17 May

Thesis defense Chloé Baron: single-cell sequencing and the development of blood cells

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During her PhD, Chloé Baron, Hubrecht researcher in the Van Oudenaarden group and co-supervised by Catherine Robin, researched the ontogeny of hematopoietic stem cells, the key cell type responsible for the production of all blood cells in an adult organism. Using single-cell transcriptomics, she aimed to unravel the molecular mechanisms involved in HSC generation and retrace the clonal history of the hematopoietic system. Baron defended her thesis successfully on May 17th, 2018.

Hematopoietic stem cells (HSCs) are responsible for the production of all mature blood cells during the life of an organism. In both man and mouse, HSCs reside in the bone marrow, and in zebrafish they are present in the kidney marrow. However, adult HSCs are initially generated during embryonic development.

HSC origin
Although the the process of HSC formation during embryogenesis is not completely understood, it is known that these originate from endothelial cells in the aorta. In mouse embryos, HSCs are detected in the aorta in the form of small, temporary groups of cells derived from and attached to the endothelia layer. In zebrafish, these clusters are not present; but instead, endothelial cells generating HSCs migrate from the aortic wall to enter the blood stream. Research in both organisms revealed that the HSC pool is then amplified before seeding the bone or kidney marrow where they will reside during life.

Molecular mechanisms involved in HSC generation
To gain insight in the molecular mechanisms involved in HSC generation in the mouse embryonic aorta, Baron used single-cell mRNA sequencing. This technology allows researchers to determine the gene expression profile of individual cells. This way, Baron studied the gene expression profile of single cells undergoing endothelial to hematopoietic transition, the process responsible for the formation of aortic clusters containing HSCs. Using this technology, Baron unraveled the complex transcriptional changes and transcription factor networks implicated in this transition. The dataset generated is an important resource for the scientific community to understand further the role of pivotal transcription factors and signaling pathways in HSC generation.

Building lineage trees
Together with colleagues of the Van Oudenaarden group, Baron developed a method that is able to characterize the origin and lineage relationships of thousands of cells from the same organism. This method, ScarTrace, is based on the affliction of genetic scars and can be applied in researching the clonal history of hematopoietic stem cells, but also for unveiling family ties between different cells from an organ or a cell type of choice.

Flow cytometry revisited
Another part of Baron’s thesis comprises the development of an algorithm to purify cell types by flow cytometry. The main advantage of this algorithm, GateID, is that it relies on general FACS properties of single cells and unbiased cell type identification using single-cell mRNA-Sequencing. Importantly, GateID allows isolation of cell types without the use of labeled antibodies or transgenic reporters. Baron demonstrates the power of this algorithm by effectively purifying different cell types from the zebrafish hematopoietic system and the human pancreas. According to Baron, single-cell mRNA sequencing, GateID and ScarTrace allow researchers to answer biological questions concerning the development and cellular biology of hematopoiesis.

Alexander van Oudenaarden is director and group leader at the Hubrecht Institute, professor of Quantitative Biology of Gene Regulation at the UMC Utrecht and the Faculty of Science at Utrecht University, and Oncode Investigator. His group uses a combination of experimental, computational, and theoretical approaches to quantitatively understand decision‐making in single cells, with a focus on questions in developmental and stem cell biology.

Catherine Robin is group leader at the Hubrecht Institute and is also appointed at the University Medical Center Utrecht. Her group focuses on the establishment of the hematopoietic system during embryonic development.