Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells.
Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells.
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DOI：
10.3791/63097
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发表时间：
2021-09-15
期刊：
影响因子：
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通讯作者：
Aguirre A
中科院分区：
文献类型：
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作者：
Lewis-Israeli YR;Volmert BD;Gabalski MA;Huang AR;Aguirre A
研究人类心脏在健康和疾病中的发育的能力受到体外模拟人类心脏复杂性的能力的高度限制。开发更高效的器官样平台，可以对复杂的体内表型进行建模，如有机物和芯片上的器官，将增强研究人类心脏发育和疾病的能力。本文描述了一种利用人类多能干细胞自组织并使用小分子抑制剂逐步激活发育途径来产生高度复杂的人类心脏有机化合物(HHO)的方案。类胚体(EBS)在96孔板中产生，带有圆底、超低附着孔，便于个性化结构的悬浮培养。EBS通过三步Wnt信号调控策略分化为hHO，其中包括最初的Wnt途径激活以诱导心脏中胚层命运，第二步Wnt抑制以建立明确的心脏谱系，第三步Wnt激活步骤以诱导心包前器官组织。这些步骤以96孔的形式进行，是高效的、可重复的，并且每次运行产生大量的有机物。从分化的第3天到第11天的免疫荧光成像分析显示，HHO的第一和第二心野规格，在第15天，hHO内的高度复杂的组织，包括具有心房和室性心肌细胞区域的心肌组织，以及内腔内排列的心内膜组织。这些器官还表现出复杂的血管网络结构和心外膜组织的外部衬里。从功能的角度来看，hHO搏动强劲，并通过Fluo-4实时成像确定具有正常的钙活性。总体而言，该方案为人体器官样心脏组织的体外研究提供了坚实的平台。在这里，我们描述了一种方案，通过自组织有效地利用人类多能干细胞来创造与发育相关的人类心脏器官(HHO)。该方案依赖于发育线索的顺序激活，并产生高度复杂的、功能相关的人类心脏组织。
The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs. The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and at day 15, highly complex tissues inside hHOs, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues. Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.
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影响因子：
20.1
作者：
Pinto AR;Ilinykh A;Ivey MJ;Kuwabara JT;D'Antoni ML;Debuque R;Chandran A;Wang L;Arora K;Rosenthal NA;Tallquist MD
通讯作者：
Tallquist MD
影响因子：
23.9
作者：
Burridge, Paul W.;Keller, Gordon;Gold, Joseph D.;Wu, Joseph C.
通讯作者：
Wu, Joseph C.
影响因子：
28.1
作者：
Richards, Dylan J.;Li, Yang;Mei, Ying
通讯作者：
Mei, Ying
影响因子：
16.6
作者：
Andersen P;Tampakakis E;Jimenez DV;Kannan S;Miyamoto M;Shin HK;Saberi A;Murphy S;Sulistio E;Chelko SP;Kwon C
通讯作者：
Kwon C
影响因子：
64.8
作者：
通讯作者：
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