1] Department of Cell Biology &Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA [2] Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Nat Cell Biol. 2015 Mar;17(3):340-9. doi: 10.1038/ncb3104. Epub 2015 Feb 9.
Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.
干细胞存在于“龛位”中，其中支持细胞为组织更新提供关键信号。培养方法模拟龛位条件，并支持干细胞在体外生长。然而，目前的功能检测方法排除了对克隆干细胞、干细胞-龛位相互作用以及单细胞及其类器官后代的遗传分析进行有统计学意义的研究。在这里，我们描述了一种“微筏阵列”（MRA），它可以促进高通量克隆培养和计算鉴定单个肠干细胞（ISC）和龛位细胞。我们使用 MRA 证明了 Paneth 细胞，一种已知的 ISC 龛位组成部分，以依赖接触的方式增强类器官的形成。MRA 便于回收早期肠类器官进行定量 PCR，以将功能特性（如肠类器官形态）与基因表达的差异相关联。MRA 具有广泛的适用性，可以检测干细胞-龛位相互作用和类器官发育，并作为高通量培养平台，在干细胞命运选择的早期阶段检测基因表达。
