BIOARTIFICIAL LIVERS FROM HEPATIC PROGENITOR CELLS

来自肝祖细胞的生物人工肝

• 批准号: 6381395
• 负责人: LOLA M REID
• 金额: $ 31.47万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-24 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381395
• 关键词: acinar cell; bioassay; bioengineering /biomedical engineering; biomarker; biomaterial development /preparation; bioreactors; biotechnology; cell differentiation; cell growth regulation; cell proliferation; deficient growth media; embryonic stem cell; flow cytometry; hematopoietic stem cells; hepatocyte growth factor; laboratory rat; liver; liver cells; magnetic resonance imaging; nuclear magnetic resonance spectroscopy; organ culture; stem cells; tissue engineering; tissue support frame

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.

大鼠肝脏含有位于每个门脉三联体的祖细胞 并产生通过单向成熟的子细胞， 分化过程终止于中央静脉。 因此，板 每个腺泡内的实质细胞（体内）是以下谱系 成熟的肝细胞具有与年龄相关的大小、倍性、生长和 差异化潜力。 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。 细胞将被播种到一个空洞中 纤维生物反应器，设计新颖，并在明确的离体培养下 完全或大部分无血清的条件，包含确定的和 纯化的细胞外基质成分作为培养基，并定义和 纯化的可溶性信号（激素、生长因子、营养素）。 已经制定了协议并定义了抗原谱 鉴定和分离肝祖细胞的三个亚群 使用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）；胆汁性和 肝细胞祖细胞；汇管周围实质细胞（假定年轻 薄壁组织）； 5) 中央周围实质细胞（假定为老细胞） 薄壁组织）。 还开发了用于命运研究的体内生物测定法，例如 允许细胞扩增的体内条件和其他驱动 每个祖亚群的分化。 老鼠 生物人工肝将从5个中的每一个中建立 通过接种形成成熟阶段的实质细胞亚群 将它们转移到涂有基质的多孔、可生物降解的微载体上 组件，进入一种新型的中空纤维生物反应器并在 适当的离体扩增条件。 一个单独的生物反应器 将建立饲养细胞并将包含两种饲养细胞类型 发现产生对生长严格要求的旁分泌信号 祖细胞：1) FACS 纯化的造血 OCAP 细胞（骨髓 带有卵圆细胞抗原 3+、OC3+）的细胞和 STO 胚胎 基质细胞系，最近被发现可以替代原代细胞 年龄和肝脏特异性基质饲养细胞培养物（E14-E16） 肝脏）。 带有饲养细胞的生物反应器将耦合在 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，无法串联生存 连接的生物反应器，饲养细胞将用丝裂霉素处理 C 并与肝祖细胞共同接种到相同的生物反应器中。 生物人工肝和对照单层培养物将是 具有胎儿和成人肝脏特异性功能和 通过 Johnson and Johnson 干玻片进行造血标记 测定，通过免疫化学、生化测定、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。

项目成果

Human hepatoblast phenotype maintained by hyaluronan hydrogels.

• DOI: 10.1002/jbm.b.30717
• 发表时间: 2007-07
• 期刊: Journal of biomedical materials research. Part B, Applied biomaterials
• 作者: W. Turner;E. Schmelzer;R. McClelland;E. Wauthier;Weiliam Chen;L. Reid

Soft, porous poly(D,L-lactide-co-glycotide) microcarriers designed for ex vivo studies and for transplantation of adherent cell types including progenitors.

柔软、多孔的聚（D,L-丙交酯-共-糖苷）微载体，设计用于离体研究和包括祖细胞在内的贴壁细胞类型的移植。

• DOI: 10.1111/j.1749-6632.2001.tb03829.x
• 发表时间: 2001
• 期刊: Annals of the New York Academy of Sciences
• 影响因子: 5.2
• 作者: Xu,AS;Reid,LM
• 通讯作者: Reid,LM

Hepatic progenitors and strategies for liver cell therapies.

肝祖细胞和肝细胞治疗策略。

• DOI: 10.1111/j.1749-6632.2001.tb03851.x
• 发表时间: 2001
• 期刊: Annals of the New York Academy of Sciences
• 影响因子: 5.2
• 作者: Susick,R;Moss,N;Kubota,H;Lecluyse,E;Hamilton,G;Luntz,T;Ludlow,J;Fair,J;Gerber,D;Bergstrand,K;White,J;Bruce,A;Drury,O;Gupta,S;Reid,LM
• 通讯作者: Reid,LM

Hepatic stem cells and hepatoblasts: identification, isolation, and ex vivo maintenance.

• DOI: 10.1016/s0091-679x(08)00008-3
• 发表时间: 2008
• 期刊: Methods in cell biology
• 作者: E. Wauthier;E. Schmelzer;W. Turner;Lili Zhang;E. LeCluyse;Joseph Ruiz;R. Turner;Mark E. Furth