Patients with myotonic dystrophy,a possible segmental progeroid syndrome,and duchenne muscular dystrophy have fibroblasts with normal limits for in vitro lifespan and growth characteristics

Myotonic dystrophy (MyD) has been suggested to be a segmental progeroid syndrome in man, as this syndrome has some clinical manifestations of premature aging. Fibroblasts from patients with other progeroid syndromes have been shown to have diminished in vitro lifespans or growth characteristics; therefore, it was of interest to study cellular senescence in fibroblasts from patients with MyD. Fibroblast cultures from patients with Duchenne muscular dystrophy (DMD) were used as additional controls, as premature aging is not associated with this genetic disorder. Primary skin fibroblast cultures obtained from patients with MyD or DMD and from age-sex matched controls were grown in DMEM plus 10% FBS. The in vitro lifespan was determined by either a 1:4 split ratio or with a constant initial inoculum of 1 × 10⁴ cells/cm², followed by determination of the final density at weekly intervals. Our results demonstrate that there is no difference in the limits of the in vitro lifespan for either the MyD or DMD fibroblast strains compared to the controls. Likewise, no difference could be detected in the growth characteristics of these cells. The only observable difference was that the pooled age-matched controls and MyD cultures had a shorter in vitro lifespan than the DMD group and their pooled controls, a finding expected because of the age of the patients in each group. Unlike the other progeroid syndromes, MyD fibroblasts have normal limits for in vitro lifespan. MyD is probably not closely related to the other premature aging syndromes, although there is an increasing phenotypic expression as a function of age.     

Gingival fibroblasts “in vitro” and Down's Syndrome

Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of cromosome 21 could alter growth regulation and biochemical mechanisms, so we examined quantitatively some DS phenotypical aspects to detect possible differences from those of controls. The growth properties of gingival fibroblast cultures from DS patients were more elevated than their ND age-matched controls. There were no differences in plasma membrane polarization and in neutral endopeptidase activity. The succinate-cytochrome C reductase activity decreases in DS fibroblasts compared with ND. Our results outline the difficulties to inusing fibroblast cultures as an in vitro system to study premature ageing Down's Syndrome.     

Aging dependent nucleolar and chromatin changes in cultivated fibroblasts

Adult fibroblasts have already at low population doubling level (PDL) a significant percentage of cells with modified chromatin as compared to embryonic cells where altered chromatin is seen only at high PDL. In fibroblast populations from a patient with Werner's syndrome, the percentage of cells with both altered chromatin and nucleoli was much higher than in the control cultures from normal donors. The data show for the first time nucleoprotein changes in cells from donors with an aging syndrome and reinforce the concept that serial replication of fibroblasts in vitro causes lesions identical to those of aging in vivo.     

Actin and tubulin in normal and cystic fibrosis fibroblasts

Actin and tubulin contents of early passage, confluent human fibroblast cultures have been determined. Actin comprised 5.87 ± 0.81% of the total protein of IMR-90 fibroblasts which was not significantly different than the actin contents of two cystic fibrosis fibroblast cultures GM0142 and GM1348 (5.64 ± 0.90% of total protein). However, a significant difference between the amount of tubulin in IMR-90 fibroblasts (7.17 ± 0.25% of total protein) and the amount of tubulin in cystic fibrosis fibroblasts (4.51 ± 0.64% of total protein) was found.     

A comparative study on efficiency of adult fibroblasts and amniotic fluid-derived stem cells as donor cells for production of hand-made cloned buffalo (Bubalus bubalis) embryos

The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical “S” shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10–15 and 8–12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.     

Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines

Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an ‘S’ shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.     

Growth of cultured cells from patients with ataxia-telangiectasia

Fibroblast cultures derived from skin biopsies of patients with ataxia-telangiectasia had doubling times (mean of five lines: 29.9 ± 0.6 hours) significantly longer than normal controls (means of ten lines: 22.4 ± 0.5 hours).     

In vitro studies of age-associated diseases.

We have carried out studies on cultured human fibroblasts in an attempt to trace the origins of age-dependent disorders to the cellular and molecular levels. Three interrelated areas are discussed. First, skin donors with diabetes mellitus (a disease complex that features inappropriate hyperglycemia) produce cultured fibroblasts with a moderate reduction in growth capacity, while two inherited disorders of inappropriate hyperglycemia and premature aging, progeria and Werner syndrome, yield fibroblast cultures with more severely impaired growth capacity. Second, there is a decreased response of progeria level and donor age; evidence is presented that this defective hormone responsiveness in aging cells may reside at the hormone receptor on the surface membrane, the cyclic AMP system, the intracellular enzymatic machinery, or all of these sites. Third, tissue factor, a procoagulant that activates the extrinsic clotting mechanism, is more abundant in cells from the premature aging syndromes of progeria and Werner syndrome. Fibroblast aging in vitro may help to explain various concomitants of normal aging and diabetes mellitus including cell dropout, impairment of hormone responsiveness, and increased atherothrombosis.     

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.     

Age-related decline in histone H1 fraction in human diploid fibroblast cultures

The age-related increase in cell volume and nuclear size of cultured human diploid fibroblasts reflected the accumulation of proteins in cytoplasm and nuclei of growth-retarded fibroblasts.Determination of the amount of nuclear proteins, which were fractionated into 0.15 M NaCl-soluble proteins, 0.4 N H₂SO₄-extractable proteins and residual acidic proteins, indicated that age-related increase in nuclear proteins was due mainly to the accumulation of residual acidic proteins.However, electrophoretic fractionation of histones from various passages of fibroblast cultures on acid urea polyacrylamide gel revealed that the relative amount of H1 fraction decreased with in vitro aging. This was further confirmed by mixing experiments examining the distribution of radioactivity of the histones from cell mixtures of young and senescent cultures labeled with [³H]lysine or [¹⁴C]lysine.A pulse label and chase experiment indicated that the observed decrease in the amount of histone H1 was mainly due to decrease in synthesis of histone H1 in senescent human fibroblast cultures.     

Growth of cultured cells from patients with Fanconi anemia

Fibroblast cultures derived from skin biopsies of patients with Fanconi anemia had doubling times (mean of five lines: 30.3 ± 0.2 hours) significantly longer than randomly selected normal controls (mean of nine lines: 22.9 ± 0.4 hours). Control cultures grew more slowly in the enriched media RPMI 1640 and McCoy's 5A than in MEM, while a culture from a patient with Fanconi anemia grew more slowly only in McCoy's 5A. Differences in growth characteristics between Fanconi anemia and normal cell cultures may be useful in analyzing the metabolic error determined by the Fanconi anemia gene.     

The mechanism of regulation of fibroblastic cell replication. I. Properties of the system

Sixty to eighty per cent of the cells in a culture of human diploid fibroblasts may be stimulated from the state of density dependent inhibition of replication to active DNA synthesis and division. The maximum response is effected by 50% serum within the pH range 7.2–8.0. The proportion of cells responding depends on the concentration of serum protein in the medium which may be effectively substituted by crystalling serum albumin. There is a differential sensitivity to the stimulus of cells in the densely packed centers of whorls and in the less dense areas between the whorls. The cell response is parasynchronous and the median durations of the various phases of the cell cycle are: G₁I 6 β ?æ^® ¿ ∞ 8 hours, G₂ = 6 hours and doubling time = 30 hours. The stimulatory effect of fresh medium is lost during contact with dense cultures so that it has only 50% of its initial capacity after 14 hours. It can be restored by dialysis against serum-free medium. The stimulus must be applied for at least ten hours to be effective in inducing DNA synthesis. During the latter half of ten hour induction period subsequent DNA synthesis becomes exquisitely sensitive to actinomycin D. After this time an increasing number of cells become irreversibly committed to replicate. The data are interpreted to indicate that during contact with serum proteins (including albumin) changes in the cell surface, if continued long enough, trigger a mechanism which involves the synthesis of a unique RNA species during the fifth to tenth hours. After this RNA has been synthesized the cells are then committed to DNA synthesis.     

Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields

Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical‐free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5–100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10⁵–1.4 ± 0.2 × 10⁵ cell/mL range. Our results suggest that IDPEFs may prove useful as a non‐chemical method for fibroblast density control in human wound healing. Biotechnol. Bioeng. 2013; 110: 1759–1768. © 2013 Wiley Periodicals, Inc.     

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.     

Donor age reflects the replicative lifespan of human fibroblasts in culture

Human fibroblasts, which have a finite lifespan in cultures, have been widely used as a model system for cellular aging, and frequently used as one model of human aging. But whether cellular aging contributes to organismal aging has been controversial. To reinvestigate this question, we cultured human fibroblasts from the skin of one individual volunteer collected at different ages. Over a period of 27 years (donor age 36 years to 62 years), we obtained skin cells four times at appropriate intervals, and established eight fibroblast lines. These human fibroblasts have presented evidence for a correlation between donor age and proliferative lifespan in vitro . This result parallels the fact that telomeric DNA size cultured fibroblasts decrease with the increase in donor age. These cell lines had a normal diploid human chromosome constitution and will be useful in studies of human biology including aging.     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Turnover of ribosomal RNA in mouse fibroblasts (3T3) in culture.

Growing and confluent cultures of mouse fibroblasts were labeled with ³H-uridine and chased with an excess of nonradioactive uridine to investigate the turnover of ribosomal RNA. Growing cultures did not turn over their 18S and 28S ribosomal RNA; however, confluent cultures did show ribosomal RNA (rRNA) turnover. If the cells were labeled while growing, and chased when confluent, 18S RNA displayed a two-component decay curve, while 28S RNA showed only single-component decay, similar in lifetime to the first component of the 18S RNA decay curve. If the cells were labeled while confluent, both the 18S and 28S RNA showed single-component decay curves with a lifetime possibly only slightly longer than the lifetime of the first component of the 18S RNA and the single component of the 28S RNA of the cultures labeled while growing.     

Variegated translocation mosaicism in human skin fibroblast cultures.

The term "variegated translocation mosaicism" is used to describe the repeated occurrence, within cultures of human skin fibroblasts, of a multiplicity of chromosomal rearrangements. With respect to the frequencies of such cytogenetically aberrant clones we found that they (1) were not detectable in routine diagnostic skin fibroblast cultures from 29 subjects with a wide variety of indications for biopsy; (2) were not detectable during in vitro aging of diploid strains with four normal individuals; (3) could be detected after rescue from bacterial contamination of a culture from an otherwise normal diploid male; (4) occurred with high frequencies in independent cultures from another apparently normal subject; (5) occurred with high frequencies in multiple biopsies obtained at autopsy from a patient with Werner's syndrome who died of sepsis; (6) were of pseudodiploid nature; and (7) involved a different spectrum of chromosomes in different individuals. A consistent association with mycoplasma contamination could not be made.     

Accelerated methylation of ribosomal RNA genes during the cellular senescence of Werner syndrome fibroblasts.

Ribosomal DNA (rDNA) metabolism has been implicated in cellular and organismal aging. The role of rDNA in premature and normal human aging was investigated by measuring rDNA gene copy number, the level of rDNA methylation, and rRNA expression during the in vitro senescence of primary fibroblasts from normal (young and old) donors and from Werner syndrome (WS) patients. In comparison to their normal counterparts, WS fibroblasts grew slowly and reached senescence after fewer doublings. The rDNA copy number did not change significantly throughout the life span of both normal and WS fibroblasts. However, in senescent WS and normal old fibroblasts, we detected rDNA species with unusually slow electrophoretic mobility. Cellular aging in Saccharomyces cerevisiae is accompanied by the formation and accumulation of rDNA circles. Our analysis revealed that the rDNA species observed in this study were longer, linear rDNA molecules attributable to the inhibition of ECO:RI cleavage by methylation. Furthermore, isoschizomeric restriction analysis confirmed that in vitro senescence of fibroblasts is accompanied by significant increases in cytosine methylation within rDNA genes. This increased methylation is maximal during the abbreviated life span of WS fibroblasts. Despite increased methylation of rDNA in senescent cells, the steady-state levels of 28S rRNA remained constant over the life span of both normal and WS fibroblasts.     

[14C]Acetate incorporation by cultured normal,familial hypercholesterolemia and Down's syndrome fibroblasts

Fibroblasts from patients with homozygous familial hypercholesterolemia (FH), a disease characterized by accelerated atherogenesis, are known to lack functional low-density-lipoprotein receptors, which ultimately results in increased cholesterol biosynthesis in the cultured cells. [¹⁴C]Acetate incorporation in these cells was compared to that of normal fibroblasts and to fibroblasts from patients with Down's syndrome, a disease in which atherosclerosis is rare. Total [¹⁴C]acetate incorporation did not differ significantly between normal and Down's fibroblasts, nor did its partitioning into the hexane-extractable and aqueous fractions of the cell hydrolysates. [¹⁴C]Acetate incorporation was much greater in FH cells in both the aqueous and hexane-extractable fractions. Preincubation in fetal bovine serum increased acetate incorporation only by FH cells, while 50 μg low-density lipoprotein/ml medium depressed acetate incorporation in all three groups. A C₂₇ sterol, identified by gas chromatography-mass spectrometry as a probable isomer of cholesterol, was present in small amounts in FH fibroblasts, but was not detectable in the normal or Down's cells. The absolute amounts of [¹⁴C]acetate incorporated into the non-sterol lipids were greater in the FH fibroblasts, indicating that these cells may have to synthesize, in addition to cholesterol, other required cellular lipids which are delivered to the normal cells by low-density lipoproteins.