Marcel Scheideler

Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation

BackgroundHuman mesenchymal stem cells (MSC) with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. However, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. In this study we asked if MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. To address this question we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC and MSC during differentiation into osteoblasts.ResultsThe results showed that MSC did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of MSC during expansion. Microarray analysis of MSC during osteogenic differentiation identified three candidate genes for further examination and functional analysis: ID4, CRYAB, and SORT1. Additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the SMAD signaling pathways by TGF-β2 and BMPs.ConclusionWith a variety of assays we could show that MSC represent a cell population which can be expanded for therapeutic applications.

miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging.

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non‐coding miRNAs are potent post‐transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age‐related and senescence‐related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8+ T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8+ T cell populations from old and young donors. Using locked nucleic acid‐based miRNA microarrays, we identified four commonly regulated miRNAs, miR‐17 down‐regulated in all seven; miR‐19b and miR‐20a, down‐regulated in six models; and miR‐106a down‐regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.

Monitoring the Switch from Housekeeping to Pathogen Defense Metabolism in Arabidopsis thaliana Using cDNA Arrays

Plants respond to pathogen attack by deploying several defense reactions. Some rely on the activation of preformed components, whereas others depend on changes in transcriptional activity. Using cDNA arrays comprising 13,000 unique expressed sequence tags, changes in the transcriptome ofArabidopsis thaliana were monitored after attempted infection with the bacterial plant pathogen Pseudomonas syringae pv. tomato carrying the avirulence geneavrRpt2. Sampling at four time points during the first 24 h after infiltration revealed significant changes in the steady state transcript levels of ∼650 genes within 10 min and a massive shift in gene expression patterns by 7 h involving ∼2,000 genes representing many cellular processes. This shift from housekeeping to defense metabolism results from changes in regulatory and signaling circuits and from an increased demand for energy and biosynthetic capacity in plants fighting off a pathogenic attack. Concentrating our detailed analysis on the genes encoding enzymes in glycolysis, the Krebs cycle, the pentose phosphate pathway, the biosynthesis of aromatic amino acids, phenylpropanoids, and ethylene, we observed interesting differential regulation patterns. Furthermore, our data showed potentially important changes in areas of metabolism, such as the glyoxylate metabolism, hitherto not suspected to be components of plant defense.

Processing and quality control of DNA array hybridization data

MOTIVATION The technology of hybridization to DNA arrays is used to obtain the expression levels of many different genes simultaneously. It enables searching for genes that are expressed specifically under certain conditions. However, the technology produces large amounts of data demanding computational methods for their analysis. It is necessary to find ways to compare data from different experiments and to consider the quality and reproducibility of the data. RESULTS Data analyzed in this paper have been generated by hybridization of radioactively labeled targets to DNA arrays spotted on nylon membranes. We introduce methods to compare the intensity values of several hybridization experiments. This is essential to find differentially expressed genes or to do pattern analysis. We also discuss possibilities for quality control of the acquired data. AVAILABILITY http://www.dkfz.de/tbi CONTACT M.Vingron@dkfz-heidelberg.de

Transcriptional profiling on all open reading frames of Saccharomyces cerevisiae

Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR‐amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR‐products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond.

PathwayExplorer: web service for visualizing high-throughput expression data on biological pathways.

While generation of high-throughput expression data is becoming routine, the fast, easy, and systematic presentation and analysis of these data in a biological context is still an obstacle. To address this need, we have developed PathwayExplorer, which maps expression profiles of genes or proteins simultaneously onto major, currently available regulatory, metabolic and cellular pathways from KEGG, BioCarta and GenMAPP. PathwayExplorer is a platform-independent web server application with an optional standalone Java application using a SOAP (simple object access protocol) interface. Mapped pathways are ranked for the easy selection of the pathway of interest, displaying all available genes of this pathway with their expression profiles in a selectable and intuitive color code. Pathway maps produced can be downloaded as PNG, JPG or as high-resolution vector graphics SVG. The web service is freely available at ; the standalone client can be downloaded at .

Oxytocin Controls Differentiation of Human Mesenchymal Stem Cells and Reverses Osteoporosis

Osteoporosis constitutes a major worldwide public health burden characterized by enhanced skeletal fragility. Bone metabolism is the combination of bone resorption by osteoclasts and bone formation by osteoblasts. Whereas increase in bone resorption is considered as the main contributor of bone loss that may lead to osteoporosis, this loss is accompanied by increased bone marrow adiposity. Osteoblasts and adipocytes share the same precursor cell and an inverse relationship exists between the two lineages. Therefore, identifying signaling pathways that stimulate mesenchymal stem cells osteogenesis at the expense of adipogenesis is of major importance for developing new therapeutic treatments. For this purpose, we identified by transcriptomic analysis the oxytocin receptor pathway as a potential regulator of the osteoblast/adipocyte balance of human multipotent adipose‐derived stem (hMADS) cells. Both oxytocin (OT) and carbetocin (a stable OT analogue) negatively modulate adipogenesis while promoting osteogenesis in both hMADS cells and human bone marrow mesenchymal stromal cells. Consistent with these observations, ovariectomized (OVX) mice and rats, which become osteoporotic and exhibit disequilibrium of this balance, have significant decreased OT levels compared to sham‐operated controls. Subcutaneous OT injection reverses bone loss in OVX mice and reduces marrow adiposity. Clinically, plasma OT levels are significantly lower in postmenopausal women developing osteoporosis than in their healthy counterparts. Taken together, these results suggest that plasma OT levels represent a novel diagnostic marker for osteoporosis and that OT administration holds promise as a potential therapy for this disease.

Activin A Plays a Critical Role in Proliferation and Differentiation of Human Adipose Progenitors.

OBJECTIVE Growth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process. RESEARCH DESIGN AND METHODS Expression of INHBA/activin A was investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression. RESULTS INHBA/activin A is expressed by adipose progenitors from various fat depots, and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes, respectively, adipocyte differentiation via the C/EBPβ-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared with the expression levels in lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue. CONCLUSIONS Altogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages that are located in adipose tissue regulate adipose progenitor self-renewal through activin A.

MicroRNA-30c promotes human adipocyte differentiation and co-represses PAI-1 and ALK2.

Obesity is characterized by excessive adipose tissue mass and associated with type 2 diabetes and cardiovascular diseases. To fight obesity and its sequels, elucidating molecular events that govern adipocyte differentiation and function is of key importance. MicroRNAs (miRNAs) are a novel class of non-coding, regulatory RNAs that have been shown to regulate crucial cellular processes, including differentiation. Several studies have already assigned miRNAs to distinct functions in murine adipocyte differentiation but only a few studies did so for humans. Here, we investigated the function of miR-30c in human adipogenesis. miR-30c expression was increased during adipogenesis of human multipotent adipose-derived stem (hMADS) cells, and miR-30c overexpression enforced adipocyte marker gene induction and triglyceride accumulation. miRNA target prediction revealed two putative direct targets of miR-30c, PAI-1 (SERPINE1) and ALK2 (ACVR1, ACTRI), both inversely regulated to miR-30c during adipogenesis and responsive to miR-30c overexpression. Luciferase reporter assays confirmed PAI-1 and ALK2 as direct miR-30c targets. Moreover, reciprocal expression between miR-30c and PAI-1 could also be demonstrated in white adipose tissue of obesity mouse models, suggesting a potential physiological role of miR-30c for PAI-1 regulation in the obese state. Validating PAI-1 and ALK-2 as miR-30c mediators in adipogenesis revealed that not single silencing of PAI-1 or ALK2, but only co-silencing of both phenocopied the pro-adipogenic miR-30c effect. Thus, miR-30c can target two, so far not interconnected genes in distinct pathways, supporting the idea that miRNAs might coordinate larger regulatory networks than previously anticipated.

Identification of differential and functionally active miRNAs in both anaplastic lymphoma kinase (ALK)+ and ALK− anaplastic large-cell lymphoma

Aberrant anaplastic lymphoma kinase (ALK) expression is a defining feature of many human cancers and was identified first in anaplastic large-cell lymphoma (ALCL), an aggressive non-Hodgkin T-cell lymphoma. Since that time, many studies have set out to identify the mechanisms used by aberrant ALK toward tumorigenesis. We have identified a distinct profile of micro-RNAs (miRNAs) that characterize ALCL; furthermore, this profile distinguishes ALK+ from ALK− subtypes, and thus points toward potential mechanisms of tumorigenesis induced by aberrant ALK. Using a nucleophosmin-ALK transgenic mouse model as well as human primary ALCL tumor tissues and human ALCL-derived cell lines, we reveal a set of overlapping deregulated miRNAs that might be implicated in the development and progression of ALCL. Importantly, ALK+ and ALK− ALCL could be distinguished by a distinct profile of “oncomirs”: Five members of the miR-17–92 cluster were expressed more highly in ALK+ ALCL, whereas miR-155 was expressed more than 10-fold higher in ALK− ALCL. Moreover, miR-101 was down-regulated in all ALCL model systems, but its forced expression attenuated cell proliferation only in ALK+ and not in ALK− cell lines, perhaps suggesting different modes of ALK-dependent regulation of its target proteins. Furthermore, inhibition of mTOR, which is targeted by miR-101, led to reduced tumor growth in engrafted ALCL mouse models. In addition to future therapeutical and diagnostic applications, it will be of interest to study the physiological implications and prognostic value of the identified miRNA profiles.