Investigation of the liver progenitor cell niche using CK19 lineage tracing

使用 CK19 谱系追踪研究肝脏祖细胞生态位

• 批准号: 8047089
• 负责人: Eugene Scott Swenson
• 金额: $ 8.28万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8047089
• 关键词: Investigation; liver; progenitor; cell; niche

DESCRIPTION (provided by applicant): Adult stem cell niches have been identified in rapidly dividing tissues, such as the small intestine, skin and esophagus. Identification of adult stem cells in quiescent organs, such as the liver, has been more challenging. The notion that the liver contains an anatomically anchored stem cell zone near the portal tracts, giving rise to arrays of hepatocytes which progressively mature toward the central vein, is known as the "streaming liver" hypothesis. This model of liver homeostasis remains controversial, but may be operative during regeneration after severe liver injury exceeding the proliferative potential of mature hepatocytes. The goal of the work proposed here is to investigate the potential stem cell niche near the portal tracts, in the smallest branches of the bile ducts. This cell population expresses cytokeratin 19 (CK19), while mature hepatocytes do not. However, CK19 is not a unique marker for liver stem cells, as it is also expressed in mature bile ducts. Following severe liver injury, immature hepatocytes weakly expressing CK19 can be seen directly adjacent to clusters of strongly CK19-positive biliary cells, suggesting that biliary CK19+ cells can act as hepatocyte progenitors under conditions of severe liver injury. With time, the weakly CK19-positive periductal hepatocytes disappear, presumably through maturation to fully functional hepatocytes. These experiments will test the hypothesis that mature hepatocytes arise from CK19- expressing progenitors, using a state-of-the-art transgenic lineage tracing approach. CK19-expressing cells and their descendants will express yellow fluorescent protein (YFP). Aim 1: Determine the role of intrahepatic CK19+ hepatic progenitor cells during normal liver growth and homestasis. During normal development, CK19+ hepatoblasts give rise to normal bile ducts and hepatocytes. In the adult, CK19 is expressed by cholangiocytes, but not hepatocytes. I will use the tightly regulated, Tamoxifen-inducible CK19-CreERT transgenic mouse model to mark CK19-expressing cells at specific times during development, using a YFP reporter. Marking of CK19+ populations during embryonic development is expected to mark all descendants of hepatoblasts, including hepatocytes and cholangiocytes. Marking of CK19+ populations in the adult mouse is expected to mark mature bile ducts and peribiliary oval cells, but likely will not mark hepatocytes due to their inherently slow turnover and replacement by existing mature hepatocytes. Aim 2: Determine the role of intrahepatic CK19+ hepatic progenitor cells during liver regeneration after injury by 1) partial hepatectomy and 2) acute acetaminophen injury. Partial hepatectomy provides the classic example of an acute liver injury which triggers liver regeneration exclusively through proliferation of residual mature hepatocytes, rather than progenitors. In contrast, acute acetaminophen intoxication induces severe, diffuse hepatocyte necrosis and a marked increase of CK19+ oval cells in the periportal region. I predict that hepatocytes arise from CK19+ progenitors and will be lineage marked with YFP after acetaminophen injury, but not partial hepatectomy. PUBLIC HEALTH RELEVANCE: The liver has a remarkable capacity to regenerate in response to injury induced by toxins or viruses, driven in some cases by immature liver cells called "progenitor" cells. It is critical to understand the contribution of liver progenitor cells to normal and regenerating liver in order to develop treatment strategies to support regeneration and suppress tumor formation. The goal of the current proposal is to investigate the contribution of liver progenitor cells in the regenerating mouse liver, using a genetic labeling approach to permanently mark liver progenitor cells and their descendants.

描述（由申请人提供）：在迅速分裂的组织（例如小肠，皮肤和食道）中已经鉴定出成年干细胞壁ni。静止器官（例如肝脏）中成年干细胞的鉴定更具挑战性。肝脏在门户区域附近包含一个解剖锚定的干细胞区域的概念，从而产生了肝细胞阵列，这些肝细胞逐渐向中静脉成熟，被称为“流肝”假设。这种肝稳态模型仍然存在争议，但在严重肝损伤后可能是在再生过程中起作用的，超过了成熟肝细胞的增殖潜力。这里提出的工作的目的是研究门户区域附近的潜在干细胞生态位，在胆管的最小分支中。该细胞群表达细胞角蛋白19（CK19），而成熟的肝细胞则没有。但是，CK19并不是肝干细胞的独特标记，因为它也在成熟的胆管中表达。严重的肝损伤后，可以直接看到弱表达CK19的未成熟肝细胞与强烈CK19阳性胆汁细胞的簇相邻，这表明在严重肝损伤的情况下，胆道CK19+细胞可以充当肝细胞祖细胞。随着时间的流逝，弱的CK19阳性肝细胞消失了，大概是通过成熟到功能齐全的肝细胞而消失的。这些实验将检验以下假设：使用最先进的转基因谱系跟踪方法，成熟的肝细胞由CK19-表达祖细胞产生。表达CK19的细胞及其后代将表达黄色荧光蛋白（YFP）。 AIM 1：确定肝内CK19+肝祖细胞在正常肝脏生长和寄宿期间的作用。在正常发育过程中，CK19+肝类母细胞产生正常的胆管和肝细胞。在成年人中，CK19由胆管细胞表达，但没有肝细胞。我将使用YFP Reporter使用特定时间来标记特定时间表达CK19的细胞的严格调节的他莫昔芬诱导的CK19-CREERT转基因小鼠模型。在胚胎发育过程中，CK19+种群的标记预计将标志着肝细胞的所有后代，包括肝细胞和胆管细胞。预计成年小鼠中CK19+种群的标记将标记成熟的胆管和周围椭圆形细胞，但由于其固有缓慢的周转率和现有成熟的肝细胞的替代，可能不会标记肝细胞。 AIM 2：确定肝内CK19+肝祖细胞在受伤后肝脏再生过程中的作用1）部分肝切除术和2）急性对乙酰氨基酚损伤。部分肝切除术提供了急性肝损伤的经典例子，该例子仅通过残留成熟的肝细胞而不是祖细胞的扩散而触发肝脏再生。相反，急性对乙酰氨基酚中毒会诱导严重的，弥漫性的肝细胞坏死，并在周围区域明显增加CK19+椭圆形细胞。我预测肝细胞由CK19+祖细胞产生，将在对乙酰氨基酚损伤后用YFP标记，但不是部分肝切除术。 公共卫生相关性：肝脏具有显着的能力，可响应毒素或病毒引起的损伤，在某些情况下由未成熟的肝细胞驱动，称为“祖细胞”细胞。了解肝脏祖细胞对正常和再生肝脏的贡献至关重要，以制定支持再生并抑制肿瘤形成的治疗策略。当前建议的目的是使用遗传标记方法永久标记肝脏祖细胞及其后代的遗传标记方法研究肝脏祖细胞在再生小鼠肝脏中的贡献。