Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential
Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential
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DOI：
10.1053/gast.2003.50064
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发表时间：
2003-02-01
期刊：
影响因子：
29.4
通讯作者：
Zern, MA
中科院分区：
文献类型：
--
作者：
Wege, H;Le, HT;Zern, MA
背景与目的：体外可扩增的人肝细胞的可用性将极大地推进肝定向细胞疗法。因此，我们研究了人胎肝细胞是否适合端粒酶介导的永生化，而不诱导转化表型和破坏其分化潜力。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着关键作用。人的体细胞，包括肝细胞，没有表现出端粒酶活性。因此，它们的端粒随着每个细胞周期逐渐缩短，直到非常短的端粒触发复制衰老。方法：在人胎肝细胞中表达端粒酶催化亚单位。转导细胞的特征在于端粒酶活性，端粒长度，增殖能力，肝细胞功能，致癌性，和它们在体内成熟的潜力。结果：人端粒酶逆转录酶的表达可恢复人胎肝细胞端粒酶活性。端粒酶重建的细胞能够保留延长的端粒，在超过300个细胞倍增期（迄今为止）的复制性衰老的培养物中繁殖，并保持它们的肝脏特异性性质，如通过一组肝生长因子、生长因子受体和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 450）表达谱、和尿素生产。此外，永生化细胞没有表现出致癌性，也没有检测到c-Myc的上调。这些细胞在具有肝细胞基因表达的免疫缺陷小鼠的肝脏中移植并存活。结论：端粒酶活性的重建诱导人胎肝细胞的无限复制，并提供了独特的机会，检查基本的生物学机制，并考虑发展稳定的细胞系，用于肝脏定向治疗。
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. Methods: 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. Results: 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 upregulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression. Conclusions: 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.