GENOMIC LINEAGE TRACING TO UNDERSTAND TISSUE REMODELING IN MURINE AND HUMAN LIVER

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Background: Non-cirrhotic liver has remarkable plasticity and regenerative capacity: In humans and mice, up to 60% of the liver can safely be removed and up to moderate stages of fibrosis are reversible if the underlying trigger is treated. Intriguingly, the cellular origin of this regenerative potential is yet poorly understood. Stem cell compartments have been reported at opposite poles of the porto-central axis and in the biliary tree, although the presence stem cells in the liver has been challenged in other reports. We think, that an important part of the scientific controversy is the remarkable plasticity of hepatocytes in vivo and ex vivo and that therefore these processes need to be studied in tissue context in a hypothesis-free manner.Aims: We aim to understand hepatocyte lineages and origins of regenerative processes in the steady state and upon fibrotic remodeling in humans and mice.Own proceeding work: The applicants apply their expertise in liver epigenetics, access to tissue and technologies for zonated single cell hepatocyte preparations in both humans and mice.Work plan: We will employ transcriptomic lineage tracing to define hepatocyte genealogies and compartments of stem cell potential in an approach, that minimizes cell handling and tissue disruption. We will analyze normal and cirrhotic human liver and control mice as follows: A) Single cell (sc) mRNASeq of isolated hepatocytes FACS sorted by ploidy and non-parenchymal cells (NPCs) and B) mRNASeq and reduced representation bisulfide sequencing (RRBS) of cryo laser capture microdissection (LCM) samples of three hepatocyte fractions along the porto-central axis will be performed. Data will be analyzed by reconstructing C) cellular genealogies from scRNASeq data in transcriptomic pseudo time and spacial mapping on the liver lobule using the LCM and ploidy information. Transcriptomic profiles will be mapped on epigenomes from the spatial RRBS data to elucidate underlying regulatory networks. D) Spatial assignment will be refined using mRNA in situ hybridization, immunohistochemistry. Conclusion: As we will provide a new approach to the definition the cellular genealogies and functional genomic programs of hepatocytes in the steady state and fibrotic remodeling, we hope to provide novel insights to therapeutically tackle liver cirrhosis. As we process human and murine cell preparations in parallel, we hope to provide a basis to develop model systems for further experimental characterization with good resemblance of the clinical situation.