Single-cell messenger RNA sequencing reveals rare intestinal cell types
Single-cell messenger RNA sequencing reveals rare intestinal cell types
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DOI：
10.1038/nature14966
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发表时间：
2015-09-10
期刊：
影响因子：
64.8
通讯作者：
van Oudenaarden, Alexander
中科院分区：
文献类型：
--
作者：
Grun, Dominic;Lyubimova, Anna;van Oudenaarden, Alexander
要了解一个器官的发育和功能，需要了解它的所有细胞类型。传统的可视化和分离细胞亚群的方法仅基于少数已知标记基因的信使、RNA或蛋白质表达。然而，明确识别特定的标记基因是一个重大挑战，特别是如果这种细胞类型很少见的话。识别罕见的细胞类型，如干细胞、短命祖细胞、癌症干细胞或循环肿瘤细胞，对于更好地了解正常或疾病的组织生物学至关重要。为了应对这一挑战，我们首先对数百个随机选择的小鼠肠道有机体细胞的转录组进行了测序(1)，培养的自组织上皮结构包含哺乳动物肠道的所有细胞谱系。器官芽，就像肠道隐窝一样，含有干细胞，这些干细胞不断分化为各种类型的细胞，其丰度差异很大。由于现有的计算方法只能解析更丰富的细胞类型，我们开发了RaceID算法，用于在复杂的单细胞种群中识别稀有细胞类型。我们证明了该算法可以解析随机抽样的类器官细胞群体中只有一个细胞代表的细胞类型。我们使用这个算法来识别Reg4作为肠内分泌细胞的新标记，肠内分泌细胞是一种罕见的产生激素的肠道细胞群(3)。接下来，我们使用Reg4的表达来丰富这些稀有细胞，并调查这个群体中的异质性。RaceID证实了已知的肠道内分泌谱系的存在，并发现了新的亚型，我们随后在体内进行了验证。在验证了RaceID之后，我们将该算法应用于体外分离的Lgr5阳性干细胞及其直接后代。我们发现，Lgr5阳性的细胞代表了大量的干细胞和稀有的Lgr5阳性的分泌细胞。我们展望了我们的方法在发现健康和疾病器官中罕见的细胞类型和相应的标记基因方面的广泛适用性。
Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids(1), cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances(2). Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells(3). Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5-positive secretory cells. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs.