Cell surface changes accompanying aging in human diploid fibroblasts : II. Two types of age-related changes revealed by concanavalin A-mediated red blood cell adsorption

Age-related changes in cell surfaces of human diploid fibroblasts (TIG-1) were investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. When ConA-coated RBCs were adsorbed to fibroblasts (RBC coating method), the amount of RBCs adsorbed per mg of fibroblast protein increased continuously from the early phases of cell passage up through cell senescence. On the other hand, when RBCs were adsorbed to ConA-coated fibroblasts (fibroblast coating method), RBC adsorption did not occur throughout phase II and increased with the advance of phase III. [³H]ConA binding to fibroblasts, however, did not change with aging to the extent that could explain the observed changes in RBC adsorption. These age-related characteristics in RBC adsorption and [³H]ConA binding were also observed for WI38 and IMR-90 cells. In addition, SV40- and ⁶⁰Co-transformed WI38 cells showed a close resemblance in their RBC adsorption capacity to early phase III cells.RBC adsorption with both the RBC and fibroblast coating methods was not a function of cell cycle phase and time spent in culture (metabolic time). Co-culturing of young cells with old or transformed cells did not affect the RBC adsorption capacity of respective cells. These results suggest that RBC adsorption with the RBC and fibroblast coating methods may represent cell surface markers for division age and senescence of aging human diploid cells.     

Cell surface and clonal proliferative property of aging human diploid fibroblasts

We have described previously how concanavalin A (conA)-coated red blood cell (RBC) adsorption to human diploid fibroblasts could serve as a marker of the in vitro aging of these cells. Since the heterogeneity in RBC adsorption and the proliferative property of young and old cell populations was observed, the correlation of this cell surface property with the proliferative behavior of individual clones was examined as a function of cell age. The RBC adsorption capacity of cells in both large and small colonies changed with the aging of the parent cell populations; the cells from early passage populations did not adsorb RBCs, but those from late passage populations adsorbed them well. Thus, the amount of RBC adsorption was not a function of colony size, but was related to the age of the culture.     

A new cell surface marker of aging in human diploid fibroblasts

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continuously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity di not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.     

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.     

Repair of X-ray-induced DNA damage in aging human diploid cells

Human diploid cells cultured in vitro provide an excellent model system for the study of aging. In this study, we examined the formation and rejoining of DNA single-strand breaks (SSBs) induced by X-rays in human lung diploid fibroblasts during senescence, by using a modified alkaline elution method. For detecting the formation and rejoining of DNA SSBs, conventional [¹⁴C]thymidine (TdR)-labeling and fluorometric methods were applied to dividing cells and to the whole cell population including non-dividing and slowly-dividing cells, respectively. We did not find any significant differences in the rejoining ability of X-ray-induced SSBs in human diploid cells at almost all population doubling levels, although only in terminally senescent cells the rejoining of SSBs seems to proceed more slowly. However, it was observed that the alkaline elution of DNA from unirradiated and X-irradiated cells seems to become faster with increasing in population doubling number, although there were no remarkable differences in the elution rates of DNA as measured by the [¹⁴C]TdR-labeling method and those measured by the fluorometric method. These results seem to suggest that the molecular size of DNA in human diploid cells in culture decreases with aging.     

X-ray-induced mutation to 6-thioguanine resistance in cultured human diploid fibroblasts

X-ray induced mutation to 6-thioguanine (6TG)-resistance was studied in early passage cultures of human diploid fibroblasts.The appearance of phenotypic induced mutants in irradiated cell populations was linearly related to the number of post-irradiation cell doublings and to the duration of the growth period prior to mutant selection; the maximum yield of X-ray induced mutants was observed when cells surviving radiation had completed 3–4 doublings (6–7 days growth_in non-selective medium.The maximum induced mutation frequency was linearly related to X-ray dose and the mutation rate was estimated to be 3.1 · 10^?7 mutations per viable cell per rad.The data obtained for X-ray induced mutations in cultured human diploid fibroblasts were compared with (a) similar experimental data obtained with established cell cultures and (b) theoretical predictions of X-ray mutation rates in human germ cells.     

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time. In particular we analysed functional responses to hormonal stimulation with lysophosphatidic acid (LPA), a signalling molecule occurring in blood plasma, with the Ca²⁺ sensor Fluo-4. Additionally, we developed an approach for analysing the Ca²⁺ responses of RBCs that allowed the quantitative characterization of single-cell signals. In RBCs, the LPA-induced Ca²⁺ influx showed substantial diversity in both kinetics and amplitude. Also the age-classification was determined for each particular RBC and consecutively analysed. While reticulocytes lack a Ca²⁺ response to LPA stimulation, old RBCs approaching clearance generated robust LPA-induced signals, which still displayed broad heterogeneity. Observing phospatidylserine exposure as an effector mechanism of intracellular Ca²⁺ revealed an even increased heterogeneity of RBC responses. The functional diversity of RBCs needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified heterogeneity in RBC responses is important for the basic understanding of RBC signalling and their contribution to numerous diseases, especially with respect to Ca²⁺ influx and the associated pro-thrombotic activity.     

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia ^1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens ⁴), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs ^{4, 5}. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions³. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers ^{6, 7}, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane ^{8, 9} and mask RBC surface antigens^{10, 11}.     

Standardization of Human Diploid Fibroblast Cultivation: Centrifugation Procedure

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

Electrical excitability and chemosensitivity of mouse neuroblastoma X mouse or human fibroblast hybrids

Intracellular microelectrode recording techniques were used to measure passive membrane properties, electrical excitability and chemosensitivity of mouse neuroblastoma cells and somatic cell hybrids formed between these cells and either L cells or human diploid fibroblasts. Different clones of the hybrid cells showed varying degrees of neuronal or fibroblastic membrane-differentiated function; a selection technique involving incubation of the cells with aminopterin gave quite homogeneous non-dividing populations of cells within a given clone of the neuroblastoma x L cell hybrids. Despite relatively uniform chromosomal numbers within a given clone, the neuroblastoma x human fibroblast hybrids were morphologically and electrophysiologically heterogeneous. The possibility is considered that this may represent the effect of variable segregation of the human chromosomal complement.     

Multiple thymidine incorporation peaks in the S phase of synchronous human diploid fibroblasts

Synchronous populations of human diploid fibroblasts, CHO cells, and HeLa cells were obtained by mitotic selection. Human diploid fibroblasts were found to have three distinct peaks of thymidine incorporation during a 10–13 h S phase. This contrasts with CHO and HeLa which have one (or, for CHO, occasionally 2) peak(s) of thymidine incorporation during a 6–8 h S phase.     

A study of the effect of ultraviolet light on the division potential of human diploid fibroblasts

Culture and UV (254 nm) irradiation conditions that are suggested as appropriate for a study of the effect of UV on the limited in vitro lifespan of a normal human diploid fibroblast (HDF) strain are first described. An inoculation density at each subcultivation of 1.8 x 10(4) viable cells/cm-2 permits the decline in proliferative capacity to occur with kinetics similar to that observed using a 1:2 split and prevents cell overlap at the time of irradiation. Doses of 5 and 10 J/m2 have only a slight effect on initial growth rates and little or no effect on cell density achieved at confluence. With these conditions populations can be irradiated several times throughout the in vitro lifespan. No effect of UV on the limited division potential was observed. In the extreme, a population irradiated 14 times, once every second passage starting at P-18 with doses of 5 or 10 J/m2 had the same lifespan as controls, as measured by lifespan determinations and thymidine labeling index. Transformed cells were not detected in the multi-irradiated populations. Evidently no accumulation in the populations of damage induced by UV that affected life span, thymidine labeling index, growth rates or confluent cell densities occurred. No selection of a population with altered sensitivity occurred. An argument that genome hits may not be a prime reason for the limited proliferative capacity of HDF populations is presented.     

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell–cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm² areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.     

Proliferative and phenotypical characteristics of human adipose tissue–derived stem cells: comparison of Ficoll gradient centrifugation and red blood cell lysis buffer treatment purification methods

Background aimsAdult human subcutaneous adipose tissue harbors a multipotent stem cell population, the so-called human adipose tissue–derived mesenchymal stromal cells (AT-MSCs). These cells are able to differentiate in vitro into various cell types and possess immunomodulatory features. Yet procedures to obtain AT-MSCs can vary significantly. The two most extensively used AT-MSC purification techniques are (i) density gradient centrifugation using Ficoll and (ii) red blood cell (RBC) lysis buffer treatment of the stromal vascular fraction. In the context of potential clinical cell therapy, the stem cell yield after purification and upon consecutive passages, as well as the purity of the obtained cell population, are of utmost importance.MethodsWe investigated the expansion capacity and purity of AT-MSCs purified by both procedures immediately after isolation and upon consecutive passages. We also investigated possible purification-dependent differences in their expression of immune-inhibitory factors and cell adhesion molecules.ResultsWe found that RBC lysis buffer treatment is a more robust and easier method to purify AT-MSCs than density gradient fractionation. However, the resulting AT-MSC-RBC population contains a significantly higher number of CD34⁺ cells, particularly during the first passages after plating. From passage 4 onward, no significant differences could be observed between both populations with respect to the immunophenotype, expansion capacity and expression of immune inhibitory factors and cell adhesion molecules.ConclusionsOur data show that RBC lysis buffer treatment may be a good alternative to density fractionation, providing a faster, more robust and easier method to purify AT-MSCs with biologically preserved characteristics.     

Adsorption of LLCMK2 cell-grown Sendai virus onto human red blood cells and its release from the virus adsorbed cells

An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [3H]leucine in LLCMK2 cells and of human red blood cells (RBCs). The efficiency of viral release from the virus-bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C. When 7.8 x 10(2) virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C. Such RBC-bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell-grown Sendai virus (LLCMK2-Sendai) particles, but not released by treatment with hemagglutinin-neuraminidase protein (Sendai-gp) isolated from egg-grown Sendai virus.     

Fibroblast cell behavior on bound and adsorbed fibronectin onto hyaluronan and sulfated hyaluronan substrates

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.     

Further characterization of the effects of alpha-1-acid glycoprotein on the passage of human erythrocytes through micropores

Effects of human alpha-1-acid glycoprotein (AG) on the passage of human red blood cell(s) (RBC) through membrane filters with micropores were examined in vitro. RBCs, with a mean major diameter of 7.2 micron, that had been suspended at 1% in physiological phosphate-buffered saline (PBS), were filtered through membrane filters of various pore diameters under positive pressure. The percentages of cells that passed through the micropores and of cells hemolyzed during filtration were determined. RBCs suspended in PBS did not pass through micropores that had an average pore diameter of 3 micron; instead hemolysis took place. Neither temperature nor applied pressure affected cell passage; but when AG at 0.1 mg/ml or above was added to an RBC-suspension, it promoted cell passage through the 3 micron micropores and reduced the degree of hemolysis. The effects of AG were dose dependent up to a concentration of 0.5 mg/ml. The addition of AG to an RBC-suspension that contained 90% human serum had the same additive effects. Washing AG-treated RBCs with normal saline produced a marked decrease in cell passage through the 3 micron pores. Fluorescence antibody staining revealed that the exogenous AG was localized on the membrane surface of the RBCs. Our results suggest that the AG bound to the surface of the RBCs acts as a lubricant between the RBCs and the wall of the micropore; this would facilitate RBC-passage through the micropores.     

Interferon Production by Human Cells In Vitro

The relative capacity of several types of human cells and tissue to produce interferon was studied. Types of cells and tissue included were fibroblasts from embryos, foreskins, and biopsied skins; amnion cells; peripheral leukocytes; established lymphoid cell lines; established heteroploid cell lines; and chorioamniotic membrane. When Newcastle disease virus was used as the inducer, fibroblasts and amnion cells produced more interferon per 10⁶ cells than leukocytes, lymphoid cells, and heteroploid cells. Only minor variations in interferon-producing capacity were observed among fibroblasts from 36 persons. Culture passage level, cell concentration, and inducer were factors that significantly affected interferon production.     

Atrial natriuretic peptide: direct effects on human red blood cell dynamics.

The ability to deform is an important feature of red blood cells (RBCs) for performing their function of oxygen delivery. Little is known about the hormonal regulation of RBC deformability. Here we report that human atrial natriuretic peptide (ANP) acts directly on human RBCs leading to the elevation of local bending fluctuations of the cell membrane. These changes are accompanied by an increase in the filterability of RBCs. These ANP effects were mimicked by cyclic GMP analogues, suggesting modulation of local membrane bending fluctuations and RBC filterability via a cyclic GMP-dependent pathway. The effect of ANP on the mechanical properties of RBCs suggests that ANP may increase the passage red blood cells through capillaries resulting in an improved oxygen delivery to the tissues.     

Stimulation of collagen production in growth-arrested myocytes and fibroblasts in culture by growth factor(s) from platelets

Human fibroblasts, rabbit aortic fibroblasts and rabbit aortic myocytes were growth-arrested in serum-free media and stimulated with material spontaneously released from human platelets in vitro. The cells increased their collagen production 2- to 3-fold with increasing platelet factor concentration, whereas the cell mass, measured by total DNA, was unaffected. An increase in [³H]thymidine incorporation into DNA was noted, however. Thus, platelets can release factors stimulating collagen synthesis, without necessarily inducing concomitant cell division.