Riitta Lahesmaa

Copy number variation and selection during reprogramming to pluripotency

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.

Exosomes with Immune Modulatory Features Are Present in Human Breast Milk

Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant’s immune system. Exosomes are nanovesicles (30–100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant’s immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-γ production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3+CD4+CD25+ T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.

Dysregulation of lipid and amino acid metabolism precedes islet autoimmunity in children who later progress to type 1 diabetes

The risk determinants of type 1 diabetes, initiators of autoimmune response, mechanisms regulating progress toward β cell failure, and factors determining time of presentation of clinical diabetes are poorly understood. We investigated changes in the serum metabolome prospectively in children who later progressed to type 1 diabetes. Serum metabolite profiles were compared between sample series drawn from 56 children who progressed to type 1 diabetes and 73 controls who remained nondiabetic and permanently autoantibody negative. Individuals who developed diabetes had reduced serum levels of succinic acid and phosphatidylcholine (PC) at birth, reduced levels of triglycerides and antioxidant ether phospholipids throughout the follow up, and increased levels of proinflammatory lysoPCs several months before seroconversion to autoantibody positivity. The lipid changes were not attributable to HLA-associated genetic risk. The appearance of insulin and glutamic acid decarboxylase autoantibodies was preceded by diminished ketoleucine and elevated glutamic acid. The metabolic profile was partially normalized after the seroconversion. Autoimmunity may thus be a relatively late response to the early metabolic disturbances. Recognition of these preautoimmune alterations may aid in studies of disease pathogenesis and may open a time window for novel type 1 diabetes prevention strategies.

High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb–3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

Genomic views of STAT function in CD4 + T helper cell differentiation

Signal transducer and activator of transcription (STAT) proteins are well known for their essential roles in transmitting cytokine-mediated signals and specifying T helper (TH) cell differentiation. Recent technological advances have revealed that STAT proteins have broad and complex roles in gene regulation and epigenetic control, including important roles as functional repressors. However, the challenge of how to link signal transduction, nucleosome biology and gene regulation remains. The relevance of tackling this problem is highlighted by genome-wide association studies that link cytokine signalling and STATs to various autoimmune or immune deficiency disorders. Defining exactly how extrinsic signals control the specification and plasticity of TH cells will provide important insights and perhaps therapeutic opportunities in these diseases.

Gene expression signatures of seven individual human embryonic stem cell lines.

Identification of molecular components that define a pluripotent human embryonic stem cell (hESC) provides the basis for understanding the molecular mechanisms regulating the maintenance of pluripotency and induction of differentiation. We compared the gene expression profiles of seven genetically independent hESC lines with those of nonlineage‐differentiated cells derived from each line. A total of 8,464 transcripts were expressed in all hESC lines. More than 45% of them have no yet‐known biological function, which indicates that a high number of unknown factors contribute to hESC pluripotency. Among these 8,464 transcripts, 280 genes were specific for hESCs and 219 genes were more than twofold differentially expressed in all hESC lines compared with nonlineage‐differentiated cells. They represent genes implicated in the maintenance of pluripotency and those involved in early differentiation. The chromosomal distribution of these hESC‐enriched genes showed over‐representation in chromosome 19 and under‐representation in chromosome 18. Although the overall gene expression profiles of the seven hESC lines were markedly similar, each line also had a subset of differentially expressed genes reflecting their genetic variation and possibly preferential differentiation potential. Limited overlap between gene expression profiles illustrates the importance of cross‐validation of results between different ESC lines.

Gene expression profiling during differentiation of human monocytes to macrophages or dendritic cells

Macrophages and dendritic cells (DC) are APC, which regulate innate and adaptive immune responses. Macrophages function locally mainly, maintaining inflammatory responses in tissues, whereas DC take up microbes, mature, and migrate to local lymph nodes to present microbial antigens to naïve T cells to elicit microbe‐specific immune responses. Blood monocytes can be differentiated in vitro to macrophages or DC by GM‐CSF or GM‐CSF + IL‐4, respectively. In the present study, we performed global gene expression analyses using Affymetrix HG‐U133A Gene Chip oligonucleotide arrays during macrophage and DC differentiation. During the differentiation process, 340 and 350 genes were up‐regulated, and 190 and 240 genes were down‐regulated in macrophages and DC, respectively. There were also more that 200 genes, which were expressed differentially in fully differentiated macrophages and DC. Macrophage‐specific genes include, e.g., CD14, CD163, C5R1, and FcγR1A, and several cell surface adhesion molecules, cytokine receptors, WNT5A and its receptor of the Frizzled family FZD2, fibronectin, and FcεR1A were identified as DC‐specific. Our results reveal significant differences in gene expression profiles between macrophages and DC, and these differences can partially explain the functional differences between these two important cell types.

A practical comparison of methods for detecting transcription factor binding sites in ChIP-seq experiments

BackgroundChromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) is increasingly being applied to study transcriptional regulation on a genome-wide scale. While numerous algorithms have recently been proposed for analysing the large ChIP-seq datasets, their relative merits and potential limitations remain unclear in practical applications.ResultsThe present study compares the state-of-the-art algorithms for detecting transcription factor binding sites in four diverse ChIP-seq datasets under a variety of practical research settings. First, we demonstrate how the biological conclusions may change dramatically when the different algorithms are applied. The reproducibility across biological replicates is then investigated as an internal validation of the detections. Finally, the predicted binding sites with each method are compared to high-scoring binding motifs as well as binding regions confirmed in independent qPCR experiments.ConclusionsIn general, our results indicate that the optimal choice of the computational approach depends heavily on the dataset under analysis. In addition to revealing valuable information to the users of this technology about the characteristics of the binding site detection approaches, the systematic evaluation framework provides also a useful reference to the developers of improved algorithms for ChIP-seq data.

Persistence of Yersinia antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3 infection with or without reactive arthritis

OBJECTIVE To assess the persistence of bacterial antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3-triggered reactive arthritis (ReA). METHODS Peripheral blood samples were obtained from 20 patients with Y. enterocolitica O:3 infection (11 with ReA and 9 without). These samples were studied by immunochemical techniques for the presence of Yersinia antigens at the beginning of infection and up to 4 years thereafter. Synovial fluid samples from 6 of the 11 ReA patients were also studied. RESULTS The Yersinia antigens lipopolysaccharide and heat-shock protein (HSP) were detected in peripheral blood mononuclear cells and polymorphonuclear phagocytes from all patients studied at the early phase of the disease. They were also found in the synovial fluid cells of patients with Yersinia-triggered ReA. At 4 years after the onset of infection, these bacterial antigens were still detected in the peripheral blood cells of most of the ReA patients studied. CONCLUSION This study has, for the first time, directly demonstrated that bacterial antigens persist for a long time in patients who develop ReA after Y. enterocolitica O:3 infection. The finding of bacterial HSP in synovial fluid cells could provide a link to the pathogenesis of ReA, since T cell responses of synovial cells have been shown to be directed against that structure. A close similarity between the bacterial and host HSP might contribute to the development of the relatively common, chronic form of this complication.

Genome-wide Profiling of Interleukin-4 and STAT6 Transcription Factor Regulation of Human Th2 Cell Programming

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.