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）：已在快速分裂的组织（如小肠、皮肤和食管）中鉴定出成体干细胞龛。在静止器官（如肝脏）中鉴定成体干细胞更具挑战性。肝脏在汇管区附近含有解剖学上锚定的干细胞区，产生向中央静脉逐渐成熟的肝细胞阵列，这一概念被称为“流动肝脏”假说。这种肝脏稳态模型仍有争议，但在严重肝损伤后超过成熟肝细胞增殖潜力的再生过程中可能有效。本文提出的工作目标是研究在胆管的最小分支中的汇管区附近的潜在干细胞龛。该细胞群表达细胞角蛋白19（CK 19），而成熟肝细胞不表达。然而，CK 19并不是肝干细胞的唯一标志物，因为它也在成熟的胆管中表达。重度肝损伤后，可观察到弱表达CK 19的未成熟肝细胞直接邻近强CK 19阳性胆管细胞簇，表明胆管CK 19+细胞可在重度肝损伤条件下充当肝细胞祖细胞。随着时间的推移，弱CK 19阳性导管周肝细胞消失，可能是通过成熟为完全功能的肝细胞。这些实验将使用最先进的转基因谱系追踪方法来检验成熟肝细胞来自表达CK 19的祖细胞的假设。CK 19表达细胞及其后代将表达黄色荧光蛋白（YFP）。目的1：确定肝内CK 19+肝祖细胞在正常肝脏生长和稳态中的作用。在正常发育过程中，CK 19+肝母细胞产生正常的胆管和肝细胞。在成人中，CK 19由胆管细胞表达，而不是肝细胞。我将使用严格调控的他莫昔芬诱导的CK 19-CreERT转基因小鼠模型，使用YFP报告基因在发育过程中的特定时间标记CK 19表达细胞。在胚胎发育过程中标记CK 19+群体预计将标记成肝细胞的所有后代，包括肝细胞和胆管细胞。预计标记成年小鼠中的CK 19+群体可标记成熟胆管和胆管周围卵圆细胞，但由于其固有的缓慢更新和被现有成熟肝细胞替代，可能不会标记肝细胞。目标二：确定肝内CK 19+肝祖细胞在1）部分肝切除术和2）急性对乙酰氨基酚损伤后肝再生过程中的作用。部分肝切除术提供了急性肝损伤的典型例子，其仅通过残余成熟肝细胞而不是祖细胞的增殖来触发肝再生。相反，急性对乙酰氨基酚中毒诱导严重的弥漫性肝细胞坏死和门静脉周围区域的CK 19+卵圆细胞显著增加。我预测，肝细胞来自CK 19+祖细胞，对乙酰氨基酚损伤后，肝细胞将被YFP标记，而不是部分肝切除术。 公共卫生相关性：肝脏具有非凡的再生能力，以应对毒素或病毒引起的损伤，在某些情况下由称为“祖”细胞的未成熟肝细胞驱动。了解肝脏祖细胞对正常和再生肝脏的贡献对于制定支持再生和抑制肿瘤形成的治疗策略至关重要。目前建议的目标是研究肝祖细胞在再生小鼠肝脏中的贡献，使用遗传标记方法永久标记肝祖细胞及其后代。