Background N6-methyladenosine (m6A) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain. Results We show that m6A modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, m6A shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more m6A sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more m6A sites compared to mice. Differential m6A sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These m6A sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased m6A methylation in a mouse model of Alzheimer’s disease, which is correlated with reduced protein levels. Conclusions Our results suggest that m6A exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, m6A controls protein levels of key genes involved in Alzheimer’s disease-associated pathways, suggesting that m6A plays an important role in aging and neurodegenerative disease.

Th type 17 (Th17) cells have been identified as a proinflammatory T-cell subset. Here, we investigated the regulation of human Th17 cells by fetal BM-derived mesenchymal stem cells (FBM-MSC). We cocultured FBM-MSC with human PBMC or CD4 1 T cells from healthy donors. FBM-MSC significantly suppressed the proliferation of CD4 1 T cells stimulated by PHA and recombinant IL-2. Significantly higher levels of IL-17 were observed in FBM-MSC cocultured with either PBMC or CD4 1 T cells than that in PBMC cultured alone or CD4 1 T cells cultured alone. Flow cytometry analysis showed that the percentage of Th17 cells in coculture of FBM-MSC and CD4 1 T cells was significantly higher than that in CD4 1 T-cell cultured alone. FBM-MSC did not express IL-17 protein. Consistent with the augmentation of Th17 cells, significantly higher levels of IL-6 and IL-1 were observed in coculture of FBM-MSC and CD4 1 T cells than that in CD4 1 T-cell culture, while the levels of IL-23 were similar between FBM-MSC 1 PBMC coculture and PBMC alone, or FBM-MSC 1 CD4 1 T-cell and CD4 1 T-cell alone. The presence of FBM-MSC decreased the percentage of Th1 cells, but minimally affected the expansion of CD4 1 CD25 1 T cells. In conclusion, our data demonstrate for the first time that FBM-MSC promote the expansion of Th17 cells and decrease IFN-c-producing Th1 cells. These data suggest that IL-6 and IL-1, instead of IL-23, may be partly involved in the expansion of Th17 cells.Key words: Fetal BM-derived mesenchymal stem cells . IL-1 . IL-6 . Th1 . Th17 IntroductionMesenchymal stem cells (MSC) are multipotent stem cells found in many tissues in the body, such as BM, muscle, adipose, synovial membrane, amniotic fluid [1][2][3][4], umbilical cord, cord blood [5] and fetal tissues [6]. Accumulating evidence has shown that MSC perform an immune regulatory function partly by suppressing the proliferation of T cells in vitro [7][8][9]. MSC-induced immunotolerance has been used therapeutically for reducing GVH disease and for modulation of autoimmune disorders [10,11].Classically, effector CD4 1 Th cells have been categorized into two subsets: Th1 and Th2. Th1 cells produce IFN-g, which is à These authors contributed equally to this work. 2840required for the clearance of certain intracellular pathogens; while Th2 cells produce IL-4, which is required for the clearance of parasites [12]. Recently, a new effector CD4 1 T-cell lineage, Th17, has been identified in an EAE model. Th17 cells are characterized by their production of a distinct profile of cytokines, including IL-17, IL-17F and IL-6, and are thought to be involved in inflammatory and autoimmune diseases [13,14]. There have been many investigations in mouse Th17 cells, but the knowledge of human Th17 cells remains limited.So far little is known about the interaction of fetal BM-derived MSC (FBM-MSC) with immune cells, especially human Th17 cells. Therefore, in this study, we cocultured FBM-MSC with human PBMC or CD4 1 T cells and demonstrated for the first time that FBM-MSC had the capability to p...

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Purpose This study evaluates the effect of a sequential culture system on the follicular development of sheep lamb ovaries, aiming to establish an available in vitro culture system for ovarian culture. Methods Lamb ovarian cortical fragments were cultured on a steel mesh with a nitrocellulose membrane pre-coated by type1 collagen. Several culture media were used for the determinations, specifically, a control medium (α-MEM), a constant medium (control medium supplemented with 75 ng/mL human recombinant EGF, 200 mIU/mL sheep FSH, 100 ng/mL human recombinant GDF-9, and 100 ng/mL human recombinant bFGF), and a sequential medium (control medium supplemented with sequential growth factors added on different days). Ovarian tissues, both fresh and cultured, were processed for histological and apoptotic assays, while spent culture media were processed for hormone assays.Results It was found that the growth of lamb primordial follicles can be initiated during culture in vitro. Compared to the control medium, sequential culture medium significantly increased the percentage of secondary follicles in cultures, while the follicle and oocyte diameters of primary and secondary follicles were also observed to increase in this medium. The constant medium was found to increase the number and diameter of secondary follicles only 18 days after culture. After this same period of time, some normal antral follicles were found in the sequential medium, while a few abnormal antral-like follicles were found in the control medium. Moreover, sequential medium appeared to significantly increase estradiol and inhibin production, especially 10-18 days after culture. The highest percentage of normal follicles and the lowest apoptotic cell rates were observed in the sequential medium, suggesting that a sequential addition style of culture can improve follicle and tissue viability. Conclusions The sequential addition of FSH, EGF, GDF-9, and bFGF can stimulate primordial follicle transmittal into the later development stages, even as far as the antral stage, improve the survival rate of follicles, and maintain follicular viability.

T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells are identified as the new subset of immune cells. This study aims to investigate the role of circulating Tfh cells (cTfh) and Tfr (cTfr) cells in the pathogenesis of non-small cell lung cancer (NSCLC). A total of 27 NSCLC patients and 19 age and sex-matched healthy controls were enrolled. The percentage of cTfh and cTfr was detected by flow cytometric analysis. Compared to healthy controls, a significantly higher percentage of both cTfh and cTfr cells were observed in NSCLC patients (for cTfh, 18.88% ± 16.84% versus 5.98% ± 3.70%, P < 0.01; for cTfr, 2.67% ± 2.20% versus 1.14% ± 0.76%, P < 0.01). Furthermore, there was a positive correlation between cTfh/cTfr ratio and age in NSCLC patients (P < 0.05). When taking age 60 as a cut-off, the percentage of both cTfh cells and cTfr cells were higher in older patients than younger patients. Moreover, our data showed there was lower percentage of cTfh cells in NSCLC patients with early stage disease (I and II) (12.10% ± 12.22%) than that in advanced stage disease (III and IV) (30.41% ± 17.87%) (P < 0.01). However, no significant relationship was observed between cTfr cells and clinical stage in NSCLC patients. A higher percentage of cTfh cells was observed in patients with squamous cell carcinoma compared with adenocarcinoma (31.70% ± 20.73% versus. 13.48% ± 11.78%, P < 0.05). Taken together, there was significantly higher percentage of cTfh and cTfr cells in NSCLC patients. cTfh and cTfr cells might play an important role in the pathogenesis of NSCLC patients.

Multipotent mesenchymal stromal cells (MSCs) are a promising cell type for cell transplantation; however, their utilization remains limited until the availability of adequate alternative sources of MSCs and the thorough understanding of the biology of MSCs isolated from various sources are realized. Fetal lung has been identified as a rich source of MSCs. To explore the therapeutic potential of passaged fetal lung MSCs (FLMSCs), the present study evaluated their growth kinetics, telomere length, karyotype, immunophenotype, and the differentiation potential during in vitro expansion. FLMSCs could be easily amplified in vitro with no significant shorting of telomere length and had a normal karyotype. No significant differences between passage 5 or passage 25 were observed in the immunophenotype analysis using flow cytometry. Moreover, flow cytometry results provided the first demonstration, to our knowledge, that FLMSCs stably expressed pluripotent markers including Oct4, Nanog, Sox2, TRA-1-60, c-Myc, and SSEA-4 through 25 passages. In vitro differentiation studies as identified by confocal microscopy, flow cytometry, RT-PCR, and immunohistochemistry showed that FLMSCs had extended capacity of differentiating into mesodermal, ectodermal, and endodermal lineages, and that their potential for adipogenic, osteogenic, and chondrogenic differentiation may be maintained over 25 passages. Furthermore, osteogenic and chondrogenic differentiation was used as an indicator of their differentiation capability in vivo, as evidenced by ectopic bone and cartilage formation. In summary, these results suggest that FLMSCs are a primitive population and that their extensive in vitro expansion does not involve significant functional modification of the cells, including morphology, growth, karyotype, immunophenotype, and mesodermal differentiation potential. Hence, FLMSCs might constitute an attractive cell resource for cell transplantation to induce regeneration of damaged tissues/organs.

Nonnegative matrix factorization is a powerful technique to realize dimension reduction and pattern recognition through single-layer data representation learning. Deep learning, however, with its carefully designed hierarchical structure, is able to combine hidden features to form more representative features for pattern recognition. In this paper, we proposed sparse deep nonnegative matrix factorization models to analyze complex data for more accurate classification and better feature interpretation. Such models are designed to learn localized features or generate more discriminative representations for samples in distinct classes by imposing L1-norm penalty on the columns of certain factors. By extending one-layer model into multi-layer one with sparsity, we provided a hierarchical way to analyze big data and extract hidden features intuitively due to nonnegativity. We adopted the Nesterov's accelerated gradient algorithm to accelerate the computing process with the convergence rate of O(1/k 2 ) after k steps iteration. We also analyzed the computing complexity of our framework to demonstrate their efficiency. To improve the performance of dealing with linearly inseparable data, we also considered to incorporate popular nonlinear functions into this framework and explored their performance. We applied our models onto two benchmarking image datasets, demonstrating our models can achieve competitive or better classification performance and produce intuitive interpretations compared with the typical NMF and competing multi-layer models.

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