Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colonyforming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled

Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells

Generation of iPSCs in bulk culture.

<p>Time axis for reprogramming of human dermal fibroblasts with episomal plasmids (<b>A</b>). Exemplary phase contrast images of bulk-cultured colonies are depicted in the course of culture-expansion. Already at day 6 the first changes in cell morphology could be noticed (arrows). Bulk cultured colonies revealed a typical ESC-like morphology after 10 passages (<b>B</b>).</p

iPSC colonies on different feeder layers.

<p>Three types of irradiated feeder layers have been used: murine embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and human bone marrow derived mesenchymal stromal cell (MSCs) (<b>A</b>). The morphology of iPSC colonies is exemplarily presented on each of these feeder layers (<b>B</b>). iPSC colonies were further characterized by immunofluorescent staining for the pluripotency markers OCT4 (green), TRA-1-60 (red) and SSEA3 (red) with nuclear counterstaining (DAPI, blue) (<b>C,D</b>). The initial colony-frequency and increase of colony-size was compared on different feeder layers: colony-frequency was significantly higher on irradiated MEFs than on HDFs (P = 0.03), or MSCs (P = 0.01; total colony counts (in four experiments): 333 with MEF, 225 with HDF, and 153 with MSC) (<b>E</b>) and the colony-size increased faster on MEFs than on HDFs or MSCs (P = 0.01 and P = 0.13, respectively) (<b>F</b>). Alternatively, induced fibroblasts were re-seeded in wells without preformed irradiated feeder cells. Non-induced fibroblasts from the parental cell preparation then grew out to provide a stromal support for iPSC colonies but their frequency was lower than using preformed irradiated feeders (total colony counts (in three experiments): 215 with HDF and 132 without feeder) (<b>G</b>). (** = P<0.01; * = P<0.05; n.s. =  not significant).</p

Gene expression profiles and <i>in vitro</i> differentiation of iPSCs.

<p>Hierarchical clustering of global gene expression profiles revealed clear separation of non-transfected fibroblasts (F1, F2; green) and iPSCs (bulk: black; clonal: red). iF2_B1 and iF2_C1 grew nicely until passage 3, but were then lost due to bacterial contamination. iF4 was only cultured clonally without bulk counterpart. (<b>A</b>). Scatterplot analysis of mean signal intensity demonstrated very similar gene expression profiles of clonal and bulk-cultured iPSCs and statistical analysis did not reveal significant differences (<b>B</b>). PluriTest analysis supported the notion that bulk-cultured as well as clonally-derived iPSCs are pluripotent as they grouped with established pluripotent cells (red area) and not with somatic samples (blue area) <b>(C)</b>. <i>In vitro</i> differentiation potential of iPSC colonies was evaluated by an embryoid body (EB) assay. After 10 days, each cell preparation revealed cystic structures (black arrows), areas with cells of epithelial morphology (white arrows) and spontaneous beating areas indicating cardiac differentiation (<b>D</b>). Up-regulation of differentiation markers AFP (endodermal marker), cTnT (mesodermal marker) and nestin (ectodermal marker) could be induced in all bulk-cultured and clonally derived iPSCs (<b>E</b>). Expression of ectodermal (GFAP, NKX6-1, PAX6, SOX1), mesodermal (PECAM1, CD34, MYH6), and endodermal markers (AFP, ALB, SOX17) was assessed by RT-qPCR: all of these genes were up-regulated upon differentiation, whereas OCT4 expression was down-regulated (<b>F</b>).</p