Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Opportunities for organoids as new models of aging.

The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans. However, the best model organisms remain expensive and time-consuming to use. More importantly, they may not reflect directly on the process of aging in people. Human cell culture provides an alternative, but many functional signs of aging occur at the level of tissues rather than cells and are therefore not readily apparent in traditional cell culture models. Organoids have the potential to effectively balance between the strengths and weaknesses of traditional models of aging. They have sufficient complexity to capture relevant signs of aging at the molecular, cellular, and tissue levels, while presenting an experimentally tractable alternative to animal studies. Organoid systems have been developed to model many human tissues and diseases. Here we provide a perspective on the potential for organoids to serve as models for aging and describe how current organoid techniques could be applied to aging research

Cell-based gene therapy for mending infarcted hearts

The goal of this study was to analyse the efficiency of a combinatorial cell/growth factor therapy to improve function of infarcted murine hearts. The Insulin-like Growth Factor-1 (IGF-1) isoform, IGF-1Ea, has been shown to reduce scar formation and decrease cell death after MI. The present study utilized P19Cl6-derived, IGF-1Ea over-expressing cardiomyocytes to achieve its goal. The P19Cl6 cells were stably transduced with IGF-1Ea using a lentiviral vector and investigated first in vitro for their feasibility for in vivo cell therapy. The engineered pluripotent cells over-expressing IGF-1Ea survived better to hypoxia-induced injury than the control cells. The cells maintained their pluripotency and efficient differentiation capacity towards ventricular cardiomyocyte lineage, generating large quantities of cardiomyocytes optimal for the transplantation study. The generated cardiomyocytes were functionally active and exhibited a mature phenotype. Transplantation of the cardiomyocytes into allogeneic wild type murine infarcted hearts conferred a tendency for maintenance of function at short-term time point. At long-term however, this effect was lost, returning to the level of the control infarcted hearts. Cell tracing assessment revealed engraftment of both IGF-1Ea- and empty-cells, although the cells failed to couple with the recipient tissue. Scar size and capillary density analyses revealed no significant difference between the cells transplanted compared to the saline treated hearts, corroborating with the long-term functional data. Interestingly, the IGF- 1Ea-cell transplanted hearts expressed significantly higher amount of VEGFa compared to the controls, albeit no change in capillary density. Further investigation revealed that the enhanced VEGFa expression in IGF-1Ea-cells transplanted hearts was associated with reduced hypertrophy, marked by reduced cell cross-sectional area at the border-zone, aSK and bMHC expression compared to the control hearts. Nonetheless, modulation of hypertrophic response and transplantation of IGF-1Ea-cells were not able to confer lasting functional preservation, possibly due to lack of sufficient engraftment and coupling of the transplanted cells

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use

In vivo delivery of transcription factors with multifunctional oligonucleotides.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury

DNA Mismatch Repair Dependent Damage Response in Human Pluripotent Stem Cells and Intestinal Organoids

The DNA mismatch repair (MMR) pathway is a very important DNA repair pathway to maintain genomic integrity. Germline mutations in the MMR genes can cause a hereditary cancer predisposition syndrome, Lynch Syndrome (LS). LS patients develop colorectal cancer as well as other extracolonic cancers at an early age. However, how the loss of DNA MMR leads to tumorigenesis remains unclear. The MMR mediated DNA damage response to the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) observed in various cancer cell lines may contribute to preventing tumorigenesis by eliminating damaged cells. In the first part of this study, we examined the MMR dependent DNA damage response in the human pluripotent stem cell (hPSC) which is a nontransformed cell model. We found that hPSCs are hypersensitive to alkylation damage which triggers massive apoptosis. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cells. In somatic cells, a permanent G2/M cell cycle arrest is induced in the second cell cycle after DNA damage. The hPSCs, however, directly undergo apoptosis in the first cell cycle. Furthermore, the signaling mechanisms of this damage response are also very different from somatic cells in that the checkpoint kinases Chk1 and Chk2 are not activated in hPSCs in response to alkylation damage, but rather p53 activation is responsible for inducing apoptosis. This response reveals that hPSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Since LS patients predominantly develop colorectal cancer and human embryonic stem cells (hESCs) can be differentiated into intestinal organoids in vitro, in the second part of this study we generated both hESCs-derived human intestinal organoids (HIOs) and adult human intestinal enteroids (HIEs) from patient colon samples to study the damage responses to alkylation damage in intestinal cells specifically. We found that the MMR pathway can direct multiple responses to DNA damage in different intestinal cell types in HIOs. Intestinal stem cells (ISCs) appear more prone to undergo apoptosis in response to DNA damage whereas more differentiated cells such as the transient amplifying cells are more likely to senesce. Both mechanisms may play an important role in tumor suppression by eliminating or halting progression of damaged cells. Therefore loss of MMR pathway function might provide an immediate selective advantage at an early stage during tumorigenesis in LS patients. Taken together, this work further reveals the MMR-dependent DNA damage response in nontransformed cell types and cell types related to LS, and provides insights into how loss of these damage responses may contribute to tumorigenesis at an early stage in LS patients

Primary skin fibroblasts as a model of Parkinson's disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues

Acute Hypersensitivity of Pluripotent Testicular Cancer-Derived Embryonal Carcinoma to Low-Dose 5-Aza Deoxycytidine Is Associated with Global DNA Damage-Associated p53 Activation, Anti-Pluripotency and DNA Demethylation

Human embryonal carcinoma (EC) cells are the stem cells of nonseminoma testicular germ cells tumors (TGCTs) and share remarkable similarities to human embryonic stem (ES) cells. In prior work we found that EC cells are hypersensitive to low nanomolar doses of 5-aza deoxycytidine (5-aza) and that this hypersensitivity partially depended on unusually high levels of the DNA methyltransferase, DNMT3B. We show here that low-dose 5-aza treatment results in DNA damage and induction of p53 in NT2/D1 cells. In addition, low-dose 5-aza results in global and gene specific promoter DNA hypomethylation. Low- dose 5-aza induces a p53 transcriptional signature distinct from that induced with cisplatin in NT2/D1 cells and also uniquely downregulates genes associated with pluripotency including NANOG, SOX2, GDF3 and Myc target genes. Changes in the p53 and pluripotency signatures with 5-aza were to a large extent dependent on high levels of DNMT3B. In contrast to the majority of p53 target genes upregulated by 5-aza that did not show DNA hypomethylation, several other genes induced with 5-aza had corresponding decreases in promoter methylation. These genes include RIN1, SOX15, GPER, and TLR4 and are novel candidate tumors suppressors in TGCTs. Our studies suggest that the hypersensitivity of NT2/D1 cells to low-dose 5-aza is multifactorial and involves the combined activation of p53 targets, repression of pluripotency genes, and activation of genes repressed by DNA methylation. Low-dose 5-aza therapy may be a general strategy to treat those tumors that are sustained by cells with embryonic stem-like properties. GEO number for the microarray data: GSE42647