Non-destructive isolation of trophoblast subtypes

滋养层亚型的无损分离

• 批准号: 9374661
• 负责人: Balaji M Rao
• 金额: $ 23.65万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9374661
• 关键词: Address; Affinity; Antibodies; Benchmarking; Binding; Binding Proteins; Biological Models; Biology; CDX2 gene; Cell Culture Techniques; Cell Separation; Cell surface; Cells; Comparative Study; Complementary DNA; Data; Development; Embryo; Ethics; Exploratory/Developmental Grant; Fluorescence-Activated Cell Sorting; Foundations; Generations; Gestational Age; Heterogeneity; Human; Legal; Libraries; Mass Spectrum Analysis; Methods; Modeling; Molecular; Mus; Outcome; Pathology; Placenta; Placentation; Population; Pre-Eclampsia; Pregnancy; Pregnancy loss; Protein Engineering; Proteins; Publishing; Reagent; Recurrence; Research; Sampling; Scaffolding Protein; Stem cells; Surface; Tissue Sample; Validation; Villous; Work; cell type; combinatorial; cytotrophoblast; experimental study; human embryonic stem cell; in vitro Model; in vivo; insight; interest; mutant; novel; self-renewal; tool; transcriptome; transcriptome sequencing; transcriptomics; trophoblast

PROJECT SUMMARY/ABSTRACT The inability to isolate pure populations of specific trophoblast (TB) subtypes from heterogeneous placental samples limits mechanistic studies on primary TB in culture, or the use of transcriptomic analysis tools for TB characterization. The central challenge in non-destructive iso- lation of TB subtypes is that the specific markers uniquely identifying them are intracellular (e.g. CDX2). There- fore, commonly used methods for isolation of specific, live cell populations, such as fluorescence activated cell sorting (FACS) or magnet-activated cell sorting (MACS), cannot be employed. To address this challenge, we pro- pose a powerful protein engineering strategy for generating non-antibody affinity reagents, which will enable isolation of specific TB subtypes from heterogeneous primary placental samples. We pro- pose to isolate CDX2+ villous cytotrophoblasts (vCTBs); this will uniquely enable the characterization of primary CDX2+ vCTBs using single-cell RNA-seq analysis, and provide insight into heterogeneity of these cells in vivo. Mechanistic studies on human TB development have been limited by the lack of an in vitro model system analogous to mouse TB stem cells; the human TB stem cell has remained elusive. In this context, TB derived from human embryonic stem cells (hESCs) has emerged as a promising model system. Isolation of specific TB populations from human placentas will enable comparison of hESC-derived TB with their in vivo counterparts. Here, single-cell RNA-seq data from primary CDX2+ vCTBs will provide a bench- mark for comparing hESC-derived CDX2+ vCTBs and primary CDX2+ vCTBs. We propose to generate non-antibody binding proteins to non-destructively isolate CDX2+ vCTBs subtypes from human placentas. Accordingly, under Aim 1, we will isolate, from a combinatorial library, affinity reagents that selectively bind to CDX2+ vCTBs, and verify that these reagents can indeed be used for separation of CDX2+ vCTBs from 6-8 wk. human placentas. Under Aim 2, we will use mass spectrometry to identify protein targets bound by the affinity reagents isolated in Aim 1. Finally, under Aim 3 we will characterize CDX2+ vCTBs from 6-8 week placentas using single-cell RNA-seq. We will also compare the transcriptomes of hESC-derived CDX2+ vCTBs and placental CDX2+ vCTBs. The significance of our proposed work relates to three expected outcomes. First, our studies will gen- erate reagents to specifically isolate specific (CDX2+) vCTB sub-populations from heterogeneous mixtures, and therefore enable their characterization using single-cell RNA-seq. Second, comparative studies on vCTBs derived from hESCs and primary placental samples will enable rigorous assessment of the suitability of hESC-derived TB as faithful models of early human TB development. Finally, our strategy can be extended to isolation of other TB subtypes, thus enabling mechanistic studies in TB biology.

项目总结/摘要 无法从哺乳动物中分离出特定滋养层细胞（TB）亚型的纯群体， 异质性胎盘样本限制了对培养中原发性TB的机制研究， 使用转录组学分析工具进行TB表征。非破坏性等离子体技术的核心挑战是 TB亚型的唯一区别在于唯一识别它们的特异性标志物是细胞内的（例如CDX 2）。在那里- 因此，分离特异性活细胞群，如荧光激活细胞， 细胞分选（FACS）或磁激活细胞分选（MACS）。为了应对这一挑战，我们... 提出了一个强大的蛋白质工程策略，用于产生非抗体亲和试剂，这将 使得能够从异质性原始胎盘样品中分离特定TB亚型。我们支持- 分离CDX 2+绒毛细胞滋养层细胞（vCTB）;这将独特地使原发性 使用单细胞RNA-seq分析来检测CDX 2 + vCTB，并提供对这些细胞在体内的异质性的洞察。 由于缺乏体外模型，对人类结核病发展机制的研究受到限制 类似于小鼠结核病干细胞的系统;人类结核病干细胞仍然难以捉摸。在这种情况下，TB 来源于人胚胎干细胞（hESC）的细胞模型已经成为一种有前景的模型系统。分离 来自人胎盘的特定TB群体将能够比较hESC衍生的TB与 它们的体内对应物。在这里，来自原代CDX 2 + vCTB的单细胞RNA-seq数据将提供一个实验台， 用于比较hESC衍生的CDX 2 + vCTB和原代CDX 2 + vCTB的标记。 我们提出产生非抗体结合蛋白以非破坏性地分离CDX 2 + vCTB 从人类胎盘中分离出来的亚型因此，在目标1下，我们将从组合文库中分离亲和亲和素。 选择性结合CDX 2 + vCTB的试剂，并验证这些试剂确实可用于分离 CDX 2 + vCTB在6-8 wk时的变化。人类胎盘。在目标2下，我们将使用质谱法鉴定蛋白质 目标1中分离的亲和试剂结合的靶。最后，在目标3下，我们将表征CDX 2 + vCTB 使用单细胞RNA-seq.我们还将比较hESC衍生的转录组， CDX 2 + vCTB和胎盘CDX 2 + vCTB。 我们拟议工作的重要性涉及三个预期成果。首先，我们的研究将- 评价试剂以从异质混合物中特异性分离特异性（CDX 2+）vCTB亚群，和 因此能够使用单细胞RNA-seq对其进行表征。第二，vCTB的比较研究， 从hESC和原代胎盘样品中分离的细胞将能够严格评估hESC衍生的细胞的适用性。 结核病是早期人类结核病发展的忠实模型。最后，我们的策略可以扩展到隔离其他 TB亚型，从而使TB生物学的机制研究成为可能。