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

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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.