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Hayflick L, Moorhead PS.. The seial cultivation of human diploid cell strains. Exp Cell Res 25: 585-621
Abstract and Figures
The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypical differentiation, inadaptability to suspended culture, non-malignant characteristics in vivo, finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed.Survival of cell strains at − 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level.With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.
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... This suggests that the aging of tissues may be due to the loss of cellular multiplication. The ability to proliferate is important for replacing damaged cells that build up over time [9]. If, on the one hand, senescence helps with tissue remodeling during development after injury, while halting the proliferation of DNAdamaged cells, on the other hand, it leads to inflammation, age-related tumorigenesis, and the progressive decline of tissue regenerative potential [10]. ...
... To understand if I3C also had a senolytics effect on humans, we treated dermal fibroblasts with 40 µM of I3C for 10 days and evaluated the effects. As me before, the peculiarity of this cell line is that it goes against the normal senescence as the passages increase [9]. ...
... To understand if I3C also had a senolytics effect on humans, we treated human dermal fibroblasts with 40 µM of I3C for 10 days and evaluated the effects. As mentioned before, the peculiarity of this cell line is that it goes against the normal senescence process as the passages increase [9]. ...
Senescence and apoptosis are two fundamental cellular processes that play crucial roles in various physiological and pathological conditions. Senescence refers to the irreversible growth arrest that cells undergo in response to various stimuli, including telomeric alterations, stress, and oncogenic signaling. Pharmacological and/or genetic removal of senescent cells, also referred to as senolysis, triggers organ rejuvenation and tissue regeneration. Indole-3-carbinol (I3C) is a natural compound contained in Brassicaceae plants and identified in multiple in vitro and in vivo studies as a well-tolerated and effective compound in cancer prevention and therapy. Its anti-cancer properties have been attributed at least in part to its inhibitory activity of proto-oncogenic HECT E3-ubiquitin ligases such as NEDD4 and WWP1. While the tumor suppressive effects of I3C in cancer cell lines have been reported in multiple studies, little is known regarding the biological effects of I3C in primary normal cells, which attain spontaneous cellular senesce over serial passaging. To this end, we used two model systems: mouse embryonic fibroblasts (MEFs) and human primary dermal fibroblasts. Here, we surprisingly show that I3C does increase cellular senescence at early passages, while dramatically reducing the number of senescent cells through the induction of apoptosis in both mouse and human primary cells. Thus, our findings support the notion that I3C acts as a senolytic compound with important therapeutic implications for the prevention and treatment of aging manifestations. The notion can be readily tested in future clinical trials in humans also in view of the high tolerability and safety previously displayed by I3C in preclinical and clinical studies.
... Although the mechanisms underlying the Hayflick limit are not fully elucidated, factors such as telomere attrition and genomic instability are thought to play significant roles in driving replicative senescence [26]. The first association between cellular senescence and aging was reported in 1961, when Hayflick and Moorhead demonstrated that human fetal cells could be cultured for longer durations before reaching replicative limits compared to adult human cells [27]. In 2012, Longo et al. introduced distinctions between two forms of cellular lifespan: cellular aging, which describes the gradual functional decline leading to increased susceptibility to cell death, and replicative aging, which refers to the phase where dividing cells undergo permanent cell cycle arrest in preparation for senescence. ...
The aging process significantly impacts lung physiology and is a major risk factor for chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, and non-IPF interstitial lung fibrosis. This narrative clinical review explores the molecular and biochemical hallmarks of aging, such as oxidative stress, telomere attrition, genomic instability, epigenetic modifications, proteostasis loss, and impaired macroautophagy, and their roles in lung senescence. Central to this process are senescent cells, which, through the senescence-associated secretory phenotype (SASP), contribute to chronic inflammation and tissue dysfunction. The review highlights parallels between lung aging and pathophysiological changes in respiratory diseases, emphasizing the role of cellular senescence in disease onset and progression. Despite promising research into modulating aging pathways with interventions like caloric restriction, mTOR inhibitors, and SIRT1 activators, clinical evidence for efficacy in reversing or preventing age-related lung diseases remains limited. Understanding the interplay between aging-related mechanisms and environmental factors, such as smoking and pollution, is critical for developing targeted therapies. This review underscores the need for future studies focusing on therapeutic strategies to mitigate aging’s detrimental effects on lung health and improve outcomes for patients with chronic respiratory conditions.
... Cellular aging, first described in vitro in 1961, has become the focus of biotechnology companies seeking to improve various human conditions. Thus, Hayflick and Moorhead demonstrated in 1961 that normal cultured human fibroblasts exhibit a limited capacity for cell division before entering an irreversible growth arrest known as replicative senescence [10]. Aging is a process in which the physiological functions necessary for life deteriorate over time. ...
Citation: Gorodilova, A.V.; Kharisova, C.B.; Osinnikova, M.N.; Kitaeva, K.V.; Filin, I.Y.; Mayasin, Y.P.; Solovyeva, V.V.; Rizvanov, A.A. The Well-Forgotten Old: Platelet-Rich Plasma in Modern Anti-Aging Therapy. Cells 2024, 13, 1755. https://doi. Abstract: Currently, approaches to personalized medicine are actively developing. For example, the use of platelet-rich plasma (PRP) is actively growing every year. As a result of activation, platelets release a wide range of growth factors, cytokines, chemokines, and angiogenic factors, after which these molecules regulate chemotaxis, inflammation, and vasomotor function and play a crucial role in restoring the integrity of damaged vascular walls, angiogenesis, and tissue regeneration. Due to these characteristics, PRP has a wide potential in regenerative medicine and gerontology. PRP products are actively used not only in esthetic medicine but also to stimulate tissue regeneration and relieve chronic inflammation. PRP therapy has a number of advantages, but the controversial results of clinical studies, a lack of standardization of the sample preparation of the material, and insufficient objective data on the evaluation of efficacy do not allow us to unambiguously look at the use of PRP for therapeutic purposes. In this review, we will examine the current clinical efficacy of PRP-based products and analyze the contribution of PRP in the therapy of diseases associated with aging.
Why and how we age are probably two of science's oldest questions, echoing personal beliefs and concerns about our own finitude. From the earliest musings of ancient philosophers to recent pharmacological trials aimed at slowing ageing and prolonging longevity, these questions have fascinated scientists across time and fields of research. Taking advantage of the natural diversity of ageing trajectories, within and across species, this interdisciplinary volume provides a comprehensive view of the recent advances in ageing and longevity through a biodemographic approach. It includes the key facts, theories, ongoing fields of investigation, big questions, and new avenues for research in ageing and longevity, as well as considerations on how extending longevity integrates into the social and environmental challenges that our society faces. This is a useful resource for students and researchers curious to unravel the mysteries of longevity and ageing, from their origins to their consequences, across species, space and time.
Gingival mesenchymal stem cells (GMSCs) are distinctive homogenous subset of mesenchymal stem cells (MSCs), which has its development from neural ectomesenchyme along with contributions from the perifollicular mesenchyme and the dental follicle proper. GMSCs stand apart from other dental MSCs owing to their ease of accessibility and availability with incredible long culture sustainability without any tumorigenic capability, and stable telomerase activity. Their capacity to differentiate into various cell lineages and inherent therapeutic effect in chronic inflammatory diseases like colitis, rheumatoid arthritis, systemic lupus erythematous (SLE) and diabetes makes them immensely valuable. The immunomodulatory and anti-inflammatory properties aid its usage in auto immune diseases and graft versus host disease. However, the differentiation, immunomodulatory and anti-inflammatory effects of GMSCs in periodontal tissue regeneration are less explored.
In this review article, we have comprehensively compiled and described several reports on GMSCs till date, including their basic properties and isolation protocols, subpopulations, spheroid GMSCs, gingiva-derived IPSCsinduced pluripotent stem cells (iPSCs), their characterization, multilineage differentiation, and immunomodulatory properties along with precise applications in periodontal regeneration and peri-implantitis.
Though the studies on GMSCs in periodontal regeneration lack superior quality random clinical trials, this review article still strengthens the view that GMSCs can be a newer source in periodontal tissue reconstruction/regeneration.
Secondary ossification and maintenance of the growth plate are crucial aspects of long bone formation. Parathyroid hormone-related protein (PTHrP) has been implicated as a key factor in maintaining the growth plate, and studies suggest that PTHrP expression in the resting zone is closely related with formation of the secondary ossification center (SOC). However, details of the relationship between resting zone PTHrP expression and preservation of the growth plate remain unclear. In this study, we aim to investigate the role of resting zone PTHrP expression on maintenance of the growth plate using a computational method. We extend an existing continuum-based particle model of tissue morphogenesis to include PTHrP and Indian hedgehog (Ihh) signaling, allowing the model to capture biochemical and mechanical regulation of individual cell activities. Our model indicates that the timing of resting zone PTHrP expression—specifically the rate of increase in production at the onset of SOC formation—is potentially a crucial mechanism for maintenance of the growth plate.
This study identifies senescent osteocytes in the femur and tibia of young rodents and explores their role in bone remodeling. The proximity of osteoclasts to senescent osteocytes was observed, which is a new finding. Cultured osteocytes, sorted using a podoplanin antibody in FACS, exhibited osteocytic characteristics and increased senescence-related genes. Senescent osteocytes secreted cytokines associated with senescence, remodeling, and inflammation. Notably, IGF1 and MMP2 were elevated in podoplanin-positive (pdpn⁺) osteocytes. Migration assays demonstrated significant osteoclast precursor migration towards senescent osteocytes, further confirmed by co-culture experiments leading to osteoclast differentiation. These findings suggest that senescent osteocytes have a pivotal role in initiating bone resorption, with recruitment of osteoclast precursors during early bone remodeling stages. In conclusion, our research enhances our understanding of complicated bone remodeling mechanisms and bone homeostasis.
Introduction
Progressive aging, or senescence, of mesenchymal stem/stromal cells (MSCs) is a major obstacle faced when trying to culture potent stem cells for use in therapy. Senescent cells are irreversibly nondividing cells that cease performing critical functional effects. Elimination of senescent cells using biochemical means, such as the use of senolytic drugs like dasatinib, may be useful in retaining the viable and proliferating populations of the cells.
Methods
An in vitro approach was used to investigate the effect of dasatinib on phenotypic, genotypic, and immunomodulatory functionality of osteogenic and adipogenic differentiated MSCs. Replicative senescence was achieved through multiple sub-culturing in vitro, then senescent and non-senescent cultures were treated with a standard dosage of dasatinib. MSCs were then differentiated into osteogenic, adipogenic or chondrogenic cultures using conditioned media to be tested for the three criteria being investigated.
Results
Significant changes were observed in these criteria, indicated by evidence gathered from proliferation and indoleamine 2,3 dioxygenase activity assays. Phenotypic results of dasatinib were shown to reduce the population of senescent MSCs while allowing non-senescent MSCs to continue differentiating and proliferating without interference from senescent cells. Genotypic results showed no change to upregulation in markers associated with osteogenic and adipogenic cells when exposed to dasatinib. Indoleamine Dioxygenase activity showed insignificant differences in cells exposed to dasatinib versus control groups, providing evidence against compromised cellular immune function.
Conclusion
This investigation provides insight into how dasatinib effects MSCs functional ability and provides a better understanding of the function of senolytic agents.
Senescence, caused by cell‐cycle arrest, is a hallmark of aging. Senescence has also been described in embryogenesis, wound healing, and acute injuries. Sepsis is characterized by a dysregulated host response to infection, leading to organ dysfunction and mortality. Most of the pathophysiology of human sepsis is recapitulated in the mouse model of polymicrobial sepsis, developed by cecal ligation and puncture (CLP). In this report, we demonstrate a rapid onset of cellular senescence in the liver of mice subjected to CLP‐induced sepsis, characterized by the upregulation of p21, p53, and other senescence markers, including SA‐βgal. Using RNAscope, confocal microscopy, and flow cytometry, we further confirm the emergence of p21‐expressing senescence phenotype in the liver 24 h after sepsis induction. Senescence was observed in several cell types in the liver, including hepatocytes, endothelial cells, and macrophages. We determined the landscape of senescence phenotype in murine sepsis by single‐cell sequencing, which further ascertained that this cell fate is not confined to any particular cell type but displays a heterogeneous distribution. Furthermore, we observed a significant reduction in mortality following sepsis when mice were treated with senolytics, a combination of dasatinib and quercetin, before the CLP surgery. Our experiments unequivocally demonstrated a rapid development of cellular senescence with sepsis and, for the first time, described the senescence landscape in the sepsis liver and the possible role of senescent cells in the worsening outcome following sepsis.
Seven vitamins have to date proved essential for the survival and multiplication of a mouse fibroblast (strain L) and a human carcinoma cell (strain HeLa) in tissue culture: choline, folic acid, nicotinamide, pantothenic acid, pyridoxal, riboflavin, and thiamin. It was necessary to cultivate the cells for 5 to 15 days in a medium lacking the specific vitamin before the deficiency became apparent in the cessation of multiplication and the development of specific cytopathogenic effects. In their early stages these changes could be reversed by the addition of the missing vitamin, an in vitro, "cure" of a vitamin deficiency. The maximally effective concentrations were in the range 107 to 108 gm. per ml.
The probability that additional vitamins not demonstrably essential under the conditions of the present experiments are nevertheless required for survival and growth is discussed in the text.
Spontaneous chromosomal aberrations may occur with a high frequency in human tissue culture cells, and the frequency increases with the age of the culture. In a series of experiments the frequency of aberrations found at anaphase and telophase increased from 3.6 per cent to 9.6 per cent as the culture aged over a period of three months. Most, or all, of the aberrations appear to be of the chromatid type, and must have originated at prophase of the nuclear cycle. There seems to be some correlation between the frequency of spontaneous chromosome aberrations and sensitivity to ionizing radiation in mammalian tissue culture cells.
This chapter is concerned primarily with the particles in Paramecium aurelia and is considered in relation to the particles in other organisms. It attempts to integrate as fully as possible the results of the various specialized approaches to the problems raised by kappa, pi, and mu, placing the particles themselves at the center of attention. The possible relations of the toxic agent to viruses, colicins, and toxins, and the nature of the specific protection against killing action are discussed from a comparative point of view, taking into account the possible fundamental similarity between kappa and mu in spite of their superficial but striking differences. As the particles, rather than the paramecia that bear them, are the center of attention, the role of kappa and mu as cytoplasmic genetic factors of Paramecium is given subordinate consideration. The chapter also aims to take a fresh, critical look at the whole corpus of knowledge about the particles themselves. This includes their morphology, development, actions, quantity, cytochemistry, reproduction, transmission, persistence, mutability, interrelations with each other and with the rest of paramecium, responses to injurious agents, and infectivity. On the basis of this survey, the status of the particles and the problems raised by them are discussed. A brief account of possibly comparable particles in other ciliates and in higher organisms provides the ground for speculation as to whether the particle system in Paramecium is a special case or an example of a widespread phenomenon in nature.
A combination of cytological and leukocyte culture techniques is described which constitutes a convenient, reliable approach for chromosome studies of humans and yields the following advantages: 1.1. Relative ease of obtaining blood and small volume required.2.2. Adequate mitotic yield from the short-term culture of leukocytes.3.3. High numbers of “exact count” quality metaphase spreads, permitting a critical analysis of chromosome morphology.
Plaques have been produced with the three types of poliomyelitis viruses on monolayer tissue cultures of monkey kidney and monkey testis. The number of plaques was proportional to the concentration of the virus. Each plaque originates, therefore, from a single virus particle, defined as the virus unit that is unseparable by dilution. The plaques are due to the specific action of the virus since they are suppressed by type-specific antiserum.
Pure virus lines were established by isolating the virus population produced in single plaques. These derived virus lines had the same morphological, serological, and pathogenic properties as the parent strain.
High titer virus stocks, with titers up to 7 x 10^8 plaque-forming particles per ml., were obtained.
1.1. Observations have been made on cell behaviour in the area between two embryonic chick heart explants, placed 0.5 to 1 mm apart in liquid medium tissue culture.2.2. The cell population in a given area in this situation rises steadily from zero as cells from both explants invade it, but then almost ceases to rise soon after the opposing outgrowths have met. There is no detectable diminution of rate of rise of population while the cells are approaching, before the outgrowths actually join.3.3. The velocity of individual cell movement after junction of the outgrowths is much slower between the explants than at either side of the explants.4.4. The direction of movements of cells in the region between the explants is predominantly outward from their respective explants until the two out-growths of cells join, when it becomes little more than randomly different from equal in all directions.5.5. It is concluded that outgrowth into the area between the two explants almost ceases soon after junction has been established between the opposing sheets of cells. After this junction, the number of cells in a radial strip joining the two explants becomes exceeded by the sum of two radial strips one at the side of each explant. Culture outgrowth therefore continues laterally after it has substantially ceased between the two explants.6.6. After junction the cells between the two explants tend to remain as a “monolayer”. The number of instances where one nucleus overlaps another is much fewer than is to be expected if the cells were randomly distributed.7.7. It is concluded that fibroblasts avoid moving over each other's surfaces. Such “contact-inhibition” of movement can explain why it is that fibroblasts normally migrate predominantly radially from an explant and that the whole culture tends rapidly to become circular in plan whatever its initial form.