Populations Of Human Fibroblasts Research Articles

Algorithm for cellular reprogramming

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes.

Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence.

SummarySenescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.

A bioenergetic profile of non-transformed fibroblasts uncovers a link between death-resistance and enhanced spare respiratory capacity

Apoptosis-resistance and metabolic imbalances are prominent features of cancer cells. We have recently reported on populations of human fibroblasts that exhibit resistance to mitochondrial-mediated apoptosis, acquired as a result of a single genotoxic exposure. The objective of the present study was to investigate the intrinsic bioenergetic profile of the death-resistant cells, as compared to the clonogenic control cells. Therefore, we analyzed the basic bioenergetic parameters including oxygen consumption and extracellular acidification rates, coupling efficiency, and spare respiratory capacity. Our data demonstrate a strong correlation between enhanced spare respiratory capacity and death-resistance, which we postulate to be indicative of the earliest stages of carcinogenesis.

Induced reprogramming of human somatic cells into pluripotency: a new way how to generate pluripotent stem cells

It is now possible to create human cells that exhibit morphology and properties of human pluripotent embryonic stem (ES) cells by induced expression of only four factors. ES cells are remarkable cells. They can self-renew and maintain pluripotency indefinitely or can differentiate into any cellular types in the body. Therefore human ES (hES) cells provide a great promise as a source of cells use for treating various diseases or for use in toxicology testing of new drugs. However, there is one huge obstacle to their use. Derivation of hES cells is achieved by destroying pre-implantation embryo, an action that presents ethical and religious issues. The question if pluripotent ES stem cells could be obtained without destroying embryos had been raised and many scientists were eager to find new ways of creating pluripotent stem cells (for a review, see Hochedlinger and Jaenisch, 2006). The breakthrough work was done by Takahashi and Yamanaka (2006) last year when they demonstrated that induced expression of only four factors, Oct-4, Sox-2, c-Myc, and Klf-4, in mouse fibroblast cells can form ES-like cells, termed as induced pluripotent stem (iPS) cells. This year Yamanaka’s group (Okita et al., 2007) published an improved protocol and they went on to demonstrate that iPS cells are truly pluripotent stem cells because they not only differentiate into a large variety of tissues but also make germline chimeras. The technique has since been reproduced by other laboratories (Maherali et al., 2007; Meissner et al., 2007; Qin et al., 2007; Wernig et al., 2007). The question remained. Could the same technique be applied in human or were more factors needed to reprogram human somatic cells into pluripotency? We did not need to wait long for the answer. It worked. Two teams, Yamanaka’s and Thompson’s, have demonstrated that human somatic cells can be reprogrammed into iPS cells (Table 1). Yamanaka’s group successfully applied the same technique they used in mice (Takahashi et al., 2007). They used human dermal fibroblasts and two other human fibroblast populations from different human donors and transduced them with retroviral vectors carrying human cDNAs of Oct-4, Sox-2, c-Myc, and Klf-4 genes. This time they did not use selection transgene as they did in their previous works and cultured cells in hES culture medium supplemented with FGF-2. Thirty days after infection, they identified, by morphology, human iPS colonies resembling hES colonies among other colonies. Colonies were further expanded and established iPS lines (10 iPS lines derived from 50,000 cells) were subjected to many assays to compare them with hES cells derived from embryos. They studied morphology, expression of cell-surface markers, epigenetic status, and their potential to differentiate in vitro and in vivo (teratoma formation) in newly derived iPS cells. Cell lines exhibited properties of hES cells, and, moreover, DNA microarray analysis revealed the similarity between the global gene expression of human iPS cells with hES cells. They also conformed the genetic origin of iPS lines with their original parental fibroblast cells, thus excluding any cross-contamination. Chromosomal analysis revealed that iPS cells had a normal karyotype; however, minor genetic alterations could not be observed using this technique. Unlike Yamanaka’s team, Thompson’s team first decided to perform a screen to identify the genes that were highly enriched in hES cells relative to myeloid precursors and compiled a list that did not contain c-myc and Klf-4. Subsequently they showed that 14 hES cellenriched genes, transduced by lentiviral vectors, reprogrammed somatic cells to iPS cells. Moreover, they showed that out of 14 genes Oct-4, Sox-2, nanog, and Lin28 are core genes able to reprogram human somatic cells with a mesenchymal phenotype to iPS cells (Yu et al., 2007). They also found that although Gabriela Durcova-Hills ( . *) Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road Cambridge CB2 1QN, U.K. Tel: 144 1223 334137 Fax: 144 1223 334089 E-mail: gd225@cam.ac.uk

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures

Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

Nested collagen matrices: A new model to study migration of human fibroblast populations in three dimensions

Fibroblast-3D collagen matrix culture provides a model system to analyze cell physiology under conditions that more closely resemble tissue than conventional 2D cell culture. Previous work has focused primarily on remodeling and contraction of collagen matrices by fibroblasts, and there has been little research on migration of cell populations within the matrix. Here, we introduce a nested collagen matrix model to analyze migration of fibroblasts in 3D collagen matrices. Nested collagen matrices were prepared by embedding contracted cell-containing matrices (also called dermal equivalents) inside cell-free matrices; migration occurred from the former to the latter. Control experiments with human dermal fragments in place of dermal equivalents confirmed the reliability of the model. Human fibroblast migration in nested collagen matrices occurred after a lag phase of 8-16 h, and cells migrating out of the inner matrices were bipolar with leading dendritic extensions. Migration was myosin II, Rho kinase and metalloproteinase-dependent but did not require plasma fibronectin. Platelet-derived growth factor but not lysophosphatidic acid or serum stimulated cell migration, although all three of these physiological agonists promote matrix remodeling and contraction. The nested collagen matrix model is a relatively easy, rapid and quantitative method to measure migration of cell populations. Our studies using this model demonstrate important differences between regulation of fibroblast migration and remodeling in collagen matrices.

Role of IFI 16 in cellular senescence of human fibroblasts.

Defects in interferon (IFN) signaling that result in loss of expression of IFN-inducible proteins are associated with cellular immortalization, an important early event in the development of human cancer. Here we report that loss of IFN-inducible IFI 16 expression in human fibroblasts allows bypass of cellular senescence. We found that levels of IFI 16 mRNA and protein were higher in human old versus young fibroblasts and immortalization of fibroblasts with telomerase resulted in decreased expression of IFI 16. Moreover, overexpression of IFI 16 in immortalized fibroblasts strongly inhibited cell proliferation. Interestingly, knockdown of IFI 16 expression in fibroblasts inhibited p53-mediated transcription, downregulated p21(WAF1) expression, and extended the proliferation potential. Importantly, treatment of immortal cell lines with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, resulted in upregulation of IFI 16. Our observations support the idea that increased levels of IFI 16 in older populations of human fibroblasts contribute to cellular senescence.

Genome reorganization during aging of dividing cells.

The study of the effect of low dose rate ionizing radiation on the long-term proliferation of fibroblasts led to the observation that radiation accentuated the growth potential of the cells, favoring events which normally take place during division. These events could be related to the genome reorganization taking place during division. Hence, it was hypothesized (Macieira-Coelho, 1979; Macieira-Coelho, 1980; Macieira-Coelho, 1981) that the long-term proliferation of fibroblasts depends upon the potential for reorganization of the genome, the latter being a self-limiting process. At each division residual quantitative and qualitative changes would accumulate in chromatin, limiting the long-term potential for further rearrangements. The hypothesis was checked looking for quantitative and qualitative changes in DNA through the in vitro lifespan of human fibroblast populations. It was found that at each population doubling in 20% of the cells there is unequal distribution of DNA between sister cells. Results show that this could be due to errors in chromosome assembly and segregation, to loss of DNA, to errors during semiconservative DNA synthesis and to multiple rounds of DNA replication at a single origin. An increased alkali- and thermo-lability of chromatin was found during in vitro aging. At the ultrastructural level after mild decondensation, chromatin fibers were spaced and shorter. After Miller's spreading, most of the chromatin of old cells had lost the nucleosome organization and was fragmented. These chromatin changes became apparent only towards the end of the life span of human embryonic fibroblasts but were already present in a significant fraction of low population doubling level (PDL) fibroblasts from human adults. Almost all cells of low-PDL fibroblasts from the Werner syndrome presented these chromatin changes. In addition, short, unbeaded DNA fragments could be seen in these fibroblasts, occasionally forming circles; they could correspond to transposable elements which detach during the division cycle and fail to reintegrate into chromosomes because of the age-related chromatin structural changes.

Probe for polyanionic regions on the cell surface.

The binding of polyuridylate to cells in the presence of proflavine may be used as a probe to provide relative estimates of exposed polyanionic regions on the external surface of the cell. This probe binds preferably to hydrophilic, polyanionic regions, and soluble polysaccharides containing either carboxylate or sulfate groups compete with the binding of this probe. Binding of the probe to protein and lipid regions is considerably weaker. Virus-transformed human fibroblasts bind 10 times less of the probe than nontransformed cells when confluent monolayers are compared. However, as the cell density is decreased, the amount of probe bound per cell increases dramatically both for transformed as well as for normal cells. In fact, human fibroblasts (a) derived from normal donors, (b) from donors with different metabolic disorders, and (c) transformed by simian virus 40, all bind about the same amount of probe when compared at the same density. Populations of human fibroblasts aged in vitro, which contain high proportions of large cells and grow only to relatively low densities in monolayers, bind disproportionately large amounts of the complex.

Studies on DNA repair in frog and human cells exposed to an acridine half-mustard (ICR 191) and to MNNG

Although ICR 191 has been reported to act as an intercalating and alkylating mutagen in prokaryotes, it is not clear that it acts as a mutagen in eukaryotes. Therefore, we looke for evidence that this acridine half-mustard induces DNA repair in haploid Rana pipiens or in diploid human fibroblast cell lines. We measured repair autoradiographically as unscheduled DNA synthesis. Concentrations of 10 −5 to 10 −7 M ICR 191 failed to induced repair as determined by two criteria. Of 400 nuclei examined for each concentration, the percentage of labelled nuclei did not increase during or up to 18 days after treatment. Secondly, a new class of lightly labelled nuclei did not appear. However, by these criteria, the mutagen and carcinogen, N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) stimulated unscheduled DNA synthesis in both frog and human fibroblast populations at the tested concentrations (10 −4 to 10 −6 M). The MNNG-induced repair indicated that this autoradiographic technique is sufficiently sensitive to detect unscheduled DNA synthesis, and that frog cells, like human cells, are competent to perform excision repair. Intracellular localization of ICR 191 was examined by fluorescence microscopy and showed preferential interaction with the lysosomes rather than the genome of haploid frog cells. The repair and fluorescence studies indicate that the primary intracellular localization of ICR 191 takes place outside the genome in these eukaryotic cells.

A rapid rise in cell variants during the senescence of populations of human fibroblasts

An attempt has been made to measure the frequency of mutations throughout the lifespan of human fibroblast strain MRC-5. A novel procedure has been used which involves staining individual cells for high levels of glucose-6-phosphate dehydrogenase. Evidence is presented that this phenotype is due to mutation. The frequency of variants was scored from passage 16 until the final phase of senescence (passage 60). There is an exponential increase of stained cells throughout this period. The results are in agreement with the general error theory, which proposes that aging is due to a breakdown in the fidelity of information transfer between macromolecules.