Gestation stage-specific frequency of adipogenic cells in Syrian hamster cell cultures

High frequencies (up to 50%) of spontaneous adipocyte differentiation are observed in cultures of 9 day gestation Syrian hamster embryos (E9 cells) within six to eight population doublings after primary culture. This is in contrast to the absence of adipogenic cells in primary cultures derived from later gestation age Syrian hamster tissue. In addition, E9 primary cultures contain a transient subpopulation of presumptive mesenchymal stem or progenitor cells that lack density dependent inhibition of growth [contact-insensitive (CS-) cells]. Analysis of the temporal pattern of expression of the CS- and adipocyte phenotypes during the proliferative life span of E9 cells demonstrates that maximal expression of the CS- phenotype precedes maximal expression of adipocyte differentiation. In addition, lipid accumulation appears to occur primarily, if not exclusively, in the contact-sensitive (CS+) cells that are derived from CS- cells. These observations suggest that primary E9 cultures contain either adipoblasts or primordial mesenchymal cells that become determined to the adipocyte lineage early during the in vitro life span of the cultures, and that the CS- phenotype may be a marker for these earlier developmental cell stages.     

Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.     

Systematic growth studies,cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts

Summary The growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 55% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence or cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.     

Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells

The replicative life span of cells in culture is thought to be determined by the gradually rising pool of senescent cells rather than by the simultaneous loss of proliferative capacity by all cells in the population. We found that early-passage cultures of human peritoneal mesothelial cells (HPMCs) contained a significant fraction of senescent-like cells. Furthermore, early-passage populations with a high percentage of senescent cells had a reduced subsequent life span in culture compared with populations consisting of the same number of apparently young cells but containing no senescent cells. The exposure of early-passage HPMCs to the conditioned medium from cultures containing senescent cells resulted in the retardation of growth and the induction of senescence-associated beta-galactosidase (SA-beta-Gal). This effect could be partly reduced by neutralizing TGF-beta1 activity. The timely treatment with N-tert-butyl-alpha-phenylnitrone (PBN) reduced oxidative stress, the number of early senescent cells, TGF-beta1 secretion, and ultimately extended the population life span. The effect was evident only when PBN was introduced at a very early, but not at a late, phase of tissue culture history. These results indicate that a sudden onset of senescence in early-passage HPMCs is related to oxidative stress and may influence the replicative life span of the population as a whole.     

Telomerase can extend the proliferative capacity of human myoblasts,but does not lead to their immortalization

Normal cells in culture display a limited capacity to divide and reach a non-proliferative state called cellular senescence. Spontaneous escape from senescence resulting in an indefinite life span is an exceptionally rare event for normal human cells and viral oncoproteins have been shown to extend the replicative life span but not to immortalize them. Telomere shortening has been proposed as a mitotic clock that regulates cellular senescence. Telomerase is capable of synthesizing telomere repeats onto chromosome ends to block telomere shortening and to maintain human fibroblasts in proliferation beyond their usual life span. However, the consequence of telomerase expression on the life span of human myoblasts and on their differentiation is unknown. In this study, the telomerase gene and the puromycin resistance gene were introduced into human satellite cells, which are the natural muscle precursors (myoblasts) in the adult and therefore, a target for cell-mediated gene therapy. Satellite cells expressing telomerase were selected, and the effects of the expression of the telomerase gene on proliferation, telomere length, and differentiation were investigated. Our results show that the telomerase-expressing cells are able to differentiate and to form multinucleated myotubes expressing mature muscle markers and do not form tumors in vivo. We also demonstrated that the expression of hTERT can extend the replicative life of muscle cells although these failed to undergo immortalization.     

Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation

Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions. Erosion of telomeric DNA has emerged as a key factor in senescence, which is antagonized during cell immortalization and transformation. To clarify the involvement of telomerase in the immortalization of keratinocytes, catalytic subunit of telomerase (hTERT) expression was restored in normal human esophageal epithelial cells (EPC2). EPC2-hTERT cells overcame senescence and were immortalized without p16INK4a genetic or epigenetic alterations. p16INK4a was expressed at moderate levels and remained functional as evidenced by induction with UV treatment and binding to cyclin-dependent kinase 4 and 6. There were no mutations in the p53 gene, and p53 was functionally intact. Importantly, senescence could be activated in the immortalized EPC2-hTERT cells by overexpression of oncogenic H-ras or p16INK4a. Furthermore, the EPC2-hTERT cells yielded basal cell hyperplasia in an innovative organotypic culture system in contrast to a normal epithelium from parental cells. These comprehensive results indicate that the expression of telomerase induces immortalization of normal human esophageal keratinocytes without inactivation of p16INK4a/pRb pathway or abrogation of the p53 pathway.     

Telomerase activity is sufficient to allow transformed cells to escape from crisis

The introduction of simian virus 40 large T antigen (SVLT) into human primary cells enables them to proliferate beyond their normal replicative life span. In most cases, this temporary escape from senescence eventually ends in a second proliferative block known as "crisis," during which the cells cease growing or die. Rare immortalization events in which cells escape crisis are frequently correlated with the presence of telomerase activity. We tested the hypothesis that telomerase activation is the critical step in the immortalization process by studying the effects of telomerase activity in two mortal SVLT-Rasval12-transformed human pancreatic cell lines, TRM-6 and betalox5. The telomerase catalytic subunit, hTRT, was introduced into late-passage cells via retroviral gene transfer. Telomerase activity was successfully induced in infected cells, as demonstrated by a telomerase repeat amplification protocol assay. In each of nine independent infections, telomerase-positive cells formed rapidly dividing cell lines while control cells entered crisis. Telomere lengths initially increased, but telomeres were then maintained at their new lengths for at least 20 population doublings. These results demonstrate that telomerase activity is sufficient to enable transformed cells to escape crisis and that telomere elongation in these cells occurs in a tightly regulated manner.     

Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation

During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies have shown that ectopic expression of the catalytic subunit of telomerase (hTERT) enables normal human cells to bypass senescence (M1) and oncogene transformed cells to avert crisis (M2) and become immortal. However, it is unclear whether hTERT expression is sufficient for normal human fibroblasts to overcome both M1 and M2 and become immortal. We have investigated the role of telomerase in immortalization by maintaining mass cultures of hTERT-transduced primary human fetal lung fibroblasts (MRC-5 cells) for very long periods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-transduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferating for more than 36 population doublings (PDLs) beyond senescence, the overall growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phenotype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERT-transduced cells expressed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures have proliferated beyond 240 PDLs, while another culture appears to be permanently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are consistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant changes. However, these investigations also indicate that hTERT-expressing cells may undergo crisis following an extended life span and that immortality is not the universal outcome of hTERT expression in normal diploid fibroblasts.     

Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains.

Reduced replicative capacity is a consistent characteristic of cells derived from patients with Werner syndrome. This premature senescence is phenotypically similar to replicative senescence observed in normal cell strains and includes altered cell morphology and gene expression patterns. Telomeres shorten with in vitro passaging of both WRN and normal cell strains; however, the rate of shortening has been reported to be faster in WRN cell strains, and the length of telomeres in senescent WRN cells appears to be longer than that observed in normal strains, leading to the suggestion that senescence in WRN cell strains may not be exclusively associated with telomere effects. We report here that the telomere restriction fragment length in senescent WRN fibroblasts cultures is within the size range observed for normal fibroblasts strains and that the expression of a telomerase transgene in WRN cell strains results in lengthened telomeres and replicative immortalization, thus indicating that telomere effects are the predominant trigger of premature senescence in WRN cells. Microarray analyses showed that mRNA expression patterns induced in senescent WRN cells appeared similar to those in normal strains and that hTERT expression could prevent the induction of most of these genes. However, substantial differences in expression were seen in comparisons of early-passage and telomerase-immortalized derivative lines, indicating that telomerase expression does not prevent the phenotypic drift, or destabilized genotype, resulting from the WRN defect.     

Effect of fibroblast donor cell age and cell cycle on development of bovine nuclear transfer embryos in vitro

The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm2. The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm2 containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm2 containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell.     

Analysis of correlation between some parameters depending on cell proliferation and life span of "stationary phase aging" cultures and maximum life span of mammals

Earlier we developed a "stationary phase aging" model and introduced a definition of life span of "stationary phase aging" cell cultures. In this model the cells grow after seeding in flasks without subcultivation and medium change. They reach cell saturation density, stop dividing, gradually degrade ("stationary phase aging") and perish. By the term "culture life span" we designate the time from cell seeding until culture death. We designate the culture as dead when the number of living cells is less than 10 per cent of their number at saturation density of cell culture. The life span of transformed Chinese hamster cells was found to be proportional to the duration of their growth from cell seeding up to saturation density, as well as to the number of cell culture doublings and to be inversely proportional to the velocity of cell culture doubling for the same growth period. Maximum life span of mammals is known to be proportional to pregnancy duration and to the age at puberty. We found that maximal life span of mammals was proportional to the number of cell population doublings and inversely proportional to the velocity of cell population doubling during embryonal period or for the time from zygote to growth termination. The dependences for cell cultures and for mammals are analogous to each other.     

Selection and characterization of bovine aortic endothelial cells

This paper reports techniques for isolation, selection and long-term passage of bovine aortic endothelium (BAE). A [3H]thymidine-selection technique was developed to limit overgrowth of cultures by contaminating smooth-muscle cells. The resulting cultures could be passaged for a replicative life span of 35 to 40 doublings and maintained a stable, normal karyotpye throughout this period. Despite the fact that these cultures reached a stable monolayer with density-inhibited growth state, postconfluent cells showed focal areas of a second growth pattern called "sprouting." This was seen only when cultures were maintained at high densities for periods of 1 to 2 weeks. Ultrastructural analysis, as well as immunofluorescence studies with markers for endothelial cells (factor VIII) and smooth-muscle cells (actin), indicates that this phenomenon is not due to overgrowth of a residual population of smooth-muscle cells, but may represent a second growth pattern of the endothelial cells themselves.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

Summary The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm². The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm² containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm² containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell. This work was supported by IIT Research Institute.     

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.     

Establishment and characterization of a tamoxifen-mediated reversible immortalized mouse dental papilla cell line

Odontoblasts are a type of non-proliferating and terminally differentiated cells that play an important role in the pulpo–dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited life span. We combined the traditional strategy of “Cre/LoxP-based reversible immortalization” with a tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line, mDPCET. mDPCs were sequentially transduced with a floxed SV40 T antigen-TK (SV40Tag-TK) and an ^ERT2Cre^ERT2-expressing plasmid. Clonal-isolated SV40Tag- and Cre-positive cells showed modified growth characteristics and a significantly extended life span. When mDPCET cells were treated with 4-hydroxytamoxifen, ^ERT2Cre^ERT2 translocated from the cytoplasm to the nucleus and caused the excision of SV40Tag-TK, which led to the reversion of mDPCETs. After the immortalization was reversed, the cells underwent replicative senescence and transitioned into a more differentiated state. Tamoxifen-mediated reversible immortalization, therefore, allows for the expansion of primary mDPCs, leads to the production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent a safe and ready-to-use method due to its simple manipulation.