A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids.
A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids.
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一种改良的人诱导多能干细胞来源的肠上皮细胞器的培养体系
DOI：
10.1016/j.stemcr.2017.11.004
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发表时间：
2018-01-09
影响因子：
5.9
通讯作者：
Kiyono H
中科院分区：
文献类型：
--
作者：
Takahashi Y;Sato S;Kurashima Y;Yamamoto T;Kurokawa S;Yuki Y;Takemura N;Uematsu S;Lai CY;Otsu M;Matsuno H;Osawa H;Mizushima T;Nishimura J;Hayashi M;Yamaguchi T;Kiyono H
肠道上皮类器官通常用于研究肠道生物学;然而，目前的培养方法不适合遗传操作，并且难以产生足够的数量用于高通量研究。在这里，我们提出了一个改进的培养系统的人诱导多能干细胞（iPSC）衍生的肠道类器官，涉及四个方法的进步。(1)我们采用慢病毒载体，以方便地建立和优化条件培养基的人肠道类器官培养。(2)我们通过补充WNT 3A和成纤维细胞生长因子2以诱导分化成定形内胚层，更有效地从人iPSC获得肠类器官。(3)使用二维培养，然后重建类器官，我们实现了外源基因在类器官中的有效转导。(4)我们研究了无支架的悬浮类器官培养，以便于收获和测定。这些技术使我们能够以低成本轻松快速地开发，维持和扩大肠道类器官，促进高通量筛选致病因素和胃肠道疾病的候选治疗方法。我们通过慢病毒感染优化了L细胞的条件培养基制备。我们修改了将iPSC分化为小肠类器官的方案。我们成功地通过2D培养将基因转导到类器官中。开发了几种用于人类诱导多能干细胞（iPSC）衍生的肠类器官的用户友好的培养方法。方法学的改进包括制备用于类器官培养的条件培养基、从iPSC有效分化、类器官中的基因转导以及悬浮培养中的生长。该系统将有助于高通量筛选致病因子和使用生理上强大的肠道类器官治疗肠道疾病。
Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases. We optimized conditioned medium preparation from L cells by lentiviral infection We modified a protocol to differentiate iPSCs into small intestinal organoids We succeeded in transducing a gene into organoids by 2D culture We showed that intestinal human organoids self-proliferate in suspension culture Takahashi et al. developed several user-friendly culture methods for human induced pluripotent stem cell (iPSC)-derived intestinal organoids. The methodological improvements include preparation of conditioned medium for organoid culture, efficient differentiation from iPSCs, gene transduction in organoids, and growth in suspension culture. This system will facilitate high-throughput screening of pathogenic factors and treatments for intestinal diseases using physiologically robust intestinal organoids.
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影响因子：
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