BIOARTIFICIAL LIVERS FROM HEPATIC PROGENITOR CELLS

来自肝祖细胞的生物人工肝

• 批准号: 2654560
• 负责人: LOLA M REID
• 金额: $ 10万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2654560
• 关键词: bioreactors; cell line; embryonic stem cell; flow cytometry; laboratory rat; liver; liver cells; organ culture

Rodent hepatic progenitor cells, purified by multiparametri fluorescence activated cell sorting, will be seeded into a hollow fiber bioreactor under defined ex vivo culture conditions to establish a bioartifical liver. all current forms of bioartifical livers are derived from cell lines that permit stability and longevity in the bioreactors (weeks to months) but have minimal differentiated function, or are derived from mature liver cells that yield a bioartificial liver that is short-lived (typically one week) but have a broad range of tissue- specific functions. A stable bioartificial liver with a broad rang of differentiated function should be achievable if hepatic progenitor cells are use, since purified populations of these cells are capable ex vivo of maturing to produce all the cell types of the liver. Stability will be achieved also by using ex vivo expansion conditions that re as defined as possible and include little or no serum; defined and purified extracelluar matrix components as substratum; and defined and purified soluble signals (hormones, growth factors, nutrients). The hypothesis will be tested using rodent ells, and if successful, a similar approach will be used to establish a human bioartificial liver. The proposal is based in numerous studies on rat liver stem cell biology demonstrating that rat liver, at all ages at least to 1 year of life, contains progenitor cells located by each of the portal triads. These progenitor cells produce daughter cells that mature through a unidirectional, differentiation process ending at the central vein. Thus, the plates of parenchymal cells within each acinus in vivo are, thus, lineages of maturing liver cells with age-dependent size, ploidy, growth and differentiative potential. Protocols have been developed, and antigenic profiles defined by which to identify and isolate three subpopulations of hepatic progenitors and two subpopulations of mature parencymal cells using a combination of panning and multiparametric fluorescence activated cell sorting (FACS): 1) hepatoblasts, pluripotent hepatic progenitors; 2) committed bile duct progenitors; 3) committed hepatocyte progenitors; 4) periportal hepatocyes (presumptive young hepatocytes); and 5) pericentral hepatocytes (presumptive old hepatocytes). In addition, we have developed in vivo bioassays for fate studies and ex vivo conditions that permit cell expansion and others that drive differentiation of each of the three subpopulations. The rate bioartificial liver will be established from each of the 5 subpopulations of parenchymal cells by seeding them into commercially available hollow fiber bioreactors and under appropriate ex vivo expansion conditions. For the hepatic progenitor subpopulations, their requisite feeder cells of ACSs-purified hemopoietic OCAP cells (myeloid cells that bear an oval cell antigen3+) and age-and liver-specific stormal feeder cells (from E14- E16 livers) will be put into separate bioreactors, and then the bioreactors with the feeder cells will be coupled in tandem with the one containing the hepatic progenitor cells. In parallel, we will try to identify the factors that are part of the paracrine signaling between these three embryonic hepatic cell types (progenitors of parenchymal, hemopoietic and mesenchymal cells) to enable us to eliminate one or both of the fewer cells as components of the vioartificial liver system. If the factors are novel, we will attempt to isolate and clone them through subtractive hybridization methods using biologically active feeders, the embryonic cells, versus inactive feeders derived from the adult counterparts to the cells. the bioreactors derived from progenitor cells will be characterized for fetal and adult liver-specific functions, and, as controls, for hemopoietic markers, by means of immunochemistry, biochemical assays, and molecular hybiridizaion assays. The fates of the cells in he bioreactors will be compared with those identified from in vivo bioassays.

多参数纯化的啮齿动物肝前体细胞 荧光激活细胞分选，将接种到中空纤维中 在限定的离体培养条件下在生物反应器中培养，以建立 生物人工肝 目前所有形式的生物人工肝都是 来自允许在生物反应器中稳定和长寿的细胞系， （数周至数月），但具有最小的分化功能，或 来源于成熟的肝细胞，产生生物人工肝， 寿命短（通常为一周），但具有广泛的组织- 具体功能。 一种稳定的生物人工肝， 如果肝祖细胞能够分化成 因为这些细胞的纯化群体能够离体地 成熟以产生肝脏的所有细胞类型。 稳定将是 也可通过使用如本文所定义的离体扩增条件来实现 尽可能少含或不含血清;定义和纯化 细胞外基质组分作为基质;并且定义和 纯化的可溶性信号（激素、生长因子、营养素）。 的 假设将使用啮齿动物细胞进行测试，如果成功，类似的 方法将用于建立人生物人工肝。 的 这项建议是基于大量的大鼠肝干细胞生物学研究 证明大鼠肝脏，在至少1岁的所有年龄， 包含位于每个门静脉三联体的祖细胞。 这些 祖细胞产生子细胞，子细胞通过 在中央静脉处结束的单向分化过程。 因此，在本发明中， 因此，体内每个腺泡内的实质细胞板， 具有年龄依赖性大小，倍性， 增长和分化潜力。 协议已经制定， 和抗原谱来确定和分离三种 肝祖细胞的两个亚群和 成熟的薄壁细胞，使用淘选和 多参数荧光激活细胞分选（FACS）：1） 成肝细胞、多能肝祖细胞; 2）定向胆管 祖细胞; 3）定向肝细胞祖细胞; 4）门静脉周围 肝细胞（推定的年轻肝细胞）;和5）中央周围 肝细胞（推定的老肝细胞）。 另外我们有 开发了用于命运研究和离体条件的体内生物测定， 允许细胞扩增和其他驱动每个细胞分化的因素， 三个亚群。 生物人工肝将在 从实质细胞的5个亚群中的每一个建立， 将它们接种到市售的中空纤维生物反应器中， 在适当的离体扩增条件下。 肝功能 祖细胞亚群，其必需的ACS纯化的饲养细胞 造血OCAP细胞（带有卵圆细胞的髓样细胞 抗原3+）和年龄和肝脏特异性的风暴饲养细胞（来自E14- E16肝脏）将被放入单独的生物反应器中，然后 具有饲养细胞的生物反应器将串联地与 一个含有肝祖细胞。 与此同时，我们将努力 确定哪些因素是旁分泌信号的一部分， 这三种胚胎肝细胞类型（实质的祖细胞， 造血和间充质细胞），使我们能够消除一个或 这两种细胞都是人工肝系统的组成部分。 如果这些因子是新的，我们将尝试分离和克隆它们 通过消减杂交方法， 饲养者，胚胎细胞，与来自 与细胞相对应的成年细胞。 源自祖细胞的生物反应器 细胞将被表征为胎儿和成人肝特异性功能， 作为对照，对于造血标志物，通过 免疫化学、生物化学测定和分子杂交 分析。 将比较生物反应器中细胞的命运， 从体内生物测定中鉴定的那些。