura(Invited speaker);Watanabe M
• 通讯作者: Watanabe M

Epithelial regeneration by cultured colonic cells expanded from a single adult Lgr5+ stem cell

由单个成体 Lgr5 干细胞扩增的培养结肠细胞实现上皮再生

• 发表时间: 2013
• 作者: 中村哲也;渡辺守;高野晴子，幡野雅彦，坂本明美，高野和儀，徳久剛史，遠藤 剛;Nakamura Tetsuya
• 通讯作者: Nakamura Tetsuya

GSK3 inhibitor induces the intestinal differentiation by the protein stabilization of Atoh1

GSK3 抑制剂通过 Atoh1 的蛋白质稳定作用诱导肠分化

• 发表时间: 2009
• 作者: Tsuchiya K;et al.
• 通讯作者: et al.