Single Cell/RNA-Seq dissection of Human iPS cell development into intestine

描述(由申请人提供)：我们建议结合两种快速发展的技术来生成驱动肠道发育的基因表达模式的单细胞分辨率图谱。我们最近描述了一种获取人类诱导多能干细胞的方法，将它们暴露在一系列生长因子中，并指导 典型的三维肠道器官。值得注意的是，图示隐窝绒毛结构和完整的分化细胞类型，以及成体隐窝干细胞。 细胞。此外，我们最近开发了一种名为SCAMP的程序，它可以对单个细胞的基因表达状态进行准确的RNA-Seq分析。我们建议使用单细胞的RNA-Seq来捕捉定义人类肠道发育的多个发育阶段的基因表达状态。将确定开始的人类多能干细胞、诱导内胚层、原始肠管和发育中的肠道器官的整体转录。通过比较重叠的图谱，将有可能精确地定义基因表达的进程。例如，通过将来自同种肠管细胞的基因表达模式与不同类型的肠道细胞，特别是成人肠道干细胞联系起来，将有可能准确地定义每一种发育程序。为了建立分化的成人血统的基线，我们还将分析从患者活检组织中分离的隐窝中的单个细胞。然后我们将使用这个 发育图谱，以询问肠道类器官，我们从诱导的多能干细胞系来自婴儿综合征(IDIS)患者的顽固性腹泻(IDIS)。这一分析将使我们能够识别正常发育过程中的扰动，这些扰动可能是IDIS上皮功能障碍的基础。原位杂交和免疫组织化学将用于验证RNA-Seq数据，定位具有不同基因表达谱的细胞，并重建发育过程的三维图像。此外，我们建议使用我们已经建立的程序，该程序允许对基因的肠道发育功能进行中等吞吐量分析。基因敲除是通过使用慢病毒驱动多能干细胞中shRNA的表达来完成的。然后，这些细胞被诱导形成有机化合物，并检查由此产生的发育异常。这一过程比在小鼠身上进行基因打靶至少快10倍。拟议工作的结果将确定成人肠上皮以及成人隐窝干细胞的多种分化细胞类型的完整分子特征。将确定这些细胞的新分子标记，并对完整的分子分化程序进行表征。将对基因进行功能分析，并更好地定义遗传调控网络。

DESCRIPTION (provided by applicant): We propose to combine two rapidly evolving technologies to generate a single cell resolution atlas of the gene expression patterns that drive intestinal development. We have recently described a method for taking human induced pluripotent stem cells, exposing them to a series of growth factors, and directing the formation of stereotypical 3-dimensional intestinal organoids. Remarkably these show crypt-villus structure and the presence of a complete repertoire of differentiated cell types, as well as adult crypt stem cells. In addition we have recently developed a procedure, designated SCAMP, which allows accurate RNA-Seq analysis of the gene expression states of single cells. We propose to use RNA-Seq of single cells to capture the gene expression states that define the multiple developmental stages of human intestine development. Global transcriptomes will be determined for the starting human pluripotent stem cells, the induced endoderm, the primitive gut tube, and developing intestinal organoids. By comparing overlapping profiles it will be possible to precisely define gene expression progressions. By connecting the gene expression patterns that proceed from homogeneous intestinal gut tube cells, for example, to distinct intestinal cell types, and in particular the adult intestinal stem cell, it will be possible to preisely define each developmental program. To establish a base line for differentiated adult lineages we will also analyze single cells from crypts isolated from patient biopsies. We will then use this developmental atlas to interrogate intestinal organoids that we generate from induced pluripotent stem cell lines derived from a patient with intractable diarrhea of infancy syndrome (IDIS). This analysis will allow us to identify perturbations in normal developmental processes that may underlie the dysfunction in IDIS epithelium. In situ hybridizations and immunohistochemistry will be used to validate the RNA-Seq data, to localize cells with distinct gene expression profiles, and to reconstruct a 3-D image of the developmental process. In addition we propose to use a procedure we have established that allows a medium throughput analysis of the intestinal developmental function of genes. Gene knockdown is accomplished by using lentivirus to drive expression of shRNA in pluripotent stem cells. These are then induced to form organoids, and resulting developmental abnormalities examined. This procedure is at least ten times faster than can be accomplished with gene targeting in mice. The results of the proposed work would define the full molecular character of the multiple differentiated cell types of the adult human intestinal epithelium, as well the adult crypt stem cells. Novel molecular markers for these cells will be identified and complete molecular differentiation programs characterized. Genes will be functionally analyzed and genetic regulatory networks better defined.