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

描述（由申请人提供）：我们建议将联合收割机两种快速发展的技术结合起来，以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方法，用于获取人类诱导多能干细胞，将其暴露于一系列生长因子，并指导诱导多能干细胞的形成。 典型的三维肠类器官。值得注意的是，这些显示了隐窝绒毛结构和分化细胞类型的完整库的存在，以及成年隐窝干细胞。 细胞此外，我们最近开发了一种程序，命名为SCAMP，它允许精确的RNA-Seq分析单细胞的基因表达状态。 我们建议使用单细胞的RNA-Seq来捕获定义人类肠道发育的多个发育阶段的基因表达状态。将确定起始人类多能干细胞、诱导内胚层、原肠管和发育中的肠类器官的全局转录组。通过比较重叠谱，将有可能精确地定义基因表达进展。通过将来自同质肠肠管细胞的基因表达模式与不同的肠细胞类型，特别是成体肠干细胞联系起来，将有可能精确地定义每个发育程序。 为了建立分化的成人谱系的基线，我们还将分析从患者活检组织中分离的隐窝中的单细胞。然后我们会用这个 开发图谱来询问我们从诱导多能干细胞系产生的肠道类器官，这些干细胞系来源于患有婴儿顽固性腹泻综合征（IDIS）的患者。这种分析将使我们能够确定在正常发育过程中的扰动，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3-D图像。 此外，我们建议使用一个程序，我们已经建立，允许一个中等通量的肠道发育功能的基因分析。基因敲减是通过使用慢病毒驱动shRNA在多能干细胞中表达来实现的。然后诱导这些细胞形成类器官，并检查由此产生的发育异常。这一过程比在小鼠中进行基因靶向至少快十倍。 这项工作的结果将确定成人肠上皮细胞和成人隐窝干细胞的多种分化细胞类型的完整分子特征。将鉴定这些细胞的新分子标记物，并表征完整的分子分化程序。基因将被功能分析，基因调控网络将被更好地定义。

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.