Engraftment and Repopulation Potential of Late Gestation Fetal Rat Hepatocytes. Rattus norvegicus

Liver transplantation is the only therapeutic option for patients with end-stage liver disease. The shortage of donor organs has led to the search for alternative therapies to restore liver function and bridge patients to transplantation. Our previous work has shown that the proliferation of late gestation E19 fetal hepatocytes is mitogen-independent. This is manifested as differences in the control of ribosome biogenesis, global translation, cell cycle progression and gene expression. In the present study, we investigated whether E19 fetal hepatocytes would engraft and repopulate an injured adult liver. Methods: Fetal hepatocytes were isolated using a monoclonal antibody against a hepatic surface protein, leucine amino peptidase (LAP). LAP+ and LAP- fractions were analyzed by immunofluorescence and microarray. Immunopurified E19 liver cells from DPPIV+ F344 rats were transplanted via splenic injection into partial hepatectomized DPPIV- rats that had been pretreated with mitomycin C. Results: Phenotypic characterization of the LAP+ fetal hepatocytes revealed that more than a third of the isolated cells expressed ductal markers. Transcriptomic analysis revealed that these dual expressing cells represent a distinct subpopulation of less well differentiated hepatocytes. Transplanted immunopurified LAP+ late gestation fetal hepatocytes formed small hepatic, endothelial and occasional ductal colonies within one month. The average size of the colonies derived from the LAP+ cells increased so that by 10 months up to 35% of the liver was repopulated by donor-derived cells. Conclusions: Our studies show that late gestation fetal hepatocytes, despite their being far along in the differentiation process, possess the capacity for extensive liver repopulation. This is likely related to the unexpected presence of a significant proportion of hepatocyte marker-positive cells maintaining a less well differentiated phenotype. Overall design: Two subpopulations of E19 fetal rat hepatocytes were isolated using monoclonal antibodies against the hepatic cell surface marker leucine aminopeptidase and the ducal marker OC.2. Adult hepatocytes were also isolated. RNA was isolated from triplicate biological replicates of fetal LAP+/OC.2- and LAP+/OC.2+ cells as well as adult hepatocytes using the mirvana kit. Affymetrix Rat ST 1.0 arrays were utilized.

肝移植（Liver transplantation）是终末期肝病患者唯一的治疗选择。供体器官短缺促使学界探寻可恢复肝功能、为患者桥接至移植的替代疗法。我们既往研究证实，妊娠晚期E19胎肝细胞的增殖不依赖有丝分裂原，其分子基础体现为核糖体生物发生、全局翻译、细胞周期进程及基因表达调控的差异。本研究旨在探究E19胎肝细胞是否可在受损成年肝脏中定植并实现再增殖。
方法：采用针对肝脏表面蛋白亮氨酸氨肽酶（leucine amino peptidase, LAP）的单克隆抗体分离胎肝细胞。通过免疫荧光与微阵列分析LAP阳性（LAP+）与LAP阴性（LAP-）细胞组分。将取自DPPIV+ F344大鼠的免疫纯化E19肝细胞，经脾内注射移植至经丝裂霉素C预处理的部分肝切除DPPIV-大鼠体内。
结果：对LAP+胎肝细胞的表型表征显示，分离获得的细胞中有超过三分之一表达胆管标志物。转录组分析表明，这类双阳性细胞代表了一类分化程度更低的独特肝细胞亚群。移植的免疫纯化LAP+妊娠晚期胎肝细胞可在1个月内形成小型肝脏、内皮及偶见的胆管集落。LAP+细胞形成的集落平均体积随时间逐渐增大，至10个月时，供体来源细胞可再填充多达35%的肝脏组织。
结论：本研究表明，尽管妊娠晚期胎肝细胞已处于较为成熟的分化阶段，仍具备广泛肝脏再增殖的能力。这一特性可能与相当比例表达肝细胞标志物的细胞仍维持低分化表型这一意外发现相关。
总体实验设计：采用针对肝脏细胞表面标志物亮氨酸氨肽酶与胆管标志物OC.2的单克隆抗体，分离得到两种E19胎鼠肝细胞亚群，同时分离成年肝细胞。使用mirvana试剂盒，从LAP+/OC.2-、LAP+/OC.2+胎肝细胞以及成年肝细胞的三份生物学重复样本中提取RNA。采用Affymetrix Rat ST 1.0芯片进行基因表达检测。