The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 × 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5–13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [³⁵S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

Tritium-arrested human diploid fibroblasts maintain balanced RNA and protein metabolism

Incorporation of ³H precursors into the protein or RNA of exponentially growing human diploid fibroblasts (WI38) inhibited DNA synthesis and cell division for a dose-related period. During this period of “tritium-arrest”, which can last for at least a month, the cells remain viable by morphologic criteria and maintain balanced RNA and protein metabolism. The cultures are eventually overgrown by a dose-related fraction of the population which retains DNA synthetic capacity. Tritium-arrested cell populations are suggested as a possible model for the study of metabolism in non-dividing cells.     

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.     

DNA-binding proteins in young and senescent normal human fibroblasts

DNA-binding proteins (DBP) from normal human diploid cells, strain WI38, were isolated by DNA-cellulose chromatography using undenatured calf thymus DNA. The DBP in the 0.15 M NaCl eluate were fractionated by polyacrylamide gel electrophoresis. Comparisons of the amounts of the DBP in different cell populations were made by labelling the cells with either ³H- or ¹⁴C-amino acid precursors for 40 h prior to pooling the cells for co-isolation of their DBP. When WI38 cells in the replicative and stationary phases were compared, five proteins, P5b (87 000 D), P6a (50 000 D), P8 (33 000 D), P9 (28 000 D) and P10 (25 000 D) were labelled to a greater extent in the replicating cells and two proteins, P5c (72 000 D) and P12 (18 000 D) were labelled to a greater extent in the stationary phase cells. In addition, several high molecular weight DBP, partially characterized as collagen and protocollagen, were preferentially labelled in the stationary phase cells. Stationary phase senescent WI38 cells at or near the end of their in vitro lifespan characteristically showed an increased proportion of protein component P8 (33 000 D) relative to stationary phase WI38 cells at early population doubling levels. Further characterization of WI38-P8 showed that it binds preferentially to single-stranded DNA and amounts to greater than 1% of the total soluble protein in young cells in growth phase. Thus WI38-P8 appears to be comparable to the P8 protein studied by Tsai & Green [27] in mouse 3T6 and human SB cells. The component which is increased in senescent or terminal phase non-dividing cell populations is judged to be the P8 protein by its position in SDS-gels and its preferential binding to single-stranded DNA.     

The influence of culture medium volume on cell density and lifespan of human diploid fibroblasts

The volume of culture medium in which WI38 cells are grown affects the maximum cell number at stationary phase and in the vitro lifespan in terms of total population doublings. The saturation density at 0.53 ml/cm² (40 ml/T-75) is consistently about 2-fold higher than at 0.26 ml/cm² (20 ml/T-75).At a constant medium volume the cell yield at stationary phase is directly dependent on the amount of serum present. Thus the increased yields from greater medium volumes is probably due to a large extent on the increased amount of serum growth factor(s) present.For maximal cell yields in non-perfused WI38 cells, we suggest that routine subcultivation be carried out in medium containing10 % (v/v) serum and at 0.53 ml/cm² medium of surface area.     

Berberine ameliorates cellular senescence and extends the lifespan of mice via regulating p16 and cyclin protein expression

Although aging and senescence have been extensively studied in the past few decades, however, there is lack of clinical treatment available for anti‐aging. This study presents the effects of berberine (BBR) on the aging process resulting in a promising extension of lifespan in model organisms. BBR extended the replicative lifespan, improved the morphology, and boosted rejuvenation markers of replicative senescence in human fetal lung diploid fibroblasts (2BS and WI38). BBR also rescued senescent cells with late population doubling (PD). Furthermore, the senescence‐associated β‐galactosidase (SA‐β‐gal)‐positive cell rates of late PD cells grown in the BBR‐containing medium were ~72% lower than those of control cells, and its morphology resembled that of young cells. Mechanistically, BBR improved cell growth and proliferation by promoting entry of cell cycles from the G₀ or G₁ phase to S/G₂‐M phase. Most importantly, BBR extended the lifespan of chemotherapy‐treated mice and naturally aged mice by ~52% and ~16.49%, respectively. The residual lifespan of the naturally aged mice was extended by 80%, from 85.5 days to 154 days. The oral administration of BBR in mice resulted in significantly improved health span, fur density, and behavioral activity. Therefore, BBR may be an ideal candidate for the development of an anti‐aging medicine.     

Synthesis of Lanostane‐Type Triterpenoid N‐Glycosides and Their Cytotoxicity against Human Cancer Cell Lines

Seventeen lanostane‐type triterpenoid derivatives ( 2 – 18 ), including 11N‐glycosides ( 8 – 18 ), were synthesized from the natural triterpenoid, lanosterol ( 1 ), and were evaluated for their cytotoxicity against the human cancer cell lines, HL‐60, A549, and MKN45, as well as the normal human lung cells, WI‐38. Among them, N‐β‐d ‐2‐acetamido‐2‐deoxyglucoside ( 10 ) showed cytotoxicity against HL‐60, A549, MKN45, and WI‐38 cells (IC₅₀ 0.0078 – 2.8 μm ). However, N‐β‐d ‐galactoside ( 12 ) showed cytotoxicity against HL‐60 and MKN45 cells (IC₅₀ 0.0021 – 4.0 μm ), but not the normal WI‐38 cells. Furthermore, Western blot analysis suggested that 12 induces apoptosis by activation of caspases‐3, 8, and 9. These results will be useful for the synthesis of other tetracyclic triterpenoids or steroid N‐glycosides to increase their cytotoxicity and apoptosis‐inducing activities.     

Aging and sister chromatid exchange : II. The effect of the in vitro passage level of human fetal lung fibroblasts on baseline and mutagen-induced sister chromatid exchange frequencies

The frequencies of baseline and mutagen-induced sister chromatid exchanges (SCE) were examined in human fetal lung fibroblasts (IMR-90, WI38) as a function of in vitro serial passage (in vitro aging). Although baseline SCE levels remained relatively constant throughout the in vitro lifespan of these cell cultures, a significant decline was observed at middle and late passage in the levels of SCE induced by mitomycin-C, ethyl methane-sulfonate and N-acetoxy-2-acetylaminofluorene. These findings indicate that cellular aging results in an altered response to certain types of induced DNA damage.     

Purification and characterisation of the in vitro colony forming cell in monkey hemopoietic tissue

Buoyant density gradient separation of Rhesus monkey bone marrow, spleen and blood leukocytes has demonstrated a reproducible and homogeneous light density distribution profile of cells capable of forming hemopoietic colonies in agar culture (in vitro colony forming cells — CFC). High resolution density gradient separation performed on a light density fraction of bone marrow produced on average a 100-fold enrichment of in vitro CFC with the most enriched fractions containing the majority of the in vitro CFC population present in the original marrow. Fractions were routinely obtained in which up to 23% of cells formed colonies and 33% were capable of proliferating to some degree upon stimulation. Tritiated thymidine suiciding showed the active proliferative status of the in vitro CFC and application of autoradiography and morphological characterisation to highly enriched density fractions has shown that the in vitro CFC in normal marrow is a transitional lymphocyte. Single cell transfer experiments have shown that in vitro CFC's formed colonies containing both granulocytes and macrophages, formally demonstrating the clonal origin of in vitro colonies and the common origin of granulocytes and macrophages.     

Age‐related changes in biological characteristics of human alveolar osteoblasts

Objectives: Age‐related changes are common in many tissues and organs. However, cell‐related causes in human alveolar bone remain unclear. This study has been carried out to explore the possibility that advancing age might change the biological characteristics of alveolar osteoblasts (AOBs) in women. Materials and methods: Alveolar osteoblasts from women donors (five women aged 33–38 years and five women aged 62–68 years) were cultured in vitro. The cells were serially passaged and maximal lifespan evaluated. Cell viability, ultramicrostructure and osteogenic differentiation ability were determined respectively, using MTT assay, transmission electron microscopy, alkaline phosphatase (ALP) activity assay and von Kossa staining assay. These parameters of the two groups of AOBs were evaluated. Results: When compared with cells from young adult donors, AOBs from elderly women exhibited lower maximal lifespan (P < 0.05). Mean rate of population doubling was lower in elderly donor cells compared to those from young adult cells (P < 0.05). Organelles from AOBs of elderly donors were much fewer than those from young donors. MTT value of elderly donor cells was significantly lower than those of young adult donors from day 2 (P < 0.05). Relative ratio of ALP activity in elderly donor cells was significantly lower than those of the young womens’ cells at 8, 12, 16 and 20 days (P < 0.05). Calcium nodules of young adult donors’ specimens were significantly more numerous and larger than those from elderly donors. Conclusions: Comparison of biological characteristics of AOBs from young adult women with those from elderly women in vitro revealed differences in proliferative capacity and bone formation functions, which decreased with aging. These data indicate that aging may play an important role in pathogenesis of human AOBs loss.     

Cellular senescence and DNA synthesis : Thymidine incorporation as a measure of population age in human diploid cells

For the human diploid cell lines WI-38 and WI-26, the percentage of cells able to incorporate ³H-TdR into their nuclei under standard conditions declines as a function of either elapsed calendar time or the number of population doublings. The rate of decline varies for each population depending on its overall lifespan. If, however, the percent of labelled nuclei is considered as a function of the percent of lifespan completed (i.e., the number of population doublings at time t/total number of population doublings attained during lifespan of the culture), a highly reproducible relationship is found. Using 10% class intervals for the incidence of labelled nuclei, it is possible to distinguish the ‘age’ of populations which differ by about 5 passages or less. With this simple technique, one can evaluate simultaneously and in a reproducible way the current age of the culture, as well as the remaining proliferative capacity of the population.     

Characterization of the cell cycle of cultured human diploid cells: Effects of aging and hydrocortisone

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G₁. The duration of S remained constant, while a small delay in G₂ was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G₁ and possibly the G₂ periods.     

Mouse sarcoma L1 cell line holoclones have a stemness signature

Accumulating data suggest that cancers contain a fraction of cells called cancer stem cells (CSCs), that may be responsible for upkeep and relapses of disease. In experimental settings, CSCs are regarded as most effective at tumour initiation in in vivo assays. Since the first isolation of cancer stem cells from acute myeloid leukaemia in 1994, cancer stem cells have been identified in human solid tumours and they have also been found in the established cell lines, based on ability of CSCs to form in vitro colonies of a specific morphology, called holoclones. Our study examined the ability of a mouse sarcoma cell line, derived from a lung metastasis of a BALB/c mouse and established as a stably growing line (L1), to produce holoclones in vitro. We aimed to verify a stemness signature of the holoclone cells. The L1 cell line was found to form holoclone colonies in vitro, which were shown to contain a percentage of CSC‐like cells. A fraction of the L1 cells was able to repopulate the original cell line, and presented an increased clonogenic and metastatic potential (18th passage). In addition, MTT assay and flow cytometry of the side population fraction revealed that these cells were more resistant to chemotherapeutic drugs than the original cell line, and over‐expressed the anti‐apoptotic genes, GRP78 and GADD153. We conclude that mouse L1 sarcoma cell line contains CSC‐like cells.     

Cell division and the stability of cellular populations

 This paper couples a general d-dimensional (d arbitrary) model for the intracellular biochemistry of a generic cell with a probabilistic division hypothesis and examines the consequence of division for stability of cell function and structure. We show rather surprisingly that cell division is capable of giving rise to a stable population of cells with respect to function and structure even if, in the absence of cell division, the underlying biochemical dynamics are unstable. In the context of a simple example, our stability condition suggests that rapid cell proliferation plays a stabilizing role for cellular populations. Received: 15 January 1996 / Revised version: 31 July 1998     

HYDROXYUREA: USE IN DETERMINING THE DURATION OF G2 IN TETRAHYMENA

Observation of division of individual cells in microdrops, plus autoradiographic studies using tritiated thymidine and standard cell cycle analysis techniques, reveal that hydroxyurea (10 DIM) reversibly arrests the normal progression of exponentially growing Tetrahymena pyriformis through the initial 92 % of S-phase while not affecting cells in the terminal 8 % and in G₂ and division. Thus the fraction of the population of cells that is in G₂ can be approximately determined by the fraction of the population able to divide in the presence of hydroxyurea. This fraction can be related to the approximate duration of G₂ by calculations which compensate for the age gradient.     

Human nerve stem cells in vitro

The growth characteristics of a fraction of the human embryonic brain Nest-positive cells cultivated in vitro are the subject of the study. Our data demonstrate a probable increase in the content of Nest-positive cells in the cell population from 44.1 to 62.5% as a response to the presence of EGF and bFGF in a growth medium supplemented with heat-inactivated serum. Further differentiation in the cell culture led to a decrease in the number of Nest-positive cells. Retinoic acid-based stimulation of neural induction causes a decrease in the content of these cells in the population by 14.1%. The results of this study will allow for the obtaining of a nerve cell population that could be more effective as a graft material suitable for therapeutic use.     

Regulatory Mechanism of Delayed-Type Hypersensitivity in Mice: III. In Vitro Analysis of Memory T Cells Involved in Augmentation of DTH Responses

The memory of delayed-type hypersensitivity (DTH), manifested by the augmented responsiveness upon challenge with alum-absorbed ovalbumin (OA), was induced in mice primed 7 days, 21 days, or 90 days previously with 1 μg of reduced and alkylated OA. The memory cells involved in the augmentation of DTH responses were analyzed in the in vitro induction system of T cells which mediate DTH against OA. Spleen cells from the primed mice generated DTH-effector T cells (DTH-Te) in a significantly accelerated fashion, compared with unprimed spleen cells, when cultured with OA. The accelerated generation of DTH-Te in vitro was induced antigen specifically and was dependent on a certain T cell population in the primed spleen. The T cell population was found in the spleen of primed mice for at least 3 months after priming, corresponding to the persistence of DTH-memory in vivo. Moreover, it was fractionated in the high-density layer by discontinuous bovine serum albumin gradient centrifugation. The high-density cell population decreased in density with increase in the time of culture and developed into DTH-Te, which were separated in the low-density layer on day 4 of culture. These results indicate that the T cells involved in the accelerated generation of DTH-Te in vitro are long-lived DTH-memory T cells, which are probably precursor cells, capable of differentiating into DTH-Te upon challenge with the antigen.     

Establishment and characterization of two cell lines derived from primary cultures of Gekko japonicus cerebral cortex

Adult Gekko japonicus is one of those vertebrates that are able to regenerate their missing or amputated tail. The most interesting feature of this animal lies in the ability of its spinal cord to regrow a functional tail. A fundamental question is whether the neuroglial cells play a different role compared with high vertebrates. Since in vitro studies using primary neuroglial cells are hampered by the limited lifespan and miscellaneous genetic background of these cells, we generated neuroglial cell lines from primary cell cultures of cerebral cortex of G. japonicus. The SV40 (simian‐virus‐40) T antigen gene was introduced into primary cell cultures. Cell cycle analysis, cell growth and proliferation, cell colony formation and contact inhibition, as well as karyotype assays were investigated. Two cell colonies, Gsn‐1 and Gsn‐3, were immunochemically characterized as glial fibrillary acidic protein and galactocerebroside‐positive respectively. Compared with parental primary cells, the Gsn cells displayed shorter population doubling time, decreased percentage of cells in the G0/G1 phase, higher cell proliferation index, and increased cell activity. In assays of colony characteristics, Gsn cells showed increased cell activity at the lower cell densities or FBS (fetal bovine serum) supplement. The karyotype of immortalized Gsn cells exhibited transformational characteristics with hyperdiploid and polyploid chromosomes. The cell lines will provide a useful in vitro model for gecko neuroglial cells and facilitate systematic studies investigating the biological functions of specific gene products related to regeneration of the central nervous system.