Mapping_embryonic_stem_cell_surface_interactions_to_transcriptional_phenotypes. Mapping_embryonic_stem_cell_surface_interactions_to_transcriptional_phenotypes

The activation of receptors displayed on the surface of cells typically initiates cytoplasmic signalling cascades that lead to changes in cellular transcriptional responses so that a cell expresses proteins that enable it to behave appropriately as a function of their position within an organism. Understanding these signals and learning how to either prevent or ectopically activate them could have many therapeutic benefits and applications. In some cases these signals are highly localised, being delivered by directly neighbouring cells that express ligands which specifically bind to and activate receptors; for example, stem cells display receptors for ligands provided by cells within their niche to both ensure continual production of cells, and that their cellular progeny are directed towards appropriate fates. While for some cell types we know something of these signals enabling the in vitro differentiation of stem cells towards certain cellular fates, the differentiation processes is often not complete and so “immature” cell types which are not fully functional are produced, implying that some important signals are missing. Here, we propose to systematically identify receptor-triggered signalling pathways by stimulating receptors on the surface of human stem cells by using a library of soluble recombinant oligomerised protein ligands and measuring the consequent transcriptional perturbations using RNA-seq. We propose to screen an existing library of ~50 oligomerised human ligands previously used in receptor protein interaction screens and first test them for their ability to bind to human stem cells to identify those ligands for which the stem cells express a cell surface receptor. For those ligands which interact with the stem cells (we estimate between 2 and 5), they will be added to stem cell cultures and their effects on transcription quantified. The budget requested is sufficient for us to include controls and test important parameters such different time points after addition of the proteins. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/细胞表面展示的受体激活通常会启动细胞质信号级联反应（cytoplasmic signalling cascades），进而引发细胞转录应答的改变，使细胞表达相应蛋白质，从而使其能够根据自身在生物体内的位置做出恰当的行为。解析这类信号并掌握阻断或异位激活（ectopically activate）它们的方法，有望带来诸多治疗益处与应用场景。在某些情况下，这类信号具有高度的局部性，由直接相邻的细胞递送，这些细胞表达的配体（ligand）能够特异性结合并激活受体；例如，干细胞会表达其所在干细胞微环境（niche）内细胞所提供的配体的受体，这既能保证细胞的持续产生，也能引导其子代细胞走向恰当的分化命运。尽管对于部分细胞类型，我们已掌握相关信号以实现干细胞在体外向特定细胞命运的分化，但该分化过程往往并不完全，会产生未完全成熟、功能不全的"未成熟"细胞类型，这意味着仍有部分关键信号尚未被发现。本研究拟通过使用可溶性重组寡聚化蛋白配体（soluble recombinant oligomerised protein ligands）文库刺激人干细胞表面的受体，并借助RNA测序（RNA-seq）检测由此产生的转录扰动，系统性地识别受体触发的信号通路。我们计划筛选一个现有文库，该文库包含约50种此前已用于受体蛋白互作筛选的寡聚化人类配体，首先测试它们结合人干细胞的能力，以鉴定出干细胞表达其细胞表面受体的配体。对于那些能与干细胞发生相互作用的配体（我们预估数量在2至5种之间），我们会将其添加至干细胞培养体系中，并定量分析其对转录的影响。本次申请的预算足以涵盖对照组设置以及测试关键参数的相关工作，例如添加蛋白质后的不同时间点样本采集。本数据属于预出版发布数据集。若想了解威康桑格研究所（Wellcome Trust Sanger Institute）共享的预出版数据的规范使用方法（包括任何出版禁运期的相关细节），请访问http://www.sanger.ac.uk/datasharing/