Effect of growth arrest on the doubling potential of human fibroblasts in vitro: a possible influence of the donor

Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi's anemia (FA). Confluent cultures were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting stage favors the expression of growth potentialities related to instability of the cells.     

IgE-enhancing activity directly and selectively affects activated B cells: evidence for a human IgE differentiation factor

T cells from highly atopic individuals spontaneously secrete in vitro a factor that specifically induces IgE synthesis from normal human B cells. We investigated the effects of such T cell supernatants derived from atopic individuals (TCSN-A) on functionally distinct B cell subsets to determine at what developmental stage B cells become responsive to this IgE-enhancing activity. B cells from normal and allergic donors were separated into subsets of small resting and large activated cells by density centrifugation or unit gravity sedimentation. When stimulated by TCSN-A, large activated B cells made more IgE than small resting B cells. The difference was as much as 3300% in comparing these subsets from allergic donors. Similarly, resting B cells stimulated by Staphylococcus aureus Cowan I (SAC) made 52 to 125% more IgE in response to TCSN-A than unstimulated small resting B cells. However, IgE production from large B cells, already activated in vivo, was not enhanced by the addition of SAC. Notably, the IgE level synthesized by in vivo large activated B cells from allergic persons was markedly greater than that seen with similar cells from normal donors, whereas resting B cells purified from allergic and normal donors produced comparable levels of IgE in response to TCSN-A. These results suggest that this enhancing activity functions as an IgE differentiation factor for activated B cells. This was further confirmed by the effects of TCSN-A on the IgM- and IgE-secreting EBV-transformed human B cell line K1D5. TCSN-A specifically enhanced IgE synthesis from these cells; TCSN from normal donors, IL 2, IFN-gamma, and BCGF did not. These results confirm that this activity functions as an IgE-specific differentiation factor, directly influencing activated B cells to synthesize IgE.     

Antiproliferative effect of interferon on a Burkitt''s lymphoma cell line

The effect of interferon (IF) on the growth of a Burkitt's lymphoma cell line was analysed. The degree of depression of cell doublings was the same if the cells were in a steady state mode of exponential growth or in a resting state (G0) when IF was added. As IF had a lag time of 24 h before decreased growth could be observed, cells in G0 did not seem to be more sensitive when growth was estimated by cell counts expressed as cell doublings. IF inhibited cells to proceed into the cell cycle and the possibility that IF may increase the escape into a G0 loop is discussed.     

Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells

Changes in fatty acid (FA) and glycerophospholipid (GPL) metabolism associated with cell cycle entry are not fully understood. In this study FA-GPL remodeling was investigated in resting and proliferating primary human T cells. Significant changes were measured in the composition and distribution of FAs in GPLs following receptor activation of human T cells. The FA distribution of proliferating T cells was very similar to that of the human Jurkat T cell line and when the stimulus was removed from proliferating T cells, they stopped proliferating and the FA distribution largely reverted back to that of resting T cells. The cellular content of saturated and monounsaturated FAs was significantly increased in proliferating cells, which was associated with an induction of FA synthase and stearoyl-CoA desaturase-1 gene expression. Additionally, cellular arachidonate was redistributed in GPLs in a distinct pattern that was unlike any other FAs. This redistribution was associated with an induction of CoA-dependent and CoA-independent remodeling. Accordingly, significant changes in the expression of several acyl-CoA synthetases, lysophospholipid acyltransferases, and phospholipase A₂ were measured. Overall, these results suggest that metabolic pathways are activated in proliferating T cells that may represent fundamental changes associated with human cell proliferation.     

Changes in the Lipid Composition of Thalassiosira pseudonana during Its Life Cycle

The lipid and fatty acid (FA) compositions of a marine diatom alga Thalassiosira pseudonana grown in culture were investigated. The relative content of separate lipid classes and their FA composition varied during of the life cycle. During the periods of active cell division and resting cell production, the proportion of polar lipids, as the structural components of cell membranes, increased. Changes in the proportion of lipid classes resulted in shifts in the FA composition of total lipids. It is suggested that the structural components of photosynthetic and cells membranes accumulate in the resting cells. Thereby, a rapid cell growth and an extensive development of the species under favorable environmental conditions is provided.     

Genetic bottlenecks and the hazardous game of population reduction in cell line based research

Established tumour cell lines are ubiquitous tools in research, but their representativity is often debated. One possible caveat is that many cell lines are derived from cells with genomic instability, potentially leading to genotype changes in vitro. We applied SNP-array analysis to an established tumour cell line (WiT49). Even though WiT49 exhibited chromosome segregation errors in 30% of cell divisions, only a single chromosome segment exhibited a shift in copy number after 20 population doublings in culture. In contrast, sub-populations derived from single cells expanded for an equal number of population doublings showed on average 5.8 and 8.9 altered segments compared to the original culture and to each other, respectively. Most copy number variants differentiating these single cell clones corresponded to pre-existing variations in the original culture. Furthermore, no sub-clonal variation was detected in any of the populations derived from single cells. This indicates that genetic bottlenecks resulting from population reduction poses a higher threat to genetic representativity than prolonged culture per se, even in cell lines with a high rate of genomic instability. Genetic bottlenecks should therefore be considered a potential caveat in all studies involving sub-cloning, transfection and other conditions leading to a temporary reduction in cell number.     

The influence of cell size on the growth rate of Thalassiosira weissflogii

Growth rate and average cell volume were measured throughoutauxospore formation in two populations of Thalassiosira weissflogii(Hustedt). In both cases, the entire population shifted fromrelatively small (800 µm³) to large cells (2800 µm³)over a 5 day interval. This shift was accompanied by a dramaticincrease in the average growth rate of the populations from1.6 to 3.4 doublings/day.     

Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix

Objectives: Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. Materials and methods: Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi‐dishes with 8‐μm pore transwell inserts. Each cell population was characterized in terms of renewal capability, specific marker expression and differentiation potential. Cryopreservation was performed on sieved cells only. Results: Cells from both areas expressed MSC and pluripotential specific markers and were able to differentiate into mesodermic and ectodermic lineages. The sieving procedure yielded two relatively homogeneous subpopulations with comparable characteristics. Surprisingly, after sieving, large intervascular and small perivascular cells were the most rapidly replicating cells [20.53 and 19.49 cell population doublings (PD) after 31 days respectively] and also showed higher fibroblast colony forming unit frequency. Unsieved cell populations were used as controls, and showed PD of 9.42(intervascular cells) and 8.54 (perivascular cells) after 31 days. Conclusions: Here, cells from UCM represented an intermediate stage between pluripotent embryonic and adult stem cells. Size‐sieving can be used to isolate more rapidly proliferating cell populations.     

Expansion and redifferentiation of adult human pancreatic islet cells

Beta-cell replacement represents the ultimate cure for type 1 diabetes, however it is limited by availability of organ donors. Adult human islets are difficult to propagate in culture, and efforts to expand them result in dedifferentiation. Here we describe conditions for expansion of adult human islet cells, as well as a way for their redifferentiation. Most cells in islets isolated from human pancreata were induced to replicate within the first week of culture in expansion medium. Cells were propagated for 16 population doublings, without a change in replication rate or noticeable cell mortality, representing an expansion of over 65,000-fold. Replication was accompanied by a decrease in expression of key beta-cell genes. Shift of the cells to differentiation medium containing betacellulin resulted in redifferentiation, as manifested by restoration of beta-cell gene expression and insulin content. These methods may allow transplantation of functional islet cells from single donors into multiple recipients.     

Effects of chloramphenicol on the physiology and fine structure ofTetrahymena pyriformis GL: Correlation between diminishing inner mitochondrial membrane and cell doubling

Summary A time-dependent loss of tubular infolding of the inner mitochondrial membrane was reported recently as an effect of the cytostatic drug, methotrexate (MTX), onTetrahymena (Nilsson 1983); this finding was interpreted as an inhibition of mitochondrial protein synthesis. In the present study, the cells were exposed to chloramphenicol (CAP), an inhibitor of mitochondrial translation, at the same concentrations (1–25 mM) as MTX; the question asked was whether the two drugs acted similarly. CAP affected cell proliferation by causing a dose-dependent prolongation of the generation time, but at 10–25 mM permitted only a limited number of cell doublings, whereas 1 mM MTX inhibits growth after 5 cell doublings. With CAP the inner mitochondrial membrane diminished gradually in accordance with the number of cell doublings at 10–25 mM, but in 2 mM CAP, for example, some tubular infoldings were still present after 17 cell doublings. The gradual loss of the inner mitochondrial membrane correlated with a gradual decrease in the cellular ATP content, irrespective of the concentration of the drug but dependent on the progress of the cells through their first cycle when exposed to the drug; in cells which continued to proliferate, the ATP content remained at a value corresponding to 80% of the control value. With respect to cell proliferation, the two drugs act differently. CAP is less toxic than MTX, reflected in a 10 times shorter recovery time for cell proliferation after removal of CAP. Hence, although the structural manifestation of the action of the two drugs on mitochondria is identical, their target site may differ.     

Genotype and the cryopreservation process affect the levels of aneuploidy and chromosome breakage in cultured human fibroblasts

Spontaneous micronucleus frequencies were measured in 11 human fibroblast strains, with early-passage cells that had never been frozen and with cells of comparable population doublings that had been cryopreserved in liquid nitrogen. The mean micronucleus frequency of the 11 strains increased from 14.0 +/- 0.7 to 20.4 +/- 1.8/1250 mononucleated cells (P = 0.002) after the freeze-thaw process. The nature of this increase in micronucleus frequency was examined using an immunodetection assay for the in situ identification of kinetochores in micronuclei. The increase in micronucleus frequency occurred primarily in the kinetochore-positive fraction, which is indicative of aneuploidy, but also by an increase in chromosome breakage in several strains. The findings were reproducible in repeat biopsies from two donors. Plating efficiencies of the 11 strains were studied during 1-9 and 10-20 population doublings from primary outgrowth, before freezing and again after freeze-thaw. The mean plating efficiency of frozen-thawed cells before nine doublings was significantly lower than that of cells of similar ages that had never been frozen (P = 0.004). The four strains that had a greater than 25% decrease in plating efficiency post freeze-thaw also had the highest aneuploidy index post freeze-thaw, suggesting that chromosomal imbalance contributes to the observed reduction in growth. We conclude that the genotype and culture manipulations of a fibroblast strain influence the outcome of the micronucleus assay.     

Alpha-fetoprotein-mediated uptake of fatty acids by human T lymphocytes.

The binding to resting and activated T lymphocytes of two radiolabelled fatty acids (oleic and arachidonic) was studied in the presence or in the absence of alpha-fetoprotein (AFP) as carrier protein. Fatty acid binding by resting and activated T lymphocytes was determined at 4 degrees C as a function of the concentration of fatty acid and AFP. Under the conditions employed, the following observations were made: (1) in the presence of AFP, fatty acids (oleic and arachidonic acid) are bound to cells by a two-component pathway; one is a saturable process, evidenced when the fatty acid to AFP (FA/AFP) molar ratio was fixed at 1 and the concentration of the fatty acid and the protein varied from 0.1 to 3.2 microM, and the second is a nonsaturable function of FA/AFP molar ratio and was linearly related to the unbound fatty acid concentration in the medium over the entire range studied; (2) in the absence of AFP, the nonsaturable process appears to be the only component of fatty acid binding; 3) at all tested concentrations of free (unbound) fatty acid in the medium, net fatty acid binding by either resting or activated T cells was considerably greater in the presence than in the absence of AFP; (4) in the presence of AFP, fatty acid binding was much higher in activated T cells than in resting T cells, whereas in the absence of AFP, nonsignificant differences were observed between activated and resting T cells; and (5) the time course of fatty acid and AFP binding at 4 degrees C revealed that, at equilibrium, the number of fatty acid molecules bound to the cell was much greater than that of AFP suggesting an accelerated dissociation of the fatty acid upon interaction of the AFP-fatty acid complex with putative cell receptors. It is concluded to the existence of an AFP/AFP-receptor pathway that facilitates the binding of fatty acids to T lymphocytes, particularly upon their blast transformation. This pathway may fulfill the increased requirement for fatty acids characteristic of proliferating cells and may serve to regulate the endocytosis of fatty acids with modulatory effects on lymphocyte function and to protect cells from their cytotoxic potential when internalized in excess.     

Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.     

The Use of Hybrid Somatic Cells as an Approach to Mitochondrial Genetics in Animals

We have studied the fate of parental mitochondrial DNA (mtDNA) in hybrid somatic cells derived by Sendai virus-induced fusion of human cells and mouse or rat cells. Many hybrid cell strains were obtained which contained sequences from both human and rodent mtDNA after 40 to 60 population doublings. Some strains were subcloned and cultured further for up to 150 doublings; a large fraction of these strains contained both parental mtDNA sequences at that time.The relation between human and rodent mtDNA sequences was tested in some of the hybrid cell strains. In a high fraction of strains tested the human and rodent mtDNA sequences were linked to each other by what are most likely covalent bonds. This linkage may be described as "recombination" of mtDNA sequences from two different animals.     

Requirements for the stimulation of allogeneic T lymphocytes by acute non-lymphoblastic leukaemia cells

Summary Blast cell populations from 32 patients with acute non-lymphoblastic leukaemia of various morphological types have been examined for their ability to stimulate allogeneic T lymphocytes from normal donors in one-way mixed leucocyte culture (MLC). At the same time, these leukaemic cell populations were examined for the amounts of major histocompatibility complex Class I and Class II antigens they expressed, and their ability to release interleukin 1 (IL1) in culture both with and without stimulation by lipopolysaccharide. The abilities of the leukaemic cell populations to stimulate in MLC, and to produce IL1, were found to be associated with the expression of morphological characteristics of monocytic differentiation, and correlated significantly. In contrast, no correlation was observed between the extent of Class I or Class II expression and MLC stimulatory ability. Many myeloblast populations of immature phenotype were unable to stimulate allogeneic T cells despite their strong expression of these antigens. This lack of stimulatory ability was not overcome by the addition of exogenous IL1. We therefore conclude that the correlation between the production of IL1 and MLC stimulatory ability does not necessarily imply a cause/effect relationship, and that the interaction between allo-antigen and the T cell receptor together with a supply of lymphokine co-stimulator is not sufficient to activate resting T lymphocytes. The failure of some Class I and II antigen positive leukaemic blasts to stimulate in MLC even in the presence of exogenous IL1 may be due to the lack of other differentiation-associated cell surface molecules necessary for stable cell-cell interaction.     

Conditional immortalization of human B cells by CD40 ligation

It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function.     

Stage-related, cell-mediated cytotoxicity with effector cell analysis and computation of cytotoxic activity of T cells and non-T cells.

Cell-mediated cytotoxicity (CMC) by lymphocytes from patients with oral squamous cell carcinoma, as well as from nonmalignant control donors, was tested by a microcytotoxicity assay against a cultured cell line derived from an oral squamous cell carcinoma. In terms of the degree of CMC, stage-related cytotoxicity was observed. A further study of the effector cell analysis revealed that the cytotoxic effects of lymphocytes from both patients and control donors were largely attributable to non-T cells. However, the effectors were also stage related, and in early stage patients, T-cell-mediated cytotoxicity was super-imposed on non-T-cell-mediated cytotoxicity. This conclusion was further supported by the evidence of elevated cytotoxic activity of T cells in early stage patients, which was computed from simultaneous equations proposed in the present paper for computing the cytotoxic activity of T cells (CTA_T) and non-T cells (CTA_non-T).     

Human fibroblasts from cancer patients: lifespan and transformed phenotype in vitro and role of mesenchyme in vivo

Human fibroblasts cultured in vitro can exhibit a different potential number of population doublings. In normal donors, the average number of population doublings is inversely related to the donor's age. An increased growth potential was detected in skin fibroblasts from breast cancer patients, independently of the donor's age. These cells responded in an abnormal way to 3 biological parameters: (1) colony formation in semisolid medium; (2) colony formation on monolayers of normal human epithelial cells; and (3) increase of saturation densities in overcrowded culture conditions. A third of these cultures, as well as skin fibroblasts from other cancer patients, at the plateau phase of growth exhibited a significant percentage of cells still synthesizing DNA. Exposure to overcrowding, limited in time, caused the selection of a cell subset which displayed new biological, biochemical and functional properties commonly found in transformed cells. The abnormal in vitro behavior of skin fibroblasts from breast cancer patients does not seem to be associated with the expression of oncofetal membrane markers (4F2, IL2 receptor) while the fibroblasts from patients with the adenomatosis of the colon and rectum (ACR) syndrome expressed the 4F2 antigen. This is the first time that the IL2 receptor is found on non-hematopoietic cells. Fibroblastic cells with abnormal characteristics, which may also present a decreased efficiency in organizing a primitive fibrin matrix, could represent in vivo an anarchistic milieu, favoring disturbed epithelial-stromal interactions and the emergence of the less structured tumor stromatic tissue.     

Stability of von Willebrand Factor secretion in different human endothelial hybrid cell lines

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. Thein vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.