In Vitro Expansion of Human Hepatocytes is Restricted by Telomere-Dependent Replicative Aging

Abstract

Currently, different techniques to expand human hepatocytes in vitro are being investigated to generate enough cells for liver-directed cell therapies. However, based on observations in fibroblasts and other cell types, telomere attrition limits the proliferative capacity of normal somatic cells. Therefore, we explored whether telomere-dependent replicative aging restricts the in vitro proliferation of human hepatocytes. Subpopulations of cells isolated from a neonatal liver and characterized as hepatocyte derived by RT-PCR and flow cytometry started to proliferate 5-7 days after plating and were termed proliferating human hepatocytes (PHH). Following retroviral-mediated transduction of the catalytic telomerase subunit, telomerase reverse transcriptase (hTERT), telomerase activity increased from almost undetectable levels to levels as high as in HepG2 and other telomerase-positive cell lines. As expected, untransduced PHH progressively lost telomeric repeats and arrested after 30-35 cell divisions with telomeres of less than 5 kilo bases. In comparison, telomerasereconstituted PHH maintained elongated telomeres and continued to proliferate as shown by colorimetric assays and cell counts. In this study, telomere stabilization extended the proliferative capacity of in vitro proliferating human neonatal hepatocytes. Therefore, telomere attrition needs to be addressed when developing techniques to expand human hepatocytes.

Serum Human Telomerase Reverse Transcriptase Messenger RNA as a Novel Tumor Marker for Hepatocellular Carcinoma

“…Because we could detect negligible amounts of lymphocyte markers after three steps of centrifugation of blood samples, the RNA extraction procedure seemed to remove lymphocytes effectively. In addition, normal or damaged hepatocytes express negligible amounts of hTERT (16,17). Furthermore, the correlation of hTERT mRNA between tumor tissue and serum was shown in Fig.…”

Telomere length dynamics in normal hematopoiesis and in disease states characterized by increased stem cell turnover

“…24,25 Moreover, experiments have shown that transfection of the hTERT gene can result in the extension of telomeres and increased proliferative lifespan of fibroblast clones of retinal pigment epithelial cells 26,27 and hepatocytes. 28,29 The 'telomerized' cells appeared indistinguishable from young normal cells and did not show signs of chromosomal aberrations or cellular transformation. 28,30,31 In summary, expression of telomerase in normal somatic cells was able to prevent replicative senescence providing compelling evidence for a direct linkage between telomere shortening and replicative lifespan of cells.…”

Regulation of telomerase activity by the p53 family member p73

“…Telomerase activity was quantified by the telomeric repeat amplification protocol (TRAP) adapted to qPCR (Wege et al, 2003). A timecourse analysis revealed a significant repression of telomerase activity as early as 24 h following infection with further reduction to less than 20% after 48 h ( Figure 1a).…”

“…Cell lysis was performed in CHAPS buffer (10 mM Tris (pH7.5), 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM PMSF, 5 mM b-ME, 0.5% CHAPS) and equal amounts of protein (2 mg) were used in each reaction. Telomerase activity was quantified by qPCR as described previously (Wege et al, 2003).…”