Primate‐specific regulation of natural killer cells

Natural killer (NK) cells are circulating lymphocytes that function in innate immunity and placental reproduction. Regulating both development and function of NK cells is an array of variable and conserved receptors that interact with major histocompatibility complex (MHC) class I molecules. Families of lectin‐like and immunoglobulin‐like receptors are determined by genes in the natural killer complex (NKC) and leukocyte receptor complex (LRC), respectively. As a consequence of the strong, varying pressures on the immune and reproductive systems, NK cell receptors and their MHC class I ligands evolve rapidly, are highly diverse and exhibit dramatic species‐specific differences. The variable, polymorphic family of killer cell immunoglobulin‐like receptors (KIR) that regulate human NK cell development and function arose recently, from a single‐copy gene during the evolution of simian primates. Our studies of KIR and MHC class I genes in representative species show how these two unlinked but functionally intertwined genetic complexes have co‐evolved. In humans, combinations of KIR and HLA class I factors are associated with infectious diseases, including HIV/AIDS, autoimmunity, reproductive success and the outcome of therapeutic transplantation. The extraordinary, and unanticipated, divergence of human NK cell receptors and MHC class I ligands from their mouse counterparts can in part explain the difficulties experienced in finding informative mouse models for human diseases. Non‐human primate models have far greater potential, but to realize their promise will first require more complete definition of the genetics and function of KIR and MHC variation in non‐human primate species, at a level comparable to that achieved for the human species.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

Engineering of human mesenchymal stem cells resistant to multiple natural killer subtypes

Mesenchymal stem cells (MSCs) as a therapeutic promise are often quickly cleared by innate immune cells of the host including natural killer (NK) cells. Efforts have been made to generate immune-escaping human embryonic stem cells (hESCs) where T cell immunity is evaded by defecting β-2-microglobulin (B2M), a common unit for human leukocyte antigen (HLA) class I, and NK cells are inhibited via ectopic expression of HLA-E or -G. However, NK subtypes vary among recipients and even at different pathologic statuses. It is necessary to dissect and optimize the efficacy of the immune-escaping cells against NK subtypes. Here, we first generated B2M knockout hESCs and differentiated them to MSCs (EMSCs) and found that NK resistance occurred with B2M^-/- EMSCs expressing HLA-E and -G only when they were transduced via an inducible lentiviral system in a dose-dependent manner but not when they were inserted into a safe harbor. HLA-E and -G expressed at high levels together in transduced EMSCs inhibited three major NK subtypes, including NKG2A⁺/LILRB1⁺, NKG2A⁺/LILRB1^-, and NKG2A^-/LILRB1⁺, which was further potentiated by IFN-γ priming. Thus, this study engineers MSCs with resistance to multiple NK subtypes and underscores that dosage matters when a transgene is used to confer a novel effect to host cells, especially for therapeutic cells to evade immune rejection.     

Sequential changes in genome-wide DNA methylation status during adipocyte differentiation

DNA methylation is an epigenetic mark on the mammalian genome. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences distributed throughout the genome. To determine the epigenetic changes during adipocyte differentiation, we investigated the sequential changes in DNA methylation status of 3T3-L1 cells at the growing, confluent, postconfluent and mature adipocyte cell stages. Treatment of 3T3-L1 cells with 5-aza-2′-deoxycytidine inhibited differentiation in a stage-dependent manner, supporting the idea that formation of accurate DNA methylation profile, consisting of methylated and unmethylated T-DMRs, may be involved in differentiation. Analysis by methylation-sensitive quantitative real-time PCR of the 65 known T-DMRs which contain NotI sites detected 8 methylations that changed during differentiation, and the changes in the patterns of these methylations were diverse, confirming that the differentiation process involves epigenetic alteration at the T-DMRs. Intriguingly, the dynamics of the methylation change vary depending on the T-DMRs and differentiation stages. Restriction landmark genomic scanning detected 32 novel T-DMRs, demonstrating that differentiation of 3T3-L1 cells involves genome-wide epigenetic changes by temporal methylation/demethylation, in addition to maintenance of a static methylated/demethylated state, and both depend on differentiation stage.     

姜黄素对db/db小鼠基因组DNA甲基化的影响

目的:观察姜黄素对2型糖尿病模型db/db小鼠糖尿病症状的改善作用,并从表观遗传角度分析其对小鼠外周血DNA甲基化水平的影响.方法:2型糖尿病模型db/db小鼠随机分为糖尿病组和姜黄素干预组(给予250 mg/kg姜黄素溶液),连续灌胃8周.OGTT检测葡萄糖耐量,ELISA法测定空腹胰岛素并计算HOMA-IR和HOM...     

多能性胚胎干细胞与DNA甲基化的关系

胚胎干细胞是一种能够维持自我更新、具有无限扩增能力的多能性干细胞。灵长类多能干细胞（iPSCs）根据其发育能力、细胞形态、基因表达谱以及表观遗传学的差异分为初始态多能干细胞（pPSCs）和原始态多能干细胞（nPSCs）。nPSCs因其容易进行基因工程处理以及体内外再生出功能组织器官等优势而在临床潜在应用上备受关注,因而有效维持ESCs的原始状态对其用于基础及临床研究具有重要意义。nPSCs的线粒体活性和自我更新能力高于pPSCs,且这两种多能性干细胞在DNA甲基化等方面都存在明显差别,DNA甲基化在nPSCs的转化及代谢中起到重要的作用。本文综述了DNA甲基化对ESCs的作用,特别是维持原始态的作用。     

Genome‐wide screen of promoter methylation analysis of ES cells and ES derived epidermal‐like cells

Embryonic stem cells (ESCs) are a population of pluripotent cells which can differentiate into different cell types. However, there are few reports with regard to differentiate ESCs into epidermal cells in vitro. In this study, we aimed to investigate differentially methylated promoters involved in process of differentiation from ESCs into epidermal‐like cells (ELCs) induced by human amnion. We successfully induced ESCs into ELCs, which expressed the surface markers of CK19, CK15 and β1‐integrin. With MeDIP‐chip arrays, we identified 3435 gene promoters to be differentially methylated, involving 894 HCP (high CpG‐containing promoter), 974 ICP (intermediate CpG‐containing promoter) and 1567 LCP (low CpG‐containing promoter) among all the 17 500 DNA methylation regions of gene promoters in both ESCs and ELCs. Gene oncology and pathway analysis demonstrated that these genes were involved in all the three categories of GO enrichment analysis, including biological process, molecular function and cellular component. All these data suggested that embryonic stem cells can differentiate into epidermal‐like cells and promoter methylation is of great importance in this process. Copyright © 2015 John Wiley & Sons, Ltd.     

Population dynamics of human activated natural killer cells in culture

The growth kinetics and population dynamics of recombinent interleukin-2 (rlL-2) stimulated human natural killer (NK) cell-enriched populations were studied in vitro. The NK-enriched populations was obtained from normal peripheral blood mononuclear cells (PBMNC) by immunomagnetic bead depletion of CD3(+) and CD5(+) T cells. The growth kinetics of NK cells, T cells, monocytes, and total cells are shown. In the absence of PBMNC accessory cells, the NK-enriched population showed limited expansion. In the presence of PBMNC accessory cells, the NK-enriched population expanded threefold more than in the absence of accessory cells due to increased NK cell growth rate and increased duration of exponential growth. Using a Transwell system, which separates two cell population by a polycarbonate membrane, the accessory cells were shown to act on the NK-enriched population via a diffusible factor. Accessory cell conditioned media was able to replace the accessory cell population to stimulate NK cell expansion. A monocyte-enriched population prepared by sheep red blood cell rosetting of T cells was extensively phenotyped and compared with the NK-enriched populations. Although the final cultured cells were phenotypically homogeneous for CD56(+)/CD3(-) NK cells, the initial NK precusor populations appear to be different. Namely, the NK cell precursors in the monocyte-enriched population were predominantly CD56(+)/CD2(-). Kinetic equations were formulated for this culture system and the effects of major culture variables are investigated.     

DNA methylation in embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent, self‐renewing cells. These cells can be used in applications such as cell therapy, drug development, disease modeling, and the study of cellular differentiation. Investigating the interplay of epigenetics, genetics, and gene expression in control of pluripotence and differentiation could give important insights on how these cells function. One of the best known epigenetic factors is DNA methylation, which is a major mechanism for regulation of gene expression. This phenomenon is mostly seen in imprinted genes and X‐chromosome inactivation where DNA methylation of promoter regions leads to repression of gene expression. Differential DNA methylation of pluripotence‐associated genes such as Nanog and Oct4/Pou5f1 has been observed between pluripotent and differentiated cells. It is clear that tight regulation of DNA methylation is necessary for normal development. As more associations between aberrant DNA methylation and disease are reported, the demand for high‐throughput approaches for DNA methylation analysis has increased. In this article, we highlight these methods and discuss recent DNA methylation studies on ESCs. J. Cell. Biochem. 109: 1–6, 2010. © 2009 Wiley‐Liss, Inc.     

DNA methylation: an identity card for brain cells

A recently published study has revealed the genome-wide dynamics of DNA methylation and hydroxymethylation patterns at single-base resolution in the human and mouse developing brain.     

DNA methylation in mouse embryonic stem cells and development

Mammalian development is associated with considerable changes in global DNA methylation levels at times of genomic reprogramming. Normal DNA methylation is essential for development but, despite considerable advances in our understanding of the DNA methyltransferases, the reason that development fails when DNA methylation is deficient remains unclear. Furthermore, although much is known about the enzymes that cause DNA methylation, comparatively little is known about the mechanisms or significance of active demethylation in early development. In this review, we discuss the roles of the various DNA methyltransferases and their likely functions in development.     

Surface glycoprotein of human natural killer cells recognized by wheat germ agglutinin

We analyzed surface glycoproteins of human natural killer (NK) cells by utilizing lectins. Among the lectins tested, wheat germ agglutinin (WGA) was found to bind preferentially to CD16(Leu11)-positive lymphocytes as determined by two-colour flow cytometry. Analysis of glycoproteins in the lysate prepared from NK cells with sodium dodecyl sulfate (SDS) gel electrophoresis followed by Western blotting and¹²⁵I labeled WGA staining revealed that a glycoprotein with anM _r of 65 kDa was strongly bound to the lectin, but no corresponding glycoprotein was detected in the lysate of T lymphocytes. This glycoprotein (GP65) gave several spots in the pI range 4.1–4.6 on 2-dimensional gel electrophoresis. Sialidase treatment of GP65 resulted in a single spot on the 2-dimensional gel, suggesting that GP65 is heterogeneous in the degree of sialylation. GP65 was shown to be exposed on the cell surface, since it was radiolabeled with¹²⁵I by the lactoperoxidase-catalyzed method. We next isolated GP65 from human peripheral blood lymphocytes by a combination of chromatography on a cation-exchange column and a WGA-agarose column and preparative SDS gel electrophoresis. It is suggested that GP65 is a novel surface glycoprotein on human NK cells.     

DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells

Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo . This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.