Wege Henning, Le Hai T, Chui Michael S, Liu Li, Wu Jian, Giri Ranjit, Malhi Harmeet, Sappal Baljit S, Kumaran Vinay, Gupta Sanjeev, Zern Mark A
Transplant Research Institute, University of California, Davis Medical Center, Sacramento 95817, USA.
Gastroenterology. 2003 Feb;124(2):432-44. doi: 10.1053/gast.2003.50064.
BACKGROUND & AIMS: The availability of in vitro expandable human hepatocytes would greatly advance liver-directed cell therapies. Therefore, we examined whether human fetal hepatocytes are amenable to telomerase-mediated immortalization without inducing a transformed phenotype and disrupting their differentiation potential. Telomerase is a ribonucleoprotein that plays a pivotal role in maintaining telomere length and chromosome stability. Human somatic cells, including hepatocytes, exhibit no telomerase activity. Consequently, their telomeres progressively shorten with each cell cycle until critically short telomeres trigger replicative senescence.
The catalytic subunit, telomerase reverse transcriptase, was expressed in human fetal hepatocytes. Transduced cells were characterized for telomerase activity, telomere length, proliferative capacity, hepatocellular functions, oncogenicity, and their in vivo maturation potential.
The expression of human telomerase reverse transcriptase restored telomerase activity in human fetal hepatocytes. Telomerase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell doublings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors, growth factor receptors, and transcription factors as well as albumin, glucose-6-phosphatase, glycogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immortalized cells exhibited no oncogenicity, and no up-regulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression.
Reconstitution of telomerase activity induces indefinite replication in human fetal hepatocytes and offers unique opportunities for examining basic biologic mechanisms and for considering development of stable cell lines for liver-directed therapies.
体外可扩增的人肝细胞的获得将极大地推动肝脏定向细胞治疗的发展。因此，我们研究了人胎儿肝细胞是否适合通过端粒酶介导的永生化，同时不诱导转化表型并破坏其分化潜能。端粒酶是一种核糖核蛋白，在维持端粒长度和染色体稳定性方面起关键作用。包括肝细胞在内的人类体细胞不表现出端粒酶活性。因此，它们的端粒在每个细胞周期中逐渐缩短，直到极短的端粒触发复制性衰老。
在人胎儿肝细胞中表达催化亚基端粒酶逆转录酶。对转导的细胞进行端粒酶活性、端粒长度、增殖能力、肝细胞功能、致癌性及其体内成熟潜能的表征。
人端粒酶逆转录酶的表达恢复了人胎儿肝细胞中的端粒酶活性。端粒酶重建的细胞能够保持延长的端粒，在培养中增殖超过复制性衰老300多次细胞倍增（迄今为止），并通过一组肝生长因子、生长因子受体、转录因子以及白蛋白、葡萄糖-6-磷酸酶、糖原合成、细胞色素P450（CYP）表达谱和尿素产生来分析维持其肝脏特异性。此外，永生化细胞没有表现出致癌性，也未检测到c-Myc的上调。这些细胞在免疫缺陷小鼠的肝脏中植入并存活，具有肝细胞基因表达。
端粒酶活性的重建诱导人胎儿肝细胞无限复制，并为研究基本生物学机制以及考虑开发用于肝脏定向治疗的稳定细胞系提供了独特的机会。
