The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions.

BACKGROUND: Articular cartilage has limited healing capacity, owing in part to poor vascularity and innervation. Once injured, it cannot be repaired, typically leading to high risk for developing osteoarthritis. Thus, cell-based and/or tissue-engineered approaches have been investigated; however, no approach has yet achieved safety and regenerative repair capacity via a simple implantation procedure. PURPOSE: To assess the safety and efficacy of using a scaffold-free tissue-engineered construct (TEC) derived from autologous synovial membrane mesenchymal stem cells (MSCs) for effective cartilage repair. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Five patients with symptomatic knee chondral lesions (1.5-3.0 cm2) on the medial femoral condyle, lateral femoral condyle, or femoral groove were included. Synovial MSCs were isolated from arthroscopic biopsy specimens and cultured to develop a TEC that matched the lesion size. The TECs were then implanted into chondral defects without fixation and assessed up to 24 months postoperatively. The primary outcome was the safety of the procedure. Secondary outcomes were self-assessed clinical scores, arthroscopy, tissue biopsy, and magnetic resonance image-based estimation of morphologic and compositional quality of the repair tissue. RESULTS: No adverse events were recorded, and self-assessed clinical scores for pain, symptoms, activities of daily living, sports activity, and quality of life were significantly improved at 24 months after surgery. Secure defect filling was confirmed by second-look arthroscopy and magnetic resonance imaging in all cases. Histology of biopsy specimens indicated repair tissue approaching the composition and structure of hyaline cartilage. CONCLUSION: Autologous scaffold-free TEC derived from synovial MSCs may be used for regenerative cartilage repair via a sutureless and simple implantation procedure. Registration: 000008266 (UMIN Clinical Trials Registry number).

IGF-1 Gene Transfer to Human Synovial MSCs Promotes Their Chondrogenic Differentiation Potential without Induction of the Hypertrophic Phenotype.

Mesenchymal stem cell- (MSC-) based therapy is a promising treatment for cartilage. However, repair tissue in general fails to regenerate an original hyaline-like tissue. In this study, we focused on increasing the expression levels for insulin-like growth factor-1 (IGF-1) to improve repair tissue quality. The IGF-1 gene was introduced into human synovial MSCs with a lentiviral vector and examined the levels of gene expression and morphological status of MSCs under chondrogenic differentiation condition using pellet cultures. The size of the pellets derived from IGF-1-MSCs were significantly larger than those of the control group. The abundance of glycosaminoglycan (GAG) was also significantly higher in the IGF-1-MSC group. The histology of the IGF-1-induced pellets demonstrated similarities to hyaline cartilage without exhibiting features of a hypertrophic chondrocyte phenotype. Expression levels for the Col2A1 gene and protein were significantly higher in the IGF-1 pellets than in the control pellets, but expression levels for Col10, MMP-13, ALP, and Osterix were not higher. Thus, IGF-1 gene transfer to human synovial MSCs led to an improved chondrogenic differentiation capacity without the detectable induction of a hypertrophic or osteogenic phenotype.

Optimization of human mesenchymal stem cell isolation from synovial membrane: Implications for subsequent tissue engineering effectiveness.

Synovium-derived mesenchymal stem cells (SDMSCs) are one of the most suitable sources for cartilage repair because of their chondrogenic and proliferative capacity. However, the isolation methods for SDMSCs have not been extensively characterized. Thus, our aim in this study was to optimize the processes of enzymatic isolation followed by culture expansion in order to increase the number of SDMSCs obtained from the original tissue. Human synovium obtained from 18 donors (1.5 g/donor) was divided into three aliquots. The samples were minced and subjected to collagenase digestion, followed by different procedures: Group 1, Tissue fragments were removed by filtering followed by removing floating tissue; Group 2, No filtering. Only floating fragments were removed; Group 3, No fragments were removed. Subsequently, each aliquot was sub-divided into two density subgroups with half. In Group 1, the cell-containing media was plated either at high (5000 cells/cm2) or low density (1000 cells/cm2). In Groups 2 and 3, the media containing cells and tissue was plated onto the same number of culture dishes as used in Group 1, either at high or low density. At every passage, the cells plated at high density were consistently re-plated at high and those plated at low density were likewise. The expanded cell yields at day 21 following cell isolation were calculated. These cell populations were then evaluated for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. The final cell yields per 0.25 g tissue in Group 1 were similar at high and low density, while those in Groups 2 and 3 exhibited higher when cultured at low density. The cell yields at low density were 0.7 ± 1.2 × 107 in Group 1, 5.7 ± 1.1 × 107 in Group 2, 4.3 ± 1.2 × 107 in Group 3 (Group 1 vs Groups 2 and 3, p < 0.05). In addition, the cells obtained in each low density subgroup exhibited equivalent osteogenic, adipogenic, and chondrogenic differentiation. Thus, it was evident that filtering leads to a loss of cells and does not affect the differentiation capacities. In conclusion, exclusion of a filtering procedure could contribute to obtain higher number of SDMSCs from synovial membrane without losing differentiation capacities.

DNA methylation is dispensable for the growth and survival of the extraembryonic lineages.

DNA methylation regulates development and many epigenetic processes in mammals, and it is required for somatic cell growth and survival. In contrast, embryonic stem (ES) cells can self-renew without DNA methylation. It remains unclear whether any lineage-committed cells can survive without DNA-methylation machineries. Unlike in somatic cells, DNA methylation is dispensable for imprinting and X-inactivation in the extraembryonic lineages. In ES cells, DNA methylation prevents differentiation into the trophectodermal fate. Here, we created triple-knockout (TKO) mouse embryos deficient for the active DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) by nuclear transfer (NT), and we examined their development. In chimeric TKO-NT and WT embryos, few TKO cells were found in the embryo proper, but they contributed to extraembryonic tissues. TKO ES cells showed increasing cell death during their differentiation into epiblast lineages, but not during differentiation into extraembryonic lineages. Furthermore, we successfully established trophoblastic stem cells (ntTS cells) from TKO-NT blastocysts. These TKO ntTS cells could self-renew, and they retained the fundamental gene expression patterns of stem cells. Our findings indicated that extraembryonic-lineage cells can survive and proliferate in the absence of DNA methyltransferases and that a cell's response to the stress of epigenomic damage is cell type dependent.

Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Immune responses to repetitive adenovirus-mediated gene transfer and restoration of gene expression by cyclophosphamide or etoposide.

BACKGROUND: One major concern about adenoviral vectors for repetitive gene delivery is the induction of an immune response to the vector, thus impeding effective gene transduction. METHODS: To assess the immune response to the adenoviral vector, repetitive gene dosing was performed into rhesus monkey cervix and C3H mouse skin using the adenoviral vector carrying the lacZ gene. Three repetitive intracervical injections of adenovirus-lacZ were done in the rhesus monkey at the intervals of 4 weeks. Gene expression on the second and third injection was completely suppressed. RESULTS: Anti-adenovirus IgG levels and neutralizing antibody titers in the rhesus monkey significantly increased after the first injection of adenovirus. In the C3H mouse, neutralizing antibody titers significantly increased after the first injection of adenovirus-lacZ at more than 10(8) plaque-forming unit (PFU). The repetitive expression of lacZ gene in the mouse skin markedly decreased when the second injection is done more than 2 weeks after the first injection. Chronic low-dose treatment with cyclophosphamide or etoposide markedly suppressed neutralizing antibody titers in the mouse serum and restored the gene expression in the mouse skin on the second and third injection. CONCLUSIONS: It is suggested that repetitive gene expression by adenovirus-mediated transfer may be reduced by circulating neutralizing antibodies and could be restored by chronic low-dose treatment with cyclophosphamide or etoposide.

The antiallergic drug oxatomide promotes human eosinophil apoptosis and suppresses IL-5-induced eosinophil survival.

BACKGROUND: Eosinophils accumulated in sites of allergic inflammation are thought to play a crucial role in the pathogenesis of allergic disorders including asthma, allergic rhinitis, and atopic dermatitis, and tissue eosinophilia is attributable to increased eosinophil survival or decreased eosinophil apoptosis. OBJECTIVE: Effects of the antiallergic, histamine H1 blocker oxatomide on viability and apoptosis of eosinophils isolated from the peripheral blood of atopic subjects were studied. METHODS: Eosinophil viability and apoptosis were evaluated by using a colorimetric assay and annexin V-labeling, caspase-3 activity, and DNA fragmentation assay. RESULTS: The viability of eosinophils increased in the presence of IL-5 (10 ng/mL), confirming that IL-5 prolongs eosinophil survival in vitro. Application of oxatomide at concentrations over 20 micromol/L for 24 hours decreased the IL-5-induced enhancement of eosinophil viability. Double staining of the cells with annexin V and propidium iodide showed that deprivation of IL-5 promoted spontaneous eosinophil apoptosis and that oxatomide facilitated apoptosis and suppressed the prolongation of eosinophil survival stimulated by IL-5. In the absence of IL-5, approximately 71% and 96% of eosinophils after 24 and 48 hours, respectively, underwent spontaneous apoptosis. IL-5 decreased the rate of eosinophil apoptosis to 38% and 52% after 24 and 48 hours, respectively. Oxatomide increased eosinophil apoptosis in a concentration-dependent manner in the presence of IL-5. Furthermore, oxatomide increased caspase-3 activity and DNA fragmentation. CONCLUSION: We demonstrated that oxatomide possesses a novel therapeutic effect of apoptosis promotion on eosinophils and prevents the antiapoptotic effects of IL-5, suggesting that oxatomide may contribute to resolution of tissue eosinophilia in allergic inflammation.

Differential regulation of nonsteroidal anti-inflammatory drug-activated gene in normal human tracheobronchial epithelial and lung carcinoma cells by retinoids.

In this study, we analyze the effect of several retinoids on the expression of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in normal human tracheobronchial epithelial (HTBE) cells and several lung carcinoma cell lines. The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) greatly enhances the expression of NAG-1 mRNA and protein in a time- and dose-dependent manner in human lung adenocarcinoma H460 cells and several other carcinoma cell lines. This induction was specific for AHPN because retinoic acid, a retinoic acid receptor-, and a retinoid X receptor pan-agonist were unable to induce NAG-1, suggesting that this induction is not mediated through activation of retinoid receptors. Although NAG-1 is a p53-responsive gene, AHPN-induced NAG-1 expression does not require p53. The induction of NAG-1 expression by AHPN is caused at least in part by an 8-fold increase in the stability of NAG-1 mRNA. In contrast to carcinoma cells, NAG-1 expression is effectively induced by retinoic acid and the RAR-selective pan-agonist in normal HTBE cells and accompanies the inhibition of squamous differentiation and the initiation of normal differentiation. In vivo, NAG-1 expression was observed in the normal tracheobronchial epithelium, whereas no expression was found in either squamous metaplastic tracheal epithelium or in sections of human lung tumors. Our results suggest that the induction of NAG-1 expression by retinoids in normal HTBE and lung carcinoma cells is regulated by distinct mechanisms and is associated with different biological processes. The linkage between AHPN treatment and NAG-1 expression revealed in this study provides a new mechanism for the antitumorigenic activity of AHPN.

High incidence of T-cell lymphomas in mice deficient in the retinoid-related orphan receptor RORgamma.

Nuclear receptors are critical regulators of many physiological processes and have been shown to be involved in a variety of disease processes, including malignant neoplasms. Our laboratory is investigating the function of the retinoid-related orphan receptor gamma (RORgamma) and its possible role in disease. Studies of mice deficient in the expression of RORgamma demonstrated that this receptor plays a crucial role in the regulation of thymopoiesis and lymph node organogenesis. In this study, we show that changes in homeostasis in the thymus of RORgamma-/- mice are associated with a high incidence of T-cell lymphomas. Over 50% of the deficient mice of mixed genetic background die within the first 4 months as a result of thymic lymphomas. A high incidence of lymphomas was also observed in RORgamma-/- 129/SvEv mice. The lymphoblastic cells metastasized frequently to spleen and liver. No other tumor types were detected in any of RORgamma-/- mice that died during the course of the experiment, and none of the heterozygous mice developed thymic lymphomas. Lymphoma formation was associated with increased cellular proliferation and an increase in the number of apoptotic cells. When placed in culture, the RORgamma-/- lymphoblastic cells underwent accelerated "spontaneous" apoptosis at a rate similar to that of RORgamma-/- thymocytes. Upon prolonged culture, several lymphoblastic cell lines could be established. Analysis of the immunophenotype of the lymphoblastic cells showed that the CD4 and CD8 subpopulations varied substantially among different lymphomas. The established cell lines consisted mostly of CD44-CD25+CD4-CD8- cells. Our studies indicate that loss of RORgamma disturbs homeostasis in the thymus by enhancing apoptosis and cellular proliferation. The latter may enhance the probability of individual cells to acquire genetic alterations that make them escape negative selection and normal differentiation programs and as a consequence lead to increased susceptibility to the development of T-cell lymphoma.