Choy-Pik Chiu

Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype

Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative lifespan. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumour formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.

Enrichment of neurons and neural precursors from human embryonic stem cells.

Human embryonic stem (hES) cells proliferate and maintain their pluripotency for over a year in vitro (M. Amit, M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P., Harris, M. A. Waknitz, J. Itskovitz-Eldor, and J. A. Thomson. 2000. Dev. Biol. 227: 271-278) and may therefore provide a cell source for cell therapies. hES cells were maintained for over 6 months in vitro (over 100 population doublings) before their ability to differentiate into the neural lineage was evaluated. Differentiation was induced by the formation of embryoid bodies that were subsequently plated onto appropriate substrates in defined medium containing mitogens. These populations contained cells that showed positive immunoreactivity to nestin, polysialylated neural cell adhesion molecule (PS-NCAM) and A2B5. After further maturation, these cells expressed additional neuron-specific antigens (such as MAP-2, synaptophysin, and various neurotransmitters). Calcium imaging demonstrated that these cells responded to neurotransmitter application. Electrophysiological analyses showed that cell membranes contained voltage-dependent channels and that action potentials were triggered by current injection. PS-NCAM and A2B5 immunoselection or culture conditions could be used to produce enriched populations (60-90%) which could be further differentiated into mature neurons. The properties of the hES-derived progenitors and neurons were found to be similar to those of cells derived from primary tissue. These data indicate that hES cells could provide a cell source for the neural progenitor cells and mature neurons for therapeutic and toxicological uses.

Generation of Hepatocyte-Like Cells from Human Embryonic Stem Cells:

Use of human hepatocytes for therapeutic and drug discovery applications is hampered by limited tissue source and the inability of hepatocytes to proliferate and maintain function long term in vitro. Human embryonic stem (hES) cells are immortal and pluripotent and may provide a cell source for functional human hepatocytes. We report here that hES cells can be induced to differentiate into hepatocyte-like cells. Treatment with sodium butyrate induced hepatic differentiation as well as significant cell death, resulting in approximately 10–15% yield of a homogeneous population of cells. The differentiated cells have morphological features similar to that of primary hepatocytes and 70–80% of the cells express liver-associated proteins (albumin, alpha-1-antitrypsin, cytokeratin 8 and 18), accumulate glycogen, have inducible cytochrome P450 activity, and do not express alpha-fetoprotein. Because of the inherent proliferative capacity of hES cells, these cells may provide a reliable source of normal human hepatocytes for research and transplantation.

Shortened telomeres in the expanded CD28- CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis

OBJECTIVE To test the hypothesis that the expanded population of non-proliferative CD28-CD8+ T cells in HIV disease have shortened telomeres, thereby providing evidence that increased rounds of CD8+ cell division occur during HIV disease, possibly leading to replicative senescence and exhaustion of CD8+ T-cell responses. DESIGN CD8+ cells play a central role in control of HIV infection. In late HIV disease, an expanded population of CD28-CD8+ cells with reduced proliferative potential has been documented. A similar population of CD28-CD8+ cells has been identified in ageing humans, where telomere length measurements have suggested that these cells have reached the irreversible state of replicative senescence. METHODS CD8+ cells from HIV-infected and control subjects were sorted by flow cytometry into CD28+ and CD28- fractions. Telomere lengths were determined as mean terminal restriction fragment (TRF) lengths by Southern hybridization. RESULTS The TRF lengths of sorted CD28-CD8+ cells in HIV-infected subjects ranged between 5 and 7 kilobases (kb) and were significantly shorter than TRF lengths of CD28-CD8+ cells in uninfected subjects (P = 0.003). The TRF length in CD28-CD8+ cells from HIV-infected subjects was the same as that observed for centenarian peripheral blood mononuclear cells and is compatible with a state of replicative senescence. CONCLUSIONS The shortened telomeres in the CD28-CD8+ cells in HIV-infected subjects and the poor proliferative potential of these cells identifies CD8+ cell replicative senescence as a newly described feature of HIV disease. Our results provide a mechanism for the loss of CD8+ cell control of viral replication that accompanies advanced HIV disease. Replicative senescence may contribute to exhaustion of the T-cell response as a result of chronic HIV disease. Whether this phenomenon occurs in other chronic viral infections is unknown.

Differential Expression of Telomerase Activity in Hematopoietic Progenitors from Adult Human Bone Marrow

The loss of telomeric DNA may serve as a mitotic clock which signals cell senescence and exit from cell cycle. Telomerase, an enzyme which synthesizes telomeric repeats de novo, is required to maintain telomere lengths. In humans, significant telomerase activity has been found in cells with essentially unlimited replicative potential such as reproductive cells in ovaries and testes, immortal cell lines and cancer tissues, but not in most normal somatic cells or tissues. We have now examined telomerase expression in subpopulations of hematopoietic cells from adult human bone marrow using a sensitive polymerase chain reaction‐based telomeric repeat amplification protocol. Telomerase activity was found at low levels in the highly enriched primitive hematopoietic cells (CD34+CD71loCD45RAlo) and was increased transiently when these cells were cultured in the presence of a mixture of cytokines. In contrast, the early progenitors (CD34+CD71+) expressed telomerase activity at a higher level which was subsequently downregulated in response to cytokines. Telomerase activity remained low in the more mature CD34− cells upon exposure to cytokines. Taken together, our results suggest that telomerase is expressed at a basal level in all hematopoietic cell populations examined, is induced in a primitive subset of hematopoietic progenitor cells and is downregulated upon further proliferation and differentiation of these cells. We have previously observed telomere shortening in cytokine‐stimulated primitive hematopoietic cells. The low and transient activation of telomerase activity described here thus appears insufficient to maintain telomere lengths in cultured hematopoietic cells.

Replicative senescence and cell immortality: the role of telomeres and telomerase.

Abstract Telomere shortening is correlated with cell senescence in vitro and cell aging in vivo. The telomere hypothesis suggests that telomere length serves as a mitotic clock for timing cellular replicative life span. Expression of telomerase stabilizes telomere length and allows for continual replication, or cell immortality. This article reviews recent evidences for the role of telomere length and telomerase in the regulation of cellular replicative life span. The therapeutic potential of manipulating telomerase expression and telomere length is also discussed.

Dendritic cells reconstituted with human telomerase gene induce potent cytotoxic T-cell response against different types of tumors

Human telomerase reverse transcriptase (hTERT) is the catalytic component of a functional telomerase complex, which is important in maintaining cell immortality. In most normal human adult cells, the expression of telomerase is very low and/or transient. In contrast, almost 90% of human tumors express a relatively high level of telomerase implying the possibility of using hTERT as a universal candidate tumor antigen. In this study, we show that human monocyte-derived dendritic cells (DCs) lack telomerase activity. Similar to other normal somatic cells, DCs express the RNA (hTR) component but not the catalytic component, hTERT. We also show that telomerase activity could be reconstituted using either lipid-mediated transfection of the hTERT plasmid DNA or transduction with an E1-, E3-deleted adenoviral vector containing the hTERT gene. However, relative to plasmid transfection, adenoviral gene transfer produced higher levels of hTERT expression. Nine of 10 AdhTERT-transduced DCs were able to generate CTL responses, while only three of nine plasmid-transfected DCs did. CTLs primed against hTERT exhibited killing of telomerase positive but not telomerase negative tumor lines of diverse tissue origins. Antigenic specificity of these T cells to telomerase was further determined by introducing hTERT gene into a telomerase negative cell line, U2OS, by adenoviral transduction. Although some antigenic specificity was directed against adenoviral epitopes, the majority of CTLs were targeted against telomerase-derived antigen(s). Thus, the hTERT gene, particularly as delivered via the recombinant adenovirus, may be useful as vaccine to induce specific T-cell-mediated tumor immunity in cancer patients. In addition, our results suggest that telomerase activity and/or telomerase expression after hTERT gene transfer have a predictive value in the success of hTERT/DC-based cancer vaccination.