1] Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan. [2] Advanced Medical Research Center, Yokohama City University, Yokohama, Japan. [3] Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Japan.
1] Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan. [2].
Nat Protoc. 2014 Feb;9(2):396-409. doi: 10.1038/nprot.2014.020. Epub 2014 Jan 23.
Generation of functional and vascularized organs from human induced pluripotent stem cells (iPSCs) will facilitate our understanding of human developmental biology and disease modeling, hopefully offering a drug-screening platform and providing novel therapies against end-stage organ failure. Here we describe a protocol for the in vitro generation of a 3D liver bud from human iPSC cultures and the monitoring of further hepatic maturation after transplantation at various ectopic sites. iPSC-derived specified hepatic cells are dissociated and suspended with endothelial cells and mesenchymal stem cells. These mixed cells are then plated onto a presolidified matrix, and they form a 3D spherical tissue mass termed a liver bud (iPSC-LB) in 1-2 d. To facilitate additional maturation, 4-d-old iPSC-LBs are transplanted in the immunodeficient mouse. Live imaging has identified functional blood perfusion into the preformed human vascular networks. Functional analyses show the appearance of multiple hepatic functions in a chronological manner in vivo.
从人类诱导多能干细胞 (iPSC) 生成功能性和血管化的器官将有助于我们理解人类发育生物学和疾病建模，有望提供药物筛选平台并为终末期器官衰竭提供新的治疗方法。在这里，我们描述了一种从人 iPSC 培养物中体外生成 3D 肝芽的方案，并监测在各种异位部位移植后的进一步肝成熟情况。iPSC 衍生的特定肝细胞与内皮细胞和间充质干细胞解离并悬浮。这些混合细胞随后被种植在预凝固的基质上，在 1-2 天内形成一个 3D 球形组织块，称为肝芽 (iPSC-LB)。为了促进进一步成熟，4 天龄的 iPSC-LB 被移植到免疫缺陷小鼠中。活体成像已鉴定出功能血液灌注到预先形成的人血管网络中。功能分析显示在体内以时间顺序出现多种肝功能。
