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

背景：非肝硬化肝脏具有显著的可塑性和再生能力：在人类和小鼠中，高达60%的肝脏可以安全地切除，如果潜在的触发因素得到治疗，高达中等阶段的纤维化是可逆的。有趣的是，这种再生潜能的细胞起源尚不清楚。虽然干细胞存在于肝脏在其他报道中受到质疑，但干细胞区室已被报道存在于门中轴的相对极和胆道树中。我们认为，科学争议的一个重要部分是肝细胞在体内和体外的显著可塑性，因此这些过程需要在组织背景下以无假设的方式进行研究。目的：我们的目的是了解肝细胞谱系和再生过程的起源在稳定状态和在人类和小鼠的纤维化重塑。自己正在进行的工作：申请人应用他们在肝脏表观遗传学方面的专业知识，获得组织和技术，用于人类和小鼠的分区单细胞肝细胞制备。工作计划：我们将采用转录组谱系追踪来定义肝细胞谱系和干细胞潜能区室，从而最大限度地减少细胞处理和组织破坏。我们将对正常、肝硬化和对照小鼠的肝脏进行如下分析：A)分离肝细胞的单细胞（sc） mRNASeq，通过倍性和非实质细胞（npc）分类的FACS和B) mRNASeq和沿门-中心轴的三个肝细胞片段的低温激光捕获显微解剖（LCM）样品的减少代表性二硫测序（RRBS）。数据将通过利用LCM和倍性信息在肝小叶上的转录组伪时空映射中重建scRNASeq数据中的C)细胞谱系来分析。转录组谱将被映射到来自空间RRBS数据的表观基因组上，以阐明潜在的调控网络。D)空间分配将通过mRNA原位杂交、免疫组织化学进行细化。结论：由于我们将提供一种新的方法来定义稳定状态和纤维化重塑的肝细胞的细胞谱系和功能基因组程序，我们希望为治疗肝硬化提供新的见解。由于我们同时处理人类和小鼠细胞制备，我们希望为开发进一步实验表征的模型系统提供基础，该模型系统与临床情况具有良好的相似性。

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.