A stochastic model for in vitro ageing II. A theory of marginotomy

A model for variation in the lifespan of mass cultures and clones of human diploid fibroblasts is analyzed. The model is based on Olovnikov's theory of marginotomy and the following four assumptions: The time between successive divisions of a given cell may be split into a portion of fixed length and a portion of random length; cells divide or lose the ability to divide independently of one another; there is a fixed probability that a cell may lose one of its important parts during DNA synthesis; there is a small but fixed probability that a cell may lose all its important parts and hence the ability to replicate further during DNA synthesis. Samples taken by Monte Carlo means at successive stages in the development of a hypothetical population are compared with clonal survival data derived experimentally. The fit between theoretical and experimental findings is within the range of variation inherent in the experimental method. Applications of the theory and suggestions for further experiments are given.     

Viability of human diploid cells as a function of in vitro age

The fraction of cell capable of division was determined for (1) population of the human diploid cell strains, WI38 after different numbers of subcultivations in vitro and (2) a single population of WI38 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in vitro lifespan reported by other investigators. Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.     

Irreversible Plastid Loss in Euglena gracilis under Physiological Conditions

Irreversible loss of the ability to develop chloroplasts in Euglena gracilis may develop following transfer from organic medium to defined medium. Requirements for the loss include the absence of light and a temperature of 30 C (the optimal temperature for multiplication) although neither darkness alone nor this temperature alone serves as the bleaching agent. The extent of bleaching of a population can approach 100% but depends heavily on the following conditions: the pH of the defined medium and its phosphate content, the age of the parent culture at transfer, and the length of time spent in the defined medium before cell divisions are permitted. Bleaching is not due to loss of nonreplicating proplastids through “dilution out” as cells divide but appears to be a more direct inactivation of chloroplast differentiation from proplastids.     

Cell Division Resets Polarity and Motility for the Bacterium Myxococcus xanthus

Links between cell division and other cellular processes are poorly understood. It is difficult to simultaneously examine division and function in most cell types. Most of the research probing aspects of cell division has experimented with stationary or immobilized cells or distinctly asymmetrical cells. Here we took an alternative approach by examining cell division events within motile groups of cells growing on solid medium by time-lapse microscopy. A total of 558 cell divisions were identified among approximately 12,000 cells. We found an interconnection of division, motility, and polarity in the bacterium Myxococcus xanthus. For every division event, motile cells stop moving to divide. Progeny cells of binary fission subsequently move in opposing directions. This behavior involves M. xanthus Frz proteins that regulate M. xanthus motility reversals but is independent of type IV pilus “S motility.” The inheritance of opposing polarity is correlated with the distribution of the G protein RomR within these dividing cells. The constriction at the point of division limits the intracellular distribution of RomR. Thus, the asymmetric distribution of RomR at the parent cell poles becomes mirrored at new poles initiated at the site of division.     

Fly meets yeast: checking the correct orientation of cell division

Cell division is generally thought to be a process that produces an exact copy of the mother cell by precisely replicating its genomic DNA, doubling organelles, and segregating them into two cells. Many cell types from bacteria to human cells divide asymmetrically, however, to generate daughter cells with distinct characteristics. Such asymmetric divisions are fundamental to the lifespan of a cell, to embryonic development, and to stem cell homeostasis. Asymmetric division requires coordination of cellular asymmetry and the cell division machinery. Accumulating evidence suggests that the basic molecular mechanisms that govern this process are conserved from yeast to humans. In this review we highlight similarities in the mechanisms of asymmetric cell division in yeast and Drosophila male germline stem cells (GSCs) in the hope of extracting common themes underlying several systems.     

Myoblast senescence in muscular dystrophy

The limited proliferative capacity of normal diploid cells predicts that the utilization of cell divisions in vivo should reduce the lifespan of cells in culture. Because of the continuing demands for muscle regeneration in muscular dystrophy, myoblasts isolated from affected muscles should thus show a decrease in the number of cell divisions they are capable of expressing in culture. This hypothesis was tested by examining the proliferative capacity of myoblasts from different muscles for normal line 412 and dystrophic line 413 chickens of various ages. Prior to approx. 2 months of age, dystrophic myoblasts exhibited a relatively normal proliferative lifespan. By 5 months of age, myoblasts from the severely affected pectoralis major showed a 40% reduction in their proliferative potential, while myoblasts from the less affected posterior latissimus dorsi muscle showed a 25% decrease in their cultured lifespan. The time course of the appearance of a decreased proliferative capacity only after the disease has been clinically manifested strongly supports it representing a secondary response rather than it being an intrinsic property of dystrophic myoblasts. A hypothesis for manipulating the pattern of stem cell division in order to increase the mass of muscle produced from a constant number of cell divisions is presented. If myoblast senescence and the consequent failure of muscle regeneration is a contributing factor in the progressive deterioration of muscle function in the disease, then this hypothesis might provide an important therapeutic strategy for ameliorating the course of muscular dystrophy.     

Cell Size Control and a Cell-intrinsic Maturation Program in Proliferating Oligodendrocyte Precursor Cells

We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G₁ proposed by “transition probability” models, may explain the random variability of cell cycle times seen within clonal cell lines in culture.     

Intramitotic and intraclonal variation in proliferative potential of human diploid cells: explained by telomere shortening.

Normal human diploid cells can only divide for a limited number of times (known as the Hayflick limit). They manifest two unique features during in vitro senescence. The division capability of individual cells in a clone, though all derived from a same ancestor, is heterogeneous with a distinct bimodal distribution. Two sister cells derived from a same parent cell can have a large difference in their doubling potentials. These two unique features have not been properly explained by any known physiological process since their observation in 1980. Here I represent a telomere-shortening model based on recent experimental measurement of telomere deletion in human cells. Using computer simulation, I show that the model satisfactorily explains the intraclonal and intramitotic variation in division capability of human diploid cells. Moreover, the simulations predict that human cells may only monitor the shortening of a few, most likely two, telomeres to regulate their proliferative potential.     

Symmetric vs. Asymmetric Stem Cell Divisions: An Adaptation against Cancer?

Traditionally, it has been held that a central characteristic of stem cells is their ability to divide asymmetrically. Recent advances in inducible genetic labeling provided ample evidence that symmetric stem cell divisions play an important role in adult mammalian homeostasis. It is well understood that the two types of cell divisions differ in terms of the stem cells'' flexibility to expand when needed. On the contrary, the implications of symmetric and asymmetric divisions for mutation accumulation are still poorly understood. In this paper we study a stochastic model of a renewing tissue, and address the optimization problem of tissue architecture in the context of mutant production. Specifically, we study the process of tumor suppressor gene inactivation which usually takes place as a consequence of two “hits”, and which is one of the most common patterns in carcinogenesis. We compare and contrast symmetric and asymmetric (and mixed) stem cell divisions, and focus on the rate at which double-hit mutants are generated. It turns out that symmetrically-dividing cells generate such mutants at a rate which is significantly lower than that of asymmetrically-dividing cells. This result holds whether single-hit (intermediate) mutants are disadvantageous, neutral, or advantageous. It is also independent on whether the carcinogenic double-hit mutants are produced only among the stem cells or also among more specialized cells. We argue that symmetric stem cell divisions in mammals could be an adaptation which helps delay the onset of cancers. We further investigate the question of the optimal fraction of stem cells in the tissue, and quantify the contribution of non-stem cells in mutant production. Our work provides a hypothesis to explain the observation that in mammalian cells, symmetric patterns of stem cell division seem to be very common.     

Variability of the limited lifespan state in ameba

Muggleton & Danielli [6] reported that the normally immortal Amoeba proteus could be switched into a state of limited lifespan by restricting their food intake for several weeks so as to preclude division. Upon return to normal feeding, these cells were reportedly no longer immortal, but were only capable of a limited, unspecified number of divisions before the resulting clone died out. Attempts to duplicate these results with our present ameba stocks have resulted in failure. It appears that the presence of this phenomenon is variable and the life-spanned state may no longer be present in these cultures. An extended period on a maintenance diet which precludes division may sometimes have effects on cell viability for several generations but it no longer results in a general limitation of lifespan. Further work on this phenomenon must involve rigorous proof that the phenomenon is indeed present in the ameba being used.     

Mitochondrial biogenesis in human fibroblasts

It is not known whether limitation of lifespan represents a programmed genetic event or is a result of environmental factors imposed by the conditions of culture. An investigation of the factors surrounding the limitedin vitro lifespan of human diploid fibroblasts has been undertaken. We have investigated the role of mitochondria in the finite lifespan of WI-38 human lung fibroblasts. Mitochondrial function was depressed in a controlled manner by treating cells with ethidium bromide and chloramphenicol both of which inhibit normal biogenesis. These antibiotics decrease cytochrome oxidase activity, change cell ultrastructure, and inhibit growth at high concentration. At lower concentrations the antibiotics do not affect cell proliferation for several generations. However, their effect is cumulative and after several generations the cells enlarge, stop dividing and die. Removal of antibiotics from the culture media before death restores proliferative capacity. At still lower concentrations cytochrome oxidase activity was decreased but continuous growth in the presence of the antibiotics caused no decrease inin vitro lifespan. Thus, the potential for oxidative metabolism appears to be in excess of that needed for cell proliferation at all stages of thein vitro lifespan of a culture. The importance of cytoplasmic protein synthesis was evaluated using cycloheximide, a specific inhibitor of this process. Cycloheximide was used to try to distinguish between the effects due to general inhibition and that due to specific inhibition of mitochondrial biogenesis. Exposure of cultures to concentrations of cycloheximide which inhibited growth drastically caused no decrease in cytochrome oxidase activity.     

Plating efficiency measurements and the experimental control of ageing of adult human skin fibroblasts in vitro.

Adult human skin fibroblasts were serially cultured by means of eleven protocols differing in inoculum size, duration of culture between passage and the ability of the medium to support cell division. Each protocol was terminated only when there were too few cells for further subculturing. The fraction of the cells of an inoculum adhering to the growth surface was unaffected by serial subculturing or by differences in protocol. The final cell count at the end of a period of culture and the plating efficiency for the next culture diminished progressively with serial subculturing. Nevertheless, the computed number of cell generations per culture period of those cells which divided was unaffected by serial passaging. The total number of cell doublings accruing during an entire protocol depended only on the duration of the period of culture between successive passages which was characteristic of that protocol. The observations can be accounted for quantitatively by the following assumptions. A cell which loses its ability to divide after a given period of culture nevertheless continues to grow in size during the next period of culture. The increase in volume of cell substance during any such period is the same whether or not a cell divides. The second postulate is that the probability of a cell being able to divide at the start of a period of culture is proportional to the probability that it will not lose this ability by the following period of culture.     

Multicellular Pollen Formation in Cultured Barley Anthers: II. THE A, B, AND C PATHWAYS

Patterns of nuclear and cellular division leading to the formationof haploid, non-haploid, and mixoploid multicellular pollengrains in cultured anthers of Hordeum vulgare cv. Sabarlis aredescribed. The vegetative cell divides either by repeated unequalhaploid divisions (A pathway) or by synchronous nuclear divisionsaccompanied by fusion and variable wall formation (non-haploidand mixoploid). The divisions are random and unorganized. Thegenerative cell degenerates or is integrated into the vegetativecomponent either before or after division has commenced. Priorfusion (C pathway) leads to binucleate or bicellular diploidunits partitioned by a straight transverse wall. These binucleateand bicellular units are cytologically indistinguishable fromother diploid units derived from spores which do not form agenerative cell (B pathway). Division of the bicellular unitsmay be random and unorganized, but in some instances the twocells divide synchronously and in a highly ordered manner yieldingembryonic-like octants of cells. The diploid binucleate unitsgenerate still higher ploidies with or without wall formation. The significance of the various segmentation patterns is discussedin relation to previous concepts of pollen-callus formation.     

Immortal phenotype of the HeLa variant D98 is recessive in hybrids formed with normal human fibroblasts

Normal human cells such as human diploid fibroblasts (HDF) have a finite proliferative lifespan in culture. Previous studies have shown that the limited lifespan phenotype is dominant in cell hybrids formed by fusion of HDF to at least 23 different kinds of immortal human cells. However, two independent studies reported that hybrid clones formed by the fusion of HDF to the HeLa variant D98 had unlimited division potential. Those results were potentially very important because they implied that a) there is a dominant mechanism for immortalization of human cells in addition to the well-documented recessive mechanism, and b) a dominant mechanism would lend itself to identification of the immortalizing gene. Consequently, we carried out more detailed studies of the behavior of D98 cells in hybrids. Our results indicate that the majority of D98 x HDF hybrid clones exhibit a clear-cut finite proliferative lifespan phenotype. In addition, these hybrid cell populations often give rise to an immortal focus of cells that can be seen to take over the population of mortal cells at the end of their lifespan. This phenomenon reconciles our data with the previous reports of immortal D98 x HDF hybrid clones and leads us to conclude that D98 cells do not express a dominant immortalizing gene.     

Cultured human fibroblasts: Distribution of cell generations and a critical limit

In circular outgrowths of human skin fibroblasts we found that mitotic cells at the circumference had consumed more of their replicative lifespan than cells located more centrally. The lifespan remaining in cells at a given radial position could be predicted by determining their generation level based on the rate at which the outgrowth expanded and the cell doubling time. The data also show that outgrowths contain a heterogeneous mixture of cells described by a linear distribution of generations which can account for the variable replicative capacity observed in clones and the exponential increase in the fraction of nondividing cells with serial passage. These results support the concept that a critical limit of cell divisions determines the replicative lifespan.     

Reoccurring neural stem cell divisions in the adult zebrafish telencephalon are sufficient for the emergence of aggregated spatiotemporal patterns

Regulation of quiescence and cell cycle entry is pivotal for the maintenance of stem cell populations. Regulatory mechanisms, however, are poorly understood. In particular, it is unclear how the activity of single stem cells is coordinated within the population or if cells divide in a purely random fashion. We addressed this issue by analyzing division events in an adult neural stem cell (NSC) population of the zebrafish telencephalon. Spatial statistics and mathematical modeling of over 80,000 NSCs in 36 brain hemispheres revealed weakly aggregated, nonrandom division patterns in space and time. Analyzing divisions at 2 time points allowed us to infer cell cycle and S-phase lengths computationally. Interestingly, we observed rapid cell cycle reentries in roughly 15% of newly born NSCs. In agent-based simulations of NSC populations, this redividing activity sufficed to induce aggregated spatiotemporal division patterns that matched the ones observed experimentally. In contrast, omitting redivisions leads to a random spatiotemporal distribution of dividing cells. Spatiotemporal aggregation of dividing stem cells can thus emerge solely from the cells’ history.

An interdisciplinary study of the rules governing cell divisions in a population of neural stem cells in the zebrafish brain reveals the existence of aggregated spatio-temporal division patterns of rapid cell cycles in stem cells, and shows that these patterns can be explained by a simple agent-based model relying solely on the cells‘ division history.     

Responses of halophytes to high salinities and low water potentials

Agrobacterium tumefaciens can induce tumors on thin slices which are excised from Jerusalem artichoke (Helianthus tuberosus) tubers and grown in culture on medium containing minerals and a carbon source. A comparative study was made of the kinetics of cell division in slices under three conditions: (a) slices which were untreated and showed only spontaneous (wound-induced) cell divisions; (b) slices treated with indoleacetic acid at several concentrations; and (c) slices treated with virulent or avirulent bacteria. The earliest spontaneous cell divisions were completed (as detected by the appearance of new daughter cell pairs) by about 3 hours. These cells divide only once. In indoleacetic acid-treated tissue, more cells divide, with the first cell pairs being detected slightly earlier than in slices not subjected to the hormone. The number of cells which divide is roughly proportional to auxin concentration. Tissue treated with virulent bacteria showed only the pattern of spontaneous cell division until about 72 hours, after which another burst of cell division commenced and continued indefinitely. The bacteria-induced growths produced the unusual amino acids which are characteristic of crown gall tumors. The percentage of slices with tumors was sharply reduced if certain avirulent A. tumefaciens strains were applied prior to virulent strains.     

Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana

Precise knowledge of spatial and temporal patterns of cell division, including number and orientation of divisions, and knowledge of cell expansion, is central to understanding morphogenesis. Our current knowledge of cell division patterns during plant and animal morphogenesis is largely deduced from analysis of clonal shapes and sizes. But such an analysis can reveal only the number, not the orientation or exact rate, of cell divisions. In this study, we have analyzed growth in real time by monitoring individual cell divisions in the shoot apical meristems (SAMs) of Arabidopsis thaliana. The live imaging technique has led to the development of a spatial and temporal map of cell division patterns. We have integrated cell behavior over time to visualize growth. Our analysis reveals temporal variation in mitotic activity and the cell division is coordinated across clonally distinct layers of cells. Temporal variation in mitotic activity is not correlated to the estimated plastochron length and diurnal rhythms. Cell division rates vary across the SAM surface. Cells in the peripheral zone (PZ) divide at a faster rate than in the central zone (CZ). Cell division rates in the CZ are relatively heterogeneous when compared with PZ cells. We have analyzed the cell behavior associated with flower primordium development starting from a stage at which the future flower comprises four cells in the L1 epidermal layer. Primordium development is a sequential process linked to distinct cellular behavior. Oriented cell divisions, in primordial progenitors and in cells located proximal to them, are associated with initial primordial outgrowth. The oriented cell divisions are followed by a rapid burst of cell expansion and cell division, which transforms a flower primordium into a three-dimensional flower bud. Distinct lack of cell expansion is seen in a narrow band of cells, which forms the boundary region between developing flower bud and the SAM. We discuss these results in the context of SAM morphogenesis.     

Standardization of Human Diploid Fibroblast Cultivation: Centrifugation Procedure

The effect of varying centrifugal forces on the growth rate, longevity, and adsorption on glass of human embryonic diploid lung fibroblasts was studied. Cells centrifuged at 120, 500, or 1,500 × g at each passage had similar growth rates but their longevity decreased slightly with increasing force. These forces had no influence on the proportion of cells attaching to the glass. When the material in the first supernatant was recentrifuged at 2,000 × g for 30 min and added to the cells precipitated in the first centrifugation, the longevity of these cells was increased by several cell divisions. Cells which were not centrifuged but added directly from the cell suspension in trypsin to the new culture grew at a slightly slower rate than the centrifuged cells and became senescent at an earlier time. However, the noncentrifuged cells adsorbed to glass better than those centrifuged.     

Clonal Attenuation In Chick Embryo Fibroblasts Experimental Data, A Model and Computer Simulations

When cells from mass cultures of chick embryo fibroblasts are grown at very low density, some cells yield large clones while others produce smaller clones, and some cells fail to divide at all. the distribution of clone sizes is related to the number of population doublings which the donor mass culture has undergone: the more doublings which have occurred, the smaller the average clone size. In this report we describe a model which analyses this phenomenon, referred to as ‘clonal attenuation’, in detail. The model is based on the concept that a cell with hypothetically unlimited replicative potential—i.e. a ‘stem’ cell—can become ‘committed’ to a programme of limited replicative potential. This event is assumed to be stochastic and to have a fixed probability per stem cell division. the parameters of the model are: P_c, the probability of commitment; N, the number of differentiative divisions; and T_c, the cell-cycle times. By computer simulation, it is shown that P_c increases roughly exponentially at each successive stem cell division. According to the model, when the daughter of a stem cell becomes committed, its progeny proceed through N obligatory divisions before becoming terminally differentiated (post-mitotic). the best-fit value of N was found to be seven. The simulations also reveal that the absolute number of stem cells in the total population increases for most of the lifespan of the culture. When P_c becomes much greater than 0.5, the number of stem cells declines rapidly to zero, and the culture nears senescence. Sensitivity analysis shows that P_c can assume only a limited range of values at each stem-cell division.