BIOARTIFICIAL LIVERS FROM HEPATIC PROGENITOR CELLS

多参数纯化的啮齿动物肝祖细胞 荧光激活的细胞分选，将种子植入中空纤维 生物反应器在确定的体外培养条件下建立 生物人工肝。目前所有形式的生物人工肝都是从 从允许生物反应器中的稳定性和寿命的细胞系 (几周到几个月)，但具有最小的区分功能，或 从成熟的肝细胞衍生而来的生物人工肝 寿命短(通常为一周)，但有广泛的组织- 具体功能。一种稳定的生物人工肝，具有广泛的 如果肝祖细胞能够实现分化功能 使用，因为这些细胞的纯化群体能够在体外 成熟后可以产生肝脏的所有类型的细胞。稳定将是 也可以通过使用定义的体外扩增条件来实现 尽可能地包括很少或不包括血清；定义和纯化 作为底物的细胞外基质成分；并定义和 纯化的可溶性信号(激素、生长因子、营养素)。这个 假说将用啮齿动物细胞进行检验，如果成功，类似的 该方法将用于建立人类生物人工肝。这个 这一建议是基于对大鼠肝脏干细胞生物学的大量研究 证明了所有年龄至少到一岁的大鼠肝脏， 包含由每个门状三联体定位的祖细胞。这些 祖细胞产生子代细胞，通过 单向的分化过程在中央静脉结束。因此， 因此，体内每个腺泡内的实质细胞板是， 成熟的肝细胞系具有年龄相关的大小，倍性， 增长和分化潜力。已经开发了协议， 和定义的抗原谱来识别和分离三个 肝祖细胞亚群和两个亚群 使用平移和组合的成熟的侧壁细胞 多参数荧光激活细胞分选(FACS)：1) 肝母细胞，多能肝祖细胞；2)空肠胆管 祖细胞；3)致力于肝细胞的祖细胞；4)门静脉周围 肝细胞(假定为年轻的肝细胞)；和5)中心周围 肝细胞(假定为老年肝细胞)。此外，我们还有 开发了用于命运研究和体外条件的体内生物检测方法 允许细胞扩张和其他促进每个细胞分化的因素 这三个亚群。生物人工肝的速度将 从5个实质细胞亚群中的每一个建立的 将它们接种到商用中空纤维生物反应器中，并 在适当的体外扩增条件下。对于肝脏来说 ACSS纯化的祖细胞亚群及其必需的饲养细胞 造血型OCAP细胞(具有卵圆形细胞的髓系细胞 抗原3+)和年龄和肝脏特异性的营养细胞(来自E14- E16肝脏)将被放入单独的生物反应器中，然后 带有饲养单元的生物反应器将与 一个含有肝祖细胞的细胞。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(实质祖细胞， 造血细胞和间充质细胞)，使我们能够消除一个或多个 这两种较少的细胞都是人工肝系统的组成部分。 如果这些因素是新的，我们将尝试分离和克隆它们 通过使用生物活性的消减杂交方法 饲养者，胚胎细胞，与来自 细胞的成年对应物。祖细胞衍生的生物反应器 细胞将被表征为胎儿和成人肝脏特有的功能， 作为对照，对于造血标记物，通过以下方式 免疫化学、生化分析和分子生物学 化验。生物反应器中细胞的命运将与 那些从活体生物检测中鉴定出来的。

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.