Single-Cell mRNA Sequencing Reveals Rare Intestinal Cell Types. Mus musculus

Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating sub-populations of cells are based on mRNA or protein expression of only 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 tumor cells is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbor stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. 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. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. Reassuringly, RaceID confirmed the existence of known enteroendocrine lineages, and moreover, discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID by this proof-of-principle experiment we then apply the algorithm to ex vivo isolated LGR5 positive cells and their direct progeny and demonstrate homogeneity of the stem cell pool. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs. Overall design: Small intestinal crypts were isolated from a single wild-type C57BL/6 mouse, a Reg4-dsRed-knock-in mouse and an Lgr5-GFP-DTR mouse. The crypts were propagated and expanded in culture as organoids. For each experiment, multiple organoids were harvested and dissociated into single cells. Each experiment was done twice, using different passage of the same organoid culture. We also included a pool-and-split control for 96 Reg4-dsRed positive intetsinal cells and a control library with 5 mouse embryonic stem cells (wells 1-5), 5 mouse embryonic fibroblasts (wells 6-10), 75 random organoid cells (wells 11-85), 5 wells without primer and without template (wells 86 and 93-96), and five wells with primer and without template (wells 87-92). We also sequenced two 96 well plates of Lgr5-EGFP positive single cells isolated ex vivo, and Lgr5 progeny collected after five days of lineage tracing. Label induction was performed using an Lgr5-Cre reporter mouse expressing YFP from Rosa26 promoter with a loxP flanked transcriptional road block in between. Five 96 well plates of YFP positive were sequenced. Sample number four also contains also unrelated samples (single cell barcode 49-96), which should be discarded.

解析器官的发育与功能，需对其所有细胞类型完成表征。传统的细胞亚群可视化与分离方法，仅依托少数已知标记基因（marker gene）的mRNA或蛋白质表达水平。然而，精准鉴定特定标记基因是一项重大挑战，当该细胞类型属于稀有细胞类型（rare cell type）时尤为如此。鉴定干细胞、短寿命祖细胞、癌症干细胞或循环肿瘤细胞等稀有细胞类型，对于深化我们对正常及病变组织生物学的理解至关重要。
为应对这一挑战，我们对小鼠肠道类器官（intestinal organoid）中数百个随机选取的细胞进行了转录组（transcriptome）测序——肠道类器官是一类可体外培养的自组织上皮结构，包含哺乳动物肠道的全部细胞谱系。与肠道隐窝类似，类器官芽体中驻留干细胞，这些干细胞可持续分化为多种细胞类型，各类细胞的丰度差异悬殊。鉴于现有计算方法仅能分辨丰度较高的细胞类型，我们开发了RaceID——一款用于在复杂单细胞（single cell）群体中识别稀有细胞类型的算法。
我们证实，该算法可在随机取样的类器官细胞群体中，分辨出仅由单个细胞代表的细胞类型。我们利用该算法鉴定出Reg4可作为肠内分泌细胞（enteroendocrine cell）的新型标记基因——肠内分泌细胞是一类稀有的激素分泌型肠道细胞群体。随后，我们通过Reg4的表达富集这类稀有细胞，并对该群体内的异质性展开探究。令人满意的是，RaceID不仅证实了已知肠内分泌细胞谱系的存在，还发现了新型亚型，并后续在体内完成了验证。
通过这一原理验证实验完成RaceID的有效性验证后，我们将该算法应用于体外分离的LGR5阳性细胞及其直接子代细胞，证实了干细胞池的均质性。我们预计该方法可广泛应用于健康与病变器官中稀有细胞类型及对应标记基因的发现。
整体实验设计：从单只野生型C57BL/6小鼠、Reg4-dsRed敲入小鼠及Lgr5-GFP-DTR小鼠中分离小肠隐窝，将隐窝体外培养并扩增为类器官。每项实验均收集多个类器官，解离为单细胞悬液。每项实验均使用同一类器官培养物的不同传代次数重复两次。我们设置了混合分装对照：96个Reg4-dsRed阳性肠道细胞，以及包含5个小鼠胚胎干细胞（孔位1-5）、5个小鼠胚胎成纤维细胞（孔位6-10）、75个随机类器官细胞（孔位11-85）、5个无引物无模板空白对照（孔位86及93-96）和5个有引物无模板对照（孔位87-92）的对照文库。此外，我们还对两批96孔板的体外分离的Lgr5-EGFP阳性单细胞，以及经过5天谱系追踪后收集的Lgr5子代细胞进行了测序。谱系追踪的标记诱导使用Lgr5-Cre报告小鼠，该小鼠在带有loxP侧翼转录阻断序列的Rosa26启动子下游表达YFP。我们对五批96孔板的YFP阳性细胞完成了测序。样本4中还包含无关样本（单细胞条形码49-96），应予以剔除。