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Endogenous barcoding for in vivo fate mapping of lineage development in the blood and immune system

Periodic Reporting for period 2 - BARCODED-CELLTRACING (Endogenous barcoding for in vivo fate mapping of lineage development in the blood and immune system)

Reporting period: 2019-01-01 to 2020-06-30

The formation of blood and immune cells from bone marrow stem cells ('hematopoiesis') has traditionally been studied by transplantation of stem cells into 'conditioned' recipients whose own immune system had been ablated by irradiation. Such reconstitution provided valuable information on post-transplantation hematopoiesis but may not necessarily recapitulate physiological functions of the system. Understanding normal hematopoiesis is interesting from a basic scientific and medical point of view because life long functioning of the blood and immune system is essential to protect animals and humans from infections, prevent autoimmunity, support cancer treatment and maintain protective immunity in an aging population, to name but a few. The overall objectives of this project are to develop novel methods that shed light on the physiological functions of blood forming stem and progenitor cells. Moreover, we are investigating the responses of the system to challenges, including infections, blood loss or immunodeficiencies. To this end, the Advanced ERC Grant Project termed “barcoded-cell tracing” develops and applies new genetic technologies for high-resolution tracing by barcoding and fate mapping of stem cell activities that underlie the generation and maintenance of cells that form a functioning blood and immune system.
Cellular differentiation can be studied by introducing an inheritable marker into stem cells that pass on this tag to their progeny during differentiation. In combination with kinetic measurements, differentiation rates can be derived. Using such approaches, we studied fundamental properties of blood forming (hematopoietic) stem cells (HSCs) in the bone marrow of mice, which led to estimates on frequencies of HSCs contributing to hematopoiesis, and on differentiation rates: Many HSCs contribute, each one is rarely active and produces under normal steady state conditions only low output. These experiments were all based on the propagation of a single color label, a fluorescent protein, which falls short of revealing which sort of lineage was generated by the HSCs. This is because all cell types can only have the same color. We therefore developed a new, highly versatile barcoding system, termed Polylox, which makes it possible to introduce several hundred thousand different genetic barcodes into cells. The advantage of this DNA recombination system is that it is inducible by Cre recombinase, which enables the barcode generation to be controlled in terms of time and tissue. Polylox can be used to determine which cell lineages arise from individual HSC clones (fate analysis), and via which routes blood and immune cells emerge (analysis of hematopoietic structure). With the aim of opening the door for the coupled analysis of fates (barcoding) and transcriptome (RNA sequencing), we have developed a new version of Polylox, termed PolyloxExpress. Here, barcodes are expressed as RNA molecules and can therefore be read together with the transcriptome. Collectively, applying these new barcoding technologies, we have generated data towards the deconvolution of stem cell functions and hematopoiesis, including analysis of the origins of highly diverse immune cell lineages under physiological and stress conditions.
Fate mapping experiments using a stem cell-specific genetic driver made it possible to study endogenous blood and immune cell formation in the absence of transplantation. This also holds true for the development of our high-resolution barcoding system, Polylox, which can be induced in cells without the need for cell isolation or transplantation. These fate mapping and barcoding technologies yielded novel data towards the deconvolution of stem cell functions and hematopoiesis, including the analysis of the origins of highly diverse immune cell lineages under physiological and stress conditions.