Understanding haematopoietic stem cell development through global single-cell gene expression analysis

造血可以被定义为一个高度调控的过程，通过使用高度专业化的多阶段单向分级系统来产生所有成熟的血细胞。它涉及形态模糊的前体细胞的分化，以产生多谱系祖细胞和谱系承诺的前体细胞。HSCs是一种异质多能干细胞，能够自我更新和分化，位于该系统的顶端。在成体中，它们主要存在于骨髓(BM)中，但在胚胎发育过程中发育。在脊椎动物个体发育过程中，最终的HSC出现在AGM中，然后重新路由到胎肝和BM(出生后)。AGM是从胚胎中胚层的腹主动脉旁内脏胸膜发育而来，据说是E10.5的确定的小鼠HSCs的位置。这一过程通过AGM的腹主动脉腹侧区域的细胞从内皮细胞到造血细胞的转变而发生，导致主动脉内形成簇，从而产生前HSCs。前HSCs、I型和II型前HSCs在AGM中的发育系统及其与生态位的相互作用尚不完全清楚。我的项目主要是分析小鼠胚胎发生过程中AGM中HSC的发育情况。由于HSCs的异质性，它们的发育特征一直是这些细胞表型特征的一个混杂因素。单细胞分析最近已成为绘制细胞异质性图的一种强有力的方法(Zilionis等人，2016)。我将使用10倍铬单细胞基因表达系统对单个转录本进行高通量、无偏见的分析，以分析HSC的发育。这项技术可以在单个细胞水平上直接测量基因表达，量化种群异质性，表征单个细胞类型和AGM中的动态细胞过渡。此外，使用单细胞技术可以更好地了解转录动力学和基因调控关系。这将允许超越传统的方法，通过分析差异基因表达来探索伴随的动态变化。重要的是，单细胞基因表达允许对细胞群体的无偏见表征，而不依赖于任何细胞亚型或标记的先验知识，从而使我们能够生成全局视图(Zilionis等人，2016)。10X系统采用微流控分割来捕获单个细胞并生成条形码cDNA文库。从腹主动脉腹侧区域产生的单细胞悬浮液的有限稀释、逆转录试剂、含有条形码寡核苷酸的凝胶珠和分配油将在10X微流控芯片上结合在一起，形成乳液中的凝胶珠(GEM)。从理论上讲，GEMS应该包含单细胞、凝胶珠和RT试剂。细胞裂解后，凝胶珠溶解，将相同条形码的RT寡核苷酸释放到溶液中，允许进行逆转录。这导致从包含相同条形码的单个细胞产生的所有cDNA在测序后被映射回其原始细胞。然后，准备好的文库将进行Illumina测序，以产生用于计算分析的结果。生成的原始读数将使用各种管道进行生物信息学分析，以对齐、绘制地图并执行QC分析。在此之后，将使用基因表达矩阵和差异基因表达谱以及基因聚类来开发基因表达的全局视图。结果将被用来描述从单细胞AGM悬液中产生的单个转录本。这将使我们能够获得更多关于HSC在发育过程中的全球和层级组织以及它们在胚胎发生期间与AGM生态位相互作用的信息。

Haematopoiesis can be defined as a highly regulated process through which all mature blood cells are generated by employing a highly specialised multi-phase unidirectional hierarchical system. It involves the differentiation of morphologically indistinct precursor cells to generate multilineage progenitors and lineage-committed precursors. HSCs are heterogeneous multipotent stem cells capable of self-renewal and differentiation that lie at the apex of this system. In the adult, they primarily reside in the bone marrow (BM), but develop during embryogenesis.During vertebrate ontogeny, definitive HSCs arise in the AGM prior to their re-routing to the fetal liver and the BM (following birth). The AGM is developed from para-aortic splanchnopleaura in the embryonic mesoderm and is said to be site of definitive murine HSCs at E10.5. This process occurs through endothelial to haematopoietic transition of cells from the ventral domain of the dorsal aorta in the AGM, resulting in the formation of intra-aortic clusters that give rise to pre-HSCs .The system through which pro-HSCs, type I and type II pre-HSCs develop in the AGM and their interaction with the niche is not fully understood. My project is primarily concerned with analysing the development of HSC in the AGM during murine embryogenesis. Due to the heterogeneous nature of HSCs, characterising their development has been a confounding factor in phenotypically profiling these cells. Single-cell analysis has recently emerged as a powerful approach for mapping cellular heterogeneity (Zilionis et al., 2016). I will use the 10X Chromium Single Cell Gene Expression system to provide a high-throughput unbiased analysis of individual transcriptomes to analyze HSC development. This technique allows for a direct measurement of gene expression at a single cell level, quantification of population heterogeneity, characterization of individual cell types and dynamic cellular transitions in the AGM. In addition, using single cell technologies enables a better understanding of transcriptional dynamics and gene regulatory relationships. This would allow for an extension beyond the traditional approaches to explore concomitant dynamic changes, by analyzing differential gene expression. Importantly, single cell gene expression allows for an unbiased characterization of cellular populations independent of any prior knowledge of cell subtypes or markers allowing us to generate a global view (Zilionis et al., 2016).The 10X system employs microfluidic partitioning in order to capture single cells and generate barcoded cDNA libraries. A limited-dilution of the single cell suspension generated from the ventral domain of the dorsal aorta, reverse transcription reagents, Gel Beads containing barcoded oligonucleotides and partitioning oil will be combined on a 10X microfluidic chip to form Gel Beads in Emulsion (GEMs). GEMs should theoretically contain a single cell, gel bead and RT reagents. Following cell lysis, the gel bead is dissolved to free the identically barcoded RT oligonucleotides into solution, allowing for reverse transcription to occur. This results in all of the cDNA generated from a single-cell containing the same barcode to be mapped back to their original cell following sequencing. The prepared libraries will then undergo Illumina sequencing to generate result for computational analysis. The raw reads generated will undergo bioinformatic analysis using a variety of pipelines to align, map and perform QC analyses. Following this, gene expression matrices and differential gene expression profiles together with gene clustering will be used to develop a global view of gene expression. The results will be complied to profile the individual transcriptomes generated from the single-cell AGM suspension. This would allow us to gain more information about the global and hierarchical organization of HSC during development and their interaction with the AGM niche during embryogenes.