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

通过基因组谱系追踪了解小鼠和人类肝脏的组织重塑

• 批准号: 402694925
• 负责人: Professor Dr. Jochen Hampe
• 金额: --
• 依托单位: Bereich Gastroenterologie und Hepatologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/402694925?language=en
• 关键词: GENOMIC; LINEAGE; TRACING; UNDERSTAND; TISSUE

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.

背景资料：非硬化性肝脏具有显着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，如果治疗潜在的触发因素，高达中度的纤维化是可逆的。有趣的是，这种再生潜力的细胞起源仍然知之甚少。干细胞室已被报道在门-中轴线的两极和胆管树中，尽管在其他报道中干细胞在肝脏中的存在受到挑战。我们认为，科学争论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：我们的目标是了解肝细胞谱系和再生过程的起源在稳态和人类和小鼠的纤维化重塑。自己的工作进展：申请人将其在肝脏表观遗传学、组织获取和人类和小鼠单细胞肝细胞制备技术方面的专业知识应用于人类和小鼠。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的分区，最大限度地减少细胞处理和组织破坏。我们将如下分析正常和肝硬化的人肝和对照小鼠：A）通过倍性和非实质细胞（NPC）分选的分离的肝细胞的单细胞（sc）mRNASeq，和B）将进行沿门-中轴沿着的三个肝细胞级分的冷冻激光捕获显微切割（LCM）样品的mRNASeq和还原代表性二硫化物测序（RRBS）。将通过使用LCM和倍性信息在肝小叶上的转录组学伪时间和空间映射中从scRNASeq数据重建C）细胞谱系来分析数据。转录组学图谱将从空间RRBS数据映射到表观基因组上，以阐明潜在的调控网络。D）将使用mRNA原位杂交、免疫组织化学来细化空间分配。结论：由于我们将提供一种新的方法来定义稳态和纤维化重塑中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。当我们平行处理人和鼠细胞制剂时，我们希望为开发模型系统提供基础，用于进一步的实验表征，具有与临床情况的良好相似性。