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

Supervisors: Alexander Medvinsky, Tamir Chandra

Project description:

Although the first blood cells in mammals appear in the yolk sac, haematopoietic stem cells (HSCs) which can self-renew and give rise to the adult haematopoietic system appear separately inside the embryo body within the aorta-gonad-mesonephros (AGM) region (Medvinsky and Dzierzak, Cell, 1996). HSCs emerge through a multi-step process, which involves sequential maturation of intermediate cell types. However, our understanding of genetic mechanisms underlying HSC specification and progression through developmental stages is limited partly due to in utero development. To overcome this problem, we have already established an analytical in vitro model system which allows us to replicate the process of HSC maturation in the AGM region (Taoudi et al., Cell Stem Cell, 2008; Rybtsov et al., Stem Cell Reports, 2014). Although we have characterized several consecutive embryonic HSC precursors by cell surface markers, these markers are shared with other blood progenitors. This hampers precise identification of cells of the developing HSC lineage and specific genes underlying step-wise HSC maturation.

Recent advancement in single-cell analysis technology have dramatically transformed our understanding of cell types and lineages. It permits exploration of heterogeneity of cell populations and importantly, enables identification of novel cell type markers and otherwise unrecognisable cell sub-types. Using highly parallel barcoding of individual cells and computational tools specifically developed for single-cell data, it is now possible to build developmental/ differentiation trees into which intermediates of cell types and cellular states can be placed (Macosko et al., Cell, 2015; Mohammed et al., Cell Reports, 2017).

In this interdisciplinary project, we aim to explore molecular mechanisms underlying embryonic HSC development in mammals. The student will explore the transcriptional heterogeneity of the developing haematopoietic system in mouse using system-wide single-cell global transcriptome analysis. Single-cell gene expression analysis will be performed using cell barcoding in a microfluidics device. Using computational biology methods the student will reconstruct the developmental tree of the haematopoietic system and will identify molecular signatures specific to clades of the developing HSC lineage. Candidate genes identified in the molecular signature that could potentially be involved in HSC development will be validated using functional in vitro and in vivo assays and their expression in the embryo will be characterised using confocal microscopy. By contrast to model organisms, analysis of human embryonic HSC development has lagged behind due to the limited availability of material, lack of an in vitro modelling system and poor characterisation of cell type markers. Our data-driven approach using cutting-edge single cell methodology in mouse is expected to be highly informative for identifying homologous cellular differentiation pathways in human and will have substantial predictive power for analysing mechanisms underlying human HSC development. This project will fill a crucial gap in our mechanistic understanding of HSC development, which could in future inform improved strategies for manipulating HSCs ex vivo.

This is a collaborative interdisciplinary project addressing important biological questions at the system-wide level, between the groups of Prof. Alexander Medvinsky, an expert in embryonic development of mouse and human HSCs and experts in single-cell biology (including computational analysis) Dr. Tamir Chandra and Dr. Kristina Kirchner. To enhance the outcome of the project we have established a collaboration with Prof. Bertie Gottgens in Cambridge, a leading expert in the analysis of blood transcriptional networks.

The student will work in a highly collaborative environment and will become proficient in advanced methods of early analysis of embryonic HSC development (embryo manipulations, fluorescence activated cell sorting and analysis, HSC culture, qRT-PCR, confocal microscopy) as well as single-cell analysis and computational biology methods. Importantly, UoE has been awarded a £650K MRC Discovery Award to implement Drop-Seq-like approaches to single cell transcriptomics. This creates an excellent opportunity for the student to become an expert in this rapidly evolving cutting-edge technology. The project will allow the student to obtain important insights into the analysis of complex biological systems and to acquire important interdisciplinary skills in systems biology approaches, which should have a highly positive impact on their future career in science.