Late Passage Human Fibroblasts Research Articles

Late Passage Human Fibroblasts Induced to Pluripotency Are Capable of Directed Neuronal Differentiation

It is possible to generate induced pluripotent stem (iPS) cells from mouse and human somatic cells by ectopic expression of defined sets of transcription factors. However, the recommendation that somatic cells should be utilized at early passages for induced reprogramming limits their therapeutic application. Here we report successful reprogramming of human fibroblasts after more than 20 passages in vitro, to a pluripotent state with four transcription factors: Oct4, Sox2, Klf4, and c-Myc. The late passage-derived human iPS cells resemble human embryonic stem cells in morphology, cell surface antigens, pluripotent gene expression profiles, and epigenetic states. Moreover, these iPS cells differentiate into cell types representative of the three germ layers in teratomas in vivo, and directed neuronal differentiation in vitro.

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20–32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37°C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.

Age-related Decline in Chaperone-mediated Autophagy

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.

Differential Regulation of Collagenase and Stromelysin mRNA in Late Passage Cultures of Human Fibroblasts

Nontransformed human fibroblast cell cultures have been extensively studied as anin vitromodel for cellular senescence. Recently there has been considerable interest in using the human fibroblast in the identification of genes relevant to the process of replicative senescence. We demonstrated that in comparison with early passage cultures the expression of collagenase and stromelysin mRNAs and proteins was increased >8× in late passage cultures of human fibroblasts and, in addition, expression of Il-1α, a cytokine that regulates collagenase and stromelysin expression, was also significantly increased in late passage cell cultures. These findings suggested the hypothesis that constitutive Il-1α expression in late passage cells may coordinately regulate the age-associated increase in the expression of collagenase and stromelysin. To test this hypothesis we examined the effects of long-term Il-1α treatment, serum starvation, and cycloheximide inhibition on collagenase and stromelysin mRNA levels in early and late passage human fibroblast cell cultures. Here we report that in late passage cell cultures, collagenase and stromelysin mRNAs respond differentially to Il-1α, serum starvation, and cycloheximide addition. Continuous exposure to Il-1α reduced the half-life of stromelysin mRNA but had little effect on the half-life of collagenase mRNA. In contrast to stromelysin, the collagenase mRNA level is dependent on serum factors. Collagenase is induced during recovery from cycloheximide inhibition, but stromelysin expression is not affected. These results establish that collagenase and stromelysin mRNAs are differentially regulated in both early and late passage human fibroblasts and suggest that the mechanisms responsible for the age-associated increase in the two mRNAs are different. In addition, these studies support the conclusion that continuous long-term exposure to Il-1α, a condition that is characteristic of late passage cells, is not the factor responsible for the high levels of collagenase expression, but may be critical for stromelysin expression.

Differential growth state-dependent regulation of plasminogen activator inhibitor type-1 expression in senescent IMR-90 human diploid fibroblasts.

The type-1 inhibitor of plasminogen activator (PAI-1) regulates pericellular proteolytic activity functioning, thereby to control matrix integrity, cell growth, and morphology. Subconfluent late-passage IMR-90 human fibroblasts and normal rat kidney (NRK) cells, both at the stage of replicative senescence accumulated 15- to 30-fold more undersurface PAI-1 protein compared to early-passage, actively-proliferating, cultures. Senescence-associated elevations in PAI-1 expression by IMR-90 cells reflected corresponding 11-fold increases in the 3.0- and 2.2-kb PAI-1 mRNA species. The 2.2-kb transcript exhibited a greater age-dependent increase (7.2-fold) compared to the 3.0-kb mRNA (3.7-fold). Since PAI-1 expression is coupled to growth activation in serum-deprived cultures (Ryan and Higgins, 1993, J. Cell. Physiol., 155:376-384), it was important to determine if PAI-1 gene regulation was altered as a function of cellular aging. In contrast to early-passage cultures, senescent IMR-90 fibroblasts did not down-regulate either PAI-1 protein expression or steady-state levels of PAI-1 mRNA transcripts upon serum-deprivation. Late-passage human fibroblasts at their proliferative end-stage, thus, appear to regulate PAI-1 mRNA levels through different mechanisms than do young, actively-proliferating, cells. PAI-1 overexpression during in vitro cellular aging, therefore, may contribute to the acquisition of specific senescence-associated phenotypic traits (e.g., enlarged cell morphology; increased adhesivity) by altering the pericellular proteolytic balance influencing, in turn, the formation or stability of cell-to-substrate attachment complexes.

Expression of 72-kDa Gelatinase and TIMP-2 in Early and Late Passage Human Fibroblasts

The expression and regulation of 72-kDa gelatinase and TIMP-2 were examined in cultures of early and late passage human fibroblasts. In contrast to collagenase expression, which was low in early passage cells and highly expressed in late passage fibroblasts, 72-kDa gelatinase mRNA expression was enhanced only slightly in late passage cultures and 72-kDa gelatinase protein expression was similar in early and late passage cultures. In contrast to published reports of decreased TIMP-1 expression, TIMP-2 mRNA and protein were increased in late passage cells. Exposure to II-1α increased the steady-state level of 72-kDa gelatinase mRNA by 3X in early passage cells but had no effect on late passage cells. Although II-1α had no significant effect on TIMP-2 mRNA or on expression of 72-kDa gelatinase, in either early or late passage cells, II-1α increased the level of a TIMP-2-72-kDa gelatinase complex. Using monoclonal antibodies to TIMP-2 and to 72-kDa gelatinase we detected two forms of TIMP-2. One form was complexed to 72-kDa gelatinase and migrated with an apparent molecular weight of 72 kDa and the other migrated at the expected molecular weight of 21 kDa. Autoradiography in conjunction with Western analysis confirmed that in the late passage cell medium the newly synthesized 72-kDa gelatinase-TIMP-2 complex was increased even though the expression of 72-kDa gelatinase did not change. The present results establish that the regulation of 72-kDa gelatinase and TIMP-2 in early and late passage cultures of human fibroblasts are different from collagenase and TIMP-1 regulation. Further, they establish that in late passage cultures the activity of 72-kDa gelatinase may be regulated through the formation of a denaturation-resistant complex.

Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts.

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF beta-receptors were found to be enhanced in old donor cell strains, whereas the PDGF alpha-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts.

Modulation of mRNA levels during human keratinocyte differentiation.

Cultures of human keratinocytes provide an excellent model system in which to study differentiation. Using the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and calcium, two agents known to induce keratinocyte differentiation in vitro, we examined the expression of the genes encoding c-fos, c-myc, and c-jun; involucrin, a protein precursor of the keratinocyte cornified envelope; and L-7, a ribosomal protein. Overall, at the doses studied, TPA induced a more rapid and profound differentiation than did calcium, as evaluated by culture morphology and northern blot analysis. Our studies showed a constant low level of c-fos and c-jun expression in unstimulated cells with no significant change after addition of either TPA or calcium except when transcript breakdown was inhibited by cycloheximide. The c-myc proto-oncogene, known to have a high constitutive expression in actively proliferating cells, was strongly downregulated by TPA, but calcium had no effect over a 32 hour period, consistent with the greater growth inhibition of TPA in this system. Involucrin was induced about ninefold by both TPA and calcium as early as 8 hours after stimulation, suggesting transcriptional regulation of this gene during differentiation. L-7, recently demonstrated to be downregulated in late passage human fibroblasts in an in vitro model of senescence, was also strongly downregulated by either TPA or calcium, consistent with an interrelationship between the basic cellular processes of aging and differentiation. These finding expand our knowledge of the differentiation process in human keratinocytes.

Protein variations associated with in vitro aging of human fibroblasts and quantitative limits on the error catastrophe hypothesis.

Two-dimensional electrophoresis was used to examine protein alterations associated with in vitro cellular aging. Patterns of cellular proteins from early and late passage human fibroblasts of two strains (normal and trisomy 21) were analyzed in silver-stained gels and autoradiograms with computerized microdensitometry. Four proteins were significantly altered in density in both cell strains. In late passage cells, these proteins were from 6 to 66% the density in early passage cells. The error catastrophe hypothesis predicts that random amino acid substitutions accumulate with cellular aging. No new proteins or satellite spots due to such substitutions, however, were detected in late passage cells. An upper bound of 2.5% was set by high resolution densitometry for the fraction of abnormal protein that could be present but undetected by these methods.

Decreased sensitivity of old and progeric human fibroblasts to a preparation of factors with insulinlike activity.

To determine whether old cells have a reduced response to a preparation of factors from human plasma with insulinlike activity (ILA), we analyzed the response to ILA of early and late passage human fibroblasts from young, old, and progeric donors in the acute stimulation of [3H]2-deoxy-D-glucose (2dG) uptake and the delayed stimulation of [3H]thymidine (TdR) incorporation into DNA. The ILA concentration required to produce equivalent, relative stimulation of TdR incorporation was increased two- to three-fold in late passage cells and cells from old and progeric donors (P less than 0.01). 50 and 95% of maximal stimulation (ILA50, ILA95) was achieved by 0.26 +/- 0.07 and 1.38 +/- 0.13 ng insulin equivalents/ml (mean +/- SD) respectively, in cells from young adults at early passage. Corresponding values were 0.54 +/- 0.05 and 2.90 +/- 0.25 in cells from old donors; greater than 0.9 +/- 0.1 and greater than 3.1 +/- 0.1 in cells from a 9-yr-old progeric donor; and 0.4 +/- 0.05 and 1.1 +/- 0.04 in cells from normal children (9-13 yr). For two cell strains from young adults, ILA50 and ILA95 were 0.30 +/- 0.02 and 1.0 +/- 0.3 ng eq/ml at 30% of their in vitro lifespan completed (%LC) and these values increased at rates of 0.005 ng eq/ml per %LC and 0.04 ng eq/ml per %LC, respectively. The mean stimulation of 2dG uptake ratio (ILA/control) decreased from early to late passage from 2.1 +/- 0.6 to 1.3 +/- 0.1 in young adult donors (P less than 0.05), but there were no significant differences between young and old donors at either early or late passage. The mean stimulation ratio in progeric cells (1.2 +/- 0.2) did not change with in vitro passage, but was significantly lower than that of age-matched normal cells (2.1 +/- 0.8, P less than 0.001). In progeria cells, the reduced stimulation of 2dG uptake upon addition of ILA was due to an increased basal rate of uptake (0.19 +/- 0.01 pmol [3H]2dG/min per mg protein vs. 0.13 +/- 0.01 in age-matched normal cells), and not to a decline in the maximal rate of uptake (0.26 +/- 0.01 vs. 0.27 +/- 0.02, respectively). Similar results were found for in vitro aging in cells from an old donor.

Thermal denaturation of DNA and chromatin of early and late passage human fibroblasts.

Studies of DNA and chromatin isolated from early, mid and late passage human fibroblasts showed that the mean temperature of denaturation (Tm) did not change with increasing generations in culture. An increase in the Tm of chromatin when studied under similar conditions of low ionic strength is a well defined characteristic of aging in vivo. These observations suggest there may be fundamental differences in the mechanisms ofaging in <i>in vivo</i> non-proliferating compared to <i>in vitro</i> proliferating systems.