Cellular and Molecular Biology of the Hepatic Stem Cell Compartment

肝干细胞室的细胞和分子生物学

• 批准号: 7965036
• 负责人: Snorri Thorgeirsson
• 金额: $ 40.7万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965036
• 关键词: AFP gene; Activation Analysis; Address; Adopted; Adult; Albumins; Biliary; Biochemical; Cell Line; Cell Lineage; Cell Transplantation; Cell fusion; Cells; Coculture Techniques; Commit; Cytokeratin-18 Staining Method; Cytokeratin-8 Staining Method; Embryo; Enhancers; Epithelial Cells; Fibroblasts; Future; Genomics; Goals; Green Fluorescent Proteins; Growth; Growth Factor; Hepatic; Hepatocyte; Human; In Vitro; Injury; Liver; Liver Stem Cell; Methods; Molecular and Cellular Biology; Morphology; Mus; Muscle Cells; Neoplastic Cell Transformation; Population; Proliferating; Protocols documentation; Regenerative Medicine; Regulation; Replacement Therapy; Reporter Genes; Research; SCID Mice; Serum; Shapes; Signal Pathway; Staging; Stem cells; Stimulus; Subfamily lentivirinae; System; Testing; Transplantation; Work; clinical application; embryonic stem cell; fetal; human embryonic stem cell; improved; in vivo; induced pluripotent stem cell; injured; interest; oval cell; progenitor; promoter; repaired; stem; stem cell niche

We have established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we established that a serum-free chemically defined medium supports both the formation of embryoid bodies (EBs) and the differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P) and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and they participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl4-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. We conclude that a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy. We have developed a protocol for efficient differentiation of human ES cells toward early stages of hepatocytic differentiation using a combination of humoral factors and co-culture methods. 5%-10% of the cells acquired a polygonal shape and expressed albumin, AFP as well as CK8 and CK18 but not CK19 consistent with their differentiation along the hepatocytic lineage. We have now established a protocol for generating induced pluripotent (iPS) cells from human fibroblasts using transduction with lentiviruses carrying four reprogramming factors Oct4, Sox2, Klf4 and Lin28. Our current and future plans will focus on: (i) detailed biochemical and genomics characterization of the in vitro differentiation of the human ES and iPS cells into hepatocytes including in vivo transplantation of the cells into 1-day old mice to formally test their ability to give rise to the differentiated hepatocytes; (ii) transformation of human hepatoblasts derived from ES and IPS cells; and (iii) transformation of adult human hepatocytes and cholandocytes.

我们建立了从小鼠胚胎干细胞(ES)中分化、扩增和分离肝祖细胞的有效系统，并评估了它们重新填充受损肝脏的能力。利用由白蛋白(ALB)增强子/启动子调控的绿色荧光蛋白(GFP)报告基因转染的小鼠ES细胞，我们建立了一种无血清的化学定义的培养液，在没有外源生长因子或饲养层的情况下，支持类胚体(EBS)的形成和肝系细胞的分化。EB培养7天后，第一批表达ALB的GFP+细胞在“跳动”的心肌细胞附近被检测到。GFP+细胞数量增加，获得肝细胞样形态和肝细胞特异性标志物(即ALB、AAT、TO和G6P)，28天时占EB细胞分离出的细胞的30%以上。FACS纯化的GFP+细胞移植到MUP-uPA/SCID小鼠体内后，未见融合迹象，可发育为有功能的肝细胞，并参与了病变肝脏的修复。ES细胞来源的肝细胞对正常的生长调节有反应，并且在CCl4诱导的肝损伤的额外生长刺激下，其增殖速度与宿主肝细胞相同。移植的GFP+细胞也可分化为胆管上皮细胞。我们的结论是，在体外可以从ES细胞产生高度丰富的承诺肝细胞前体细胞群，以用于有效的细胞替代治疗。我们开发了一种方案，利用体液因素和共培养方法的组合，有效地将人类ES细胞分化为肝细胞分化的早期阶段。5%~10%的细胞呈多角形，表达白蛋白、甲胎蛋白以及CK8和CK18，但不表达CK19，与其沿肝细胞分化方向一致。我们现在已经建立了一种方法，通过携带四个重编程因子Oct4、Sox2、Klf4和Lin28的慢病毒转导，从人成纤维细胞中产生诱导多能(IPS)细胞。我们目前和未来的计划将集中在：(I)详细的生化和基因组学鉴定人ES和iPS细胞体外分化为肝细胞，包括体内移植细胞到1日龄小鼠体内，以正式测试其分化为肝细胞的能力；(Ii)从ES和IPS细胞来源的人成肝细胞的转化；以及(Iii)成人肝细胞和胆管细胞的转化。