Population Doubling Potential Research Articles

The doubling potential of T lymphocytes allows clinical-grade production of a bank of genetically modified monoclonal T-cell populations

Background aimsTo produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited. MethodsWe used clinical-grade conditions and two donors who were homozygous and identical for all HLA-alleles except HLA-DP. After mixed lymphocyte culture and transduction, we obtained 14 HLA-DP–specific T-cell clones transduced with the HSV-TK suicide gene. Clones were then selected on the basis of their specificity and functional characteristics and evaluated for their doubling potential. ResultsAfter these steps of selection the clone NAT-DP4(TK), specific for HLA-DPB1*04:01/04:02, which produced high levels of interferon-γ (IFNγ), tumor necrosis factor (TNF), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was fully sequenced. It has two copies of the HSV-TK suicide transgene whose localizations were determined. Four billion NAT-DP4(TK) cells were frozen after 50 PDs. Thawed NAT-DP4(TK) cells retain the potential to undergo 50 additional PDs, a potential very far beyond that required to produce a biological effect. This PD potential was confirmed on 6/16 additional different T-cell clones. This type of well-defined clone can also support a second genetic modification with CAR constructs. ConclusionThe possibility of choosing rare donors and exploiting the natural proliferative potential of T lymphocytes may dramatically reduce the clinical and immunologic complexity of adoptive transfer protocols that rely on the use of third-party T-cell populations.

Label-Free Morphology-Based Prediction of Multiple Differentiation Potentials of Human Mesenchymal Stem Cells for Early Evaluation of Intact Cells

Precise quantification of cellular potential of stem cells, such as human bone marrow–derived mesenchymal stem cells (hBMSCs), is important for achieving stable and effective outcomes in clinical stem cell therapy. Here, we report a method for image-based prediction of the multiple differentiation potentials of hBMSCs. This method has four major advantages: (1) the cells used for potential prediction are fully intact, and therefore directly usable for clinical applications; (2) predictions of potentials are generated before differentiation cultures are initiated; (3) prediction of multiple potentials can be provided simultaneously for each sample; and (4) predictions of potentials yield quantitative values that correlate strongly with the experimental data. Our results show that the collapse of hBMSC differentiation potentials, triggered by in vitro expansion, can be quantitatively predicted far in advance by predicting multiple potentials, multi-lineage differentiation potentials (osteogenic, adipogenic, and chondrogenic) and population doubling potential using morphological features apparent during the first 4 days of expansion culture. In order to understand how such morphological features can be effective for advance predictions, we measured gene-expression profiles of the same early undifferentiated cells. Both senescence-related genes (p16 and p21) and cytoskeleton-related genes (PTK2, CD146, and CD49) already correlated to the decrease of potentials at this stage. To objectively compare the performance of morphology and gene expression for such early prediction, we tested a range of models using various combinations of features. Such comparison of predictive performances revealed that morphological features performed better overall than gene-expression profiles, balancing the predictive accuracy with the effort required for model construction. This benchmark list of various prediction models not only identifies the best morphological feature conversion method for objective potential prediction, but should also allow clinicians to choose the most practical morphology-based prediction method for their own purposes.

Does Aging Need Its Own Program, or Is the Program of Development Quite Sufficient for It? Stationary Cell Cultures as a Tool to Search for Anti-Aging Factors

According to our conception, the aging process is caused by cell proliferation restriction-induced accumulation of various macromolecular defects (mainly DNA damage) in cells of a mature organism or in a cell population. In the case of cell cultures, the proliferation restriction is related to so-called contact inhibition and to the Hayflick's limit, while in the case of multicellular organisms, it is related to the appearance, in the process of differentiation, of organs and tissues consisting of postmitotic and very slowly dividing cells. It is assumed that the proliferation of intact cells prevents accumulation of various errors in a cell population. However, the continuous propagation of all the cells in a multicellular organism is absolutely incompatible with its normal functioning. Thus, the program of development, when it generates postmitotic or slowly dividing cells, automatically leads also to the onset of the aging process (mortality increase with age). Therefore, any additional special program for aging simply becomes unnecessary. This, however, doesn't reject, for some organisms, the reasonability of programmed death, which makes possible the elimination of harmful, from the species point of view, individuals. It is also very important to emphasize that increase or decrease of an organism's lifespan under the effects of various external factors is not always necessarily related to modification of the aging process, though the experimental results in the field are usually interpreted in just this way. I called the experimental-gerontological models similar to the Hayflick's model "correlative", since they are based on some correlations only and not related necessarily to the gist of the aging phenomenon. So, for the Hayflick's model, it is the relationship between population doubling level and donor age, between population doubling potential and species lifespan, between some cell changes in vivo and in vitro, and so forth. If the rationale of the "Hayflick phenomenon" is used, we can't explain why we age. Nevertheless, many authors virtually put a sign of equality between aging in vitro and aging in vivo, which generates conclusions that are of quite doubtful accuracy. A classic illustration of this is the telomere concept of aging. Originally, the principle of shortening end-segments of DNA (telomeres) during each cell division was formulated at the beginning of seventies by the Russian scientist Aleksey Olovnikov and used by him to explain the limited "proliferative" lifespan in vitro of normal cells. Subsequently, the existence of this phenomenon was confirmed by the results of many research reports, the culmination of which was a publication in which the authors demonstrated the possibility of increasing the proliferative potential of normal cells by introducing the enzyme telomerase to them, thus restoring the lost telomere segments. At the moment it looks like the telomere shortening contributes to aging in vitro only, but not to aging in vivo because an organism never realizes the full proliferative potential of its cells. Besides, the most "responsive to aging" are the organs and tissues consisting of postmitotic cells, for which the concept of proliferative potential loses any meaning in practical terms. We developed another "correlative" model--a model for testing of geroprotectors and geropromoters--the "cell kinetics model." It is based on the well-known correlation between the "age" of cultured cells (age of their donor) and their saturation density. The model allowed us to perform preliminary testing of a lot of different compounds and factors that are interesting from a gerontological point of view, but it revealed no information about the real mechanisms of aging. However, the second model we use in our studies--the "stationary phase aging" model--obviously, is a "gist" model. It is based on the assumption that in the cells of stationary cultures various intracellular changes similar to those of an aging organism can be observed. The proliferation restriction in the case is provided, as a rule, just by contact inhibition. Many experimental results confirming this assumption were obtained. "Age-related" changes that are well known from organismal studies were shown to really occur in our experimental stationary cell culture model. Besides, such experiments can be carried out on nearly any type of cells from various biological species. Thus, the evolutionary approach to analysis of the data is provided. Moreover, the changes in the stationary cell cultures become detectable very soon--as a rule, in 2 to 3 weeks after beginning the experiment. All this allows us to suppose that the "stationary phase aging" model should provide a very effective approach to testing of different substances and their cocktails on their activities in terms of accelerating or retarding aging--of course, if their effect is realized on the cell level only.

Cytogerontology at the beginning of the third millenium: from "correlative" to "gist" models

For the most part, research in the area of cytogerontology, i.e., investigation of the mechanisms of aging in the experiments on cultured cells, is carried out using the "Hayflick's model". More than forty years have passed since the appearance of that model, and during this period of time, very much data were obtained on its basis. These data contributed significantly to our knowledge of the behavior of both animal and human cultured cells. Specifically, we already know of the mechanisms underlying the aging in vitro. On the other hand, in my opinion, little has changed in our knowledge of the aging of the whole organism. In all likelihood, this can be explained by that the Hayflick's model is, like many others used in the experimental gerontology, correlative, i.e. based on a number of detected correlations. In the case of Hayflick's model, these are correlations between the mitotic potential of cells (cell population doubling potential) and some "gerontological" parameters and indices: species life-span, donor age, evidence of progeroid syndromes, etc., as well as various changes of normal (diploid) cells during long-term cultivation and during aging of the organism. It is, however, well known that very frequently a good correlation has nothing to do with the essence (gist) of the phenomenon. For example, we do know that the amount of gray hair correlates quite well with the age of an individual but is in no way related to the mechanisms of his/her aging and probability of death. In this case, the absence of cause-effect relationships is evident, which are, at the same time, indispensable for the development of gist models. These models, as distinct from the correlative ones, are based on a certain concept of aging. In the case of Hayflick's model, such a concept is absent: we cannot explain, using the "Hayflick's limit", why our organism ages. This conclusion was convincingly confirmed by the discovery of telomere mechanism which determines the aging of cells in vitro. That discovery initiated the appearance of theories attempting to explain the process of aging in vivo also on its basis. However, it has become clear that the mechanisms of aging of the entire organism, located, apparently, in its postmitotic cells, such as neurons or cardiomyocytes, cannot be explained in the framework of this approach. Hence, we believe that it is essential to develop "gist" models of aging using cultured cells. The mechanisms of cell aging in such models should be similar to the mechanisms of cell aging in the entire organism. Our "stationary phase aging" model could be one of such models, which is based on the assumption of the leading role of cell proliferation restriction in the processes of aging. We assume that the accumulation of "senile" damage is caused by the restriction of cell proliferation either due to the formation of differentiated cell populations during development (in vivo) or to the existence of saturation density phenomenon (in vitro). Cell proliferation changes themselves do not induce aging, they only lead to the accumulation of macromolecular defects, which, in turn, lead to the deterioration of tissues, organs, and, eventually, of the entire organism, increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of various types of damage identical to the damage arising in senescing multicellular organism. And, finally, it is essential to determine how the cell is dying and what the death of the cell is. These definitions will help to draw real parallels between the "genuine" aging of cells (i.e., increasing probability of their death with "age") and the aging of multicellular organisms.

Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.

Fibroblasts from cancer patients display a mixture of both foetal and adult-like phenotypic characteristics.

We have previously reported that foetal and adult fibroblasts display distinctive migratory phenotypes when cultured on three-dimensional collagen gels. Both skin and tumour-derived fibroblasts from a significant proportion of patients with breast cancer were subsequently observed to display foetal-like migratory behaviour. In the accompanying paper concerned with the biochemical basis of these observations, we presented evidence that foetal fibroblasts and the foetal-like fibroblasts of cancer patients produce a soluble migration-stimulating factor (MSF) not made by normal adult cells. Data are presented here indicating that: (1) the spontaneous foetal-to-adult transition in migratory phenotype that foetal fibroblasts undergo after approximately 50-55 population doublings in vitro is correlated with a cessation of MSF production; (2) breast cancer patient fibroblasts do not undergo such a phenotypic transition and continue to produce MSF for their entire in vitro lifespan. These foetal-like cancer patient fibroblasts do, however, resemble normal adult cells by a number of other criteria, including population doubling potential, enhanced migration in the presence of serum compared to platelet-poor plasma, saturation cell density and morphology in confluent culture. These data indicate that the fibroblasts of breast cancer patients express a mixture of both foetal and adult phenotypic characteristics. Such a finding is consistent with published information indicating that foetal-to-adult transitions in various fibroblast phenotypic characteristics occur in a temporally disparate fashion during normal development, and further imply that cancer patient fibroblasts have undergone only certain of these transitions.

Asbestos-associated chromosomal changes in human mesothelial cells.

Replicative cultures of human pleural mesothelial cells were established from noncancerous adult donors. The cells exhibited normal mesothelial cell characteristics including keratin, hyaluronic acid mucin, and long branched microvilli, and they retained the normal human karyotype until senescence. The mesothelial cells were 10 and 100 times more sensitive to the cytotoxic effects of asbestos fibers than normal human bronchial epithelial or fibroblastic cells, respectively. In addition, cultures of mesothelial cells that survived two cytotoxic exposures of amosite fibers were aneuploid with consistent specific chromosomal losses indicative of clonal origin. These aneuploid cells exhibit both altered growth control properties and a population doubling potential of greater than 50 divisions beyond the culture life span (30 doublings) of the control cells.

Effects of nickel sulfate on growth and differentiation of normal human bronchial epithelial cells.

Epidemiological studies have shown that inhalation of nickel compounds enhances the risk of human respiratory cancer. Cultures of normal human bronchial epithelial cells were continuously exposed to a dose (5-20 micrograms/ml) of NiSO4 that reduced their colony forming efficiency by 30-80%. After 40 days of incubation, the cultures consisted of large, squamous cells; mitotic cells were not observed. The cells were then maintained in medium without NiSO4. After 40-75 total days of incubation, colonies of mitotic cells appeared at a rate of 1 colony per 100 000 cells originally at risk; no colonies appeared in control cultures or in cultures exposed to less than 5 micrograms NiSO4/ml for 90 days. Twelve NiSO4-altered cell cultures isolated from five experiments have been expanded into mass cultures. Most of the cell lines have an increased population doubling potential (greater than 50 divisions). Some exhibit aberrations in the squamous (terminal) differentiation process whereas others have lost the requirement for epidermal growth factor for clonal growth. Aneuploidy and marker chromosomes have also been noted. However, none of these NiSO4-altered cell cultures are anchorage independent nor do they produce tumors upon injection into athymic nude mice.

Replicative potential of individual cell hybrids derived from young and old donor human skin fibroblasts.

Neonatal, adult, and aged donor human skin fibroblast cell cultures have been characterized for the population doubling potential and in vitro-in vivo relationship. Hybrid cells derived from individual whole-cell fusions of replicating GRC 387 (aged) and CSC 303 (neonatal) cells demonstrated that an intermediate mode of replication between that of the two parental cell lines occurred; therefore the longevity of the aged fibroblast cells was enhanced. The GRC 387 cells have an extended post-replicative phase-out period in comparison with the CSC 303 cells, and the experimental hybrids demonstrated a 20-25% increase of the period over that of the GRC 387 cells.

Recent advances in the cell biology of aging

Cultured normal human and animal cells are predestined to undergo irreversible functional decrements that mimic age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 ± 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occurs in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.

Modifications of doubling potential of cultured human diploid cells by ionizing radiation and hydrocortisone

Cells possessing the ability to multiply into colonies have advanced successively to the next colony formation, which makes it possible to estimate the maximum doubling potential in the human diploid cell population. Attempts were made to modify the population doubling potential and the maximum doubling potential of human diploid cells with external agents. X-ray irradiation at high dose rates reduced the population doubling potential (Ban et al., 1980), while hydrocortisone had the advantage of extending the population life span, as has neen reported. The magnitude of extension acquired was equivalent to about a quarter of the non-treated population's life span. However, X-irradiation and hydrocortisone treatment failed to change the maximum doubling potential. This means that ionizing radiation might accelerate the progression of cells to non-cycling state, while hydrocortisone might decelerate it. Our results support the hypothesis that the growth and death of cultured human diploid cells might reflect cell differentiation in vitro (Bell et al., 1978).

The Cell Biology of Aging

Cultured normal human and animal cells are predestinued to undergo irreversible functional decrements that mimick age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 +/- 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occur in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.

Progress in cytogerontology

The finite in vitro lifetime of cultured normal cells is interpreted to be aging at the cellular level. In addition to the inverse relationship between donor age and population doubling potential (PDP), a number of biochemical and physiological increments and decrements occur prior to the cessation of cell division. The reconstruction of replicating normal human cells from the nuclei of “young” cells and the cytoplasm of “old” cells (and the reverse) suggests that the nucleus governs PDP. Several morphological changes were found to occur in late phase III cells held for up to one year in culture. Autoradiography studies show that (1) a cell population may be composed of several subpopulations, each of which is at a different stage in its life history and (2) lipid synthesis is affected much less as cells age than is DNA, RNA and protein synthesis. Changes occurring in the genetic program of individual cells seem to be the most tenable hypothesis to explain fundamental causes of aging.

Nuclear control of cellular aging demonstrated by hybridization of anucleate and whole cultured normal human fibroblasts

Summary Cytoplasmic hybridization was achieved by fusing anucleate cytoplasms to iodoacetate-treated, whole, normal human fibroblasts. The enzymes in the untreated anucleate cytoplasms replaced inactivated enzymes in the treated whole cells and permitted survival of the hybrids. The population doubling potential of cytoplasmic hybrids made between young and old cells was compared with those made between young/young or old/old controls. The results suggest that nuclear rather than cytoplasmic factors are involved in the control of in vitro cellular senescence.

The regulation of cellular aging by nuclear events in cultured normal human fibroblasts (WI-38).

Since the development of an invitro model for aging in 1961, interest in the biology of vertebrate senescence at the cellular level has increased greatly. Previously it had been widely believed that normal vertebrate cells were immortal (1, 2), and consequently that biological aging was not the result of fundamental changes in the genome of individual cells. In 1961 we (3) demonstrated that normal human fetal lung fibroblasts have a limited proliferative capacity of 50 ± 10 population doublings, a finding that has since been confirmed in many laboratories. This limited proliferative capacity, and the physiological decrements that precede it, have been interpreted by us to be the invitro counterpart of those processes that occur as an organism ages (3–6). Support for this hypothesis has come from many different studies demonstrating such phenomena as 1) an inverse correlation between population doubling potential of cultured cells invitro and donor age (4, 7, 8), 2) a possible direct correlation between mean maximun species lifespan and invitro proliferative capacity (9–15), and 3) a limited proliferative capacity for normal cells serially transplanted invivo in syngeneic hosts (16–19).

Mitotic index in vitro of embryonic heart fibroblasts of different donor ages

Cultured normal human and animal cells are predestined to undergo irreversible functional decrements that mimick age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 ± 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occur in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.