BIOARTIFICIAL LIVERS FROM HEPATIC PROGENITOR CELLS

大鼠肝脏含有位于每个门静脉三联体的祖细胞 并产生子细胞，通过单向的， 分化过程终止于中央脉。 因此， 每个腺泡内的实质细胞（体内）是 成熟肝细胞具有年龄依赖性大小、倍性、生长和 分化潜能 我们建议使用这些祖细胞， 通过多参数荧光激活细胞分选纯化， 建立生物人工肝。 这些细胞将被接种到一个中空的 新型设计的纤维生物反应器，在规定的离体培养条件下 完全或大部分不含血清的条件，含有定义的和 纯化的细胞外基质成分作为基质，并定义和 纯化的可溶性信号（激素、生长因子、营养素）。 已经制定了方案，并定义了抗原谱， 鉴定和分离肝祖细胞的三个亚群， 使用以下组合的成熟实质细胞的两个亚群： 淘选和多参数荧光激活细胞分选（FACS）： 成肝细胞（多能肝祖细胞）;定向胆管和 肝细胞祖细胞;门静脉周围实质细胞（假定年轻 5）中央周围的实质细胞（推测的老年细胞） 薄壁组织）。 还开发了用于命运研究的体内生物测定法， 允许细胞扩增的体内条件和其他驱动 每个祖细胞亚群的分化。 大鼠 生物人工肝将建立从每5个 通过接种的成熟阶段的实质细胞亚群 将它们转移到用基质包被的多孔的、可生物降解的微载体上 组件，进入中空纤维生物反应器的新形式和下 合适的离体扩增条件。 一种单独的生物反应器， 将建立饲养细胞，并将包含两种饲养细胞类型 发现产生旁分泌信号是生长的严格要求 1）FACS-纯化的造血OCAP细胞（骨髓 携带卵圆细胞抗原3+，OC 3+的细胞）和STO胚胎 基质细胞系，最近发现它可以取代原代 培养年龄和肝脏特异性基质饲养细胞（来自E14-E16 肝脏）。 具有饲养细胞的生物反应器将与 与含有肝祖细胞的细胞串联;如果 饲养者产生的因子太不稳定， 连接的生物反应器，饲养细胞将用丝裂霉素处理 C，并与肝祖细胞共接种到相同的生物反应器中。 将生物人工肝和对照单层培养物 其特征在于胎儿和成人肝脏特异性功能， 用约翰逊和约翰逊干玻片法测定造血标志物 免疫化学、生物化学、分子杂交 测定和流式细胞术分析。此外，生物反应器 将使用核磁共振技术进行非侵入性表征 和磁共振成像。细胞的命运 生物反应器将与体内鉴定的生物反应器进行比较。 生物测定

Rat liver contains progenitor cells located by each of the portal triads and which 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 lineages of maturing liver cells with age-dependent size, ploidy, growth and differentiative potential. We propose to use these progenitor cells, purified by multiparametric fluorescence activated cell sorting, to establish a bioartificial liver. The cells will be seeded into a hollow fiber bioreactor, of novel design, and under defined ex vivo culture conditions that are entirely or mostly serum-free, contain defined and purified extracellular matrix components as substratum, and defined and purified soluble signals (hormones, growth factors, nutrients). Protocols have been developed, and antigenic profiles defined by which to identify and isolate three subpopulations of hepatic progenitors and two subpopulations of mature parenchymal cells using a combination of panning and multiparametric fluorescence activated cell sorting (FACS): hepatoblasts (pluripotent hepatic progenitors); committed biliary and hepatocytic progenitors; periportal parenchymal cells (presumptive young parenchyma); and 5) pericentral parenchymal cells (presumptive old parenchyma). Also developed are in vivo bioassays for fate studies, ex vivo conditions that permit cell expansion and others that drive differentiation of each of the progenitor subpopulations. The rat bioartificial liver will be established from each of the 5 subpopulations of maturationally staged parenchymal cells by seeding them onto porous, biodegradable mircocarriers coated with matrix components, into a novel form of hollow fiber bioreactor and under appropriate ex vivo expansion conditions. A separate bioreactor for feeder cells will be established and will contain two feeder cell types found to yield paracrine signals that are strict requirements for growth of the progenitors: 1) FACS-purified hemopoietic OCAP cells (myeloid cells that bear an oval cell antigen 3+, OC3+) and the STO embryonic stromal cell line, that has recently been found to replace primary cultures of age- and liver-specific stromal feeder cells (from E14-E16 livers). The bioreactors with the feeder cells will be coupled in tandem with the ones containing the hepatic progenitor cells; if the factors produce by the feeders are too labile to survive the tandemly connected bioreactors, the feeder cells will be treated with mitomycin C and co-seeded into the same bioreactors with the hepatic progenitors. The bioartifical livers and control monolayer cultures will be characterized for fetal and adult liver-specific functions and for hemopoietic markers by means of the Johnson and Johnson dry slide assays, by immunochemistry, biochemical assays, molecular hybridization assays and by flow cytometric analyses. In addition, the bioreactors will be characterized non-invasively using nuclear magnetic resonance and magnetic resonance imaging. The fates of the cells in the bioreactors will be compared with those identified from in vivo bioassays.