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Phosphoglycerate kinase acts as a futile cycle at high temperature.

Abstract (Expand)

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate … to glyceraldehyde-3-phosphate at 30 degrees C and at 70 degrees C. At 30 degrees C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 degrees C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 degrees C and at 70 degrees C, however, at 70 degrees C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

Authors: T. Kouril, J. J. Eicher, B. Siebers, J. L. Snoep

Date Published: 7th Oct 2017

Publication Type: Not specified

Systems biology of the modified branched Entner-Doudoroff pathway in Sulfolobus solfataricus.

Abstract (Expand)

Sulfolobus solfataricus is a thermoacidophilic Archaeon that thrives in terrestrial hot springs (solfatares) with optimal growth at 80 degrees C and pH 2-4. It catabolizes specific carbon sources, such … as D-glucose, to pyruvate via the modified Entner-Doudoroff (ED) pathway. This pathway has two parallel branches, the semi-phosphorylative and the non-phosphorylative. However, the strategy of S.solfataricus to endure in such an extreme environment in terms of robustness and adaptation is not yet completely understood. Here, we present the first dynamic mathematical model of the ED pathway parameterized with quantitative experimental data. These data consist of enzyme activities of the branched pathway at 70 degrees C and 80 degrees C and of metabolomics data at the same temperatures for the wild type and for a metabolic engineered knockout of the semi-phosphorylative branch. We use the validated model to address two questions: 1. Is this system more robust to perturbations at its optimal growth temperature? 2. Is the ED robust to deletion and perturbations? We employed a systems biology approach to answer these questions and to gain further knowledge on the emergent properties of this biological system. Specifically, we applied deterministic and stochastic approaches to study the sensitivity and robustness of the system, respectively. The mathematical model we present here, shows that: 1. Steady state metabolite concentrations of the ED pathway are consistently more robust to stochastic internal perturbations at 80 degrees C than at 70 degrees C; 2. These metabolite concentrations are highly robust when faced with the knockout of either branch. Connected with this observation, these two branches show different properties at the level of metabolite production and flux control. These new results reveal how enzyme kinetics and metabolomics synergizes with mathematical modelling to unveil new systemic properties of the ED pathway in S.solfataricus in terms of its adaptation and robustness.

Authors: A. S. Figueiredo, T. Kouril, D. Esser, P. Haferkamp, P. Wieloch, D. Schomburg, P. Ruoff, B. Siebers, J. Schaber

Date Published: 12th Jul 2017

Publication Type: Not specified

Oxidative Stickland reactions in an obligate aerobic organism - amino acid catabolism in the Crenarchaeon Sulfolobus solfataricus

Abstract (Expand)

The thermoacidophilic Crenarchaeon Sulfolobus solfataricus is a model organism for archaeal adaptation to extreme environments and renowned for its ability to degrade a broad variety of substrates. It … has been well characterised concerning the utilisation of numerous carbohydrates as carbon source. However, its amino acid metabolism, especially the degradation of single amino acids, is not as well understood. In this work, we performed metabolic modelling as well as metabolome, transcriptome and proteome analysis on cells grown on caseinhydrolysate as carbon source in order to draw a comprehensive picture of amino acid metabolism in S. solfataricus P2. We found that 10 out of 16 detectable amino acids are imported from the growth medium. Overall, uptake of glutamate, methionine, leucine, phenylalanine and isoleucine was the highest of all observed amino acids. Our simulations predict an incomplete degradation of leucine and tyrosine to organic acids, and in accordance with this, we detected the export of branched-chain and aromatic organic acids as well as amino acids, ammonium and trehalose into the culture supernatants. The branched-chain amino acids as well as phenylalanine and tyrosine are degraded to organic acids via oxidative Stickland reactions. Such reactions are known for prokaryotes capable of anaerobic growth, but so far have never been observed in an obligate aerobe. Also, 3-methyl-2-butenoate and 2-methyl-2-butenoate are for the first time found as products of modified Stickland reactions for the degradation of branched-chain amino acids. This work presents the first detailed description of branched-chain and aromatic amino acid catabolism in S. solfataricus.

Authors: Helge Stark, Jacqueline Wolf, Andreas Albersmeier, Trong K. Pham, Julia D. Hofmann, Bettina Siebers, Jörn Kalinowski, Phillip C. Wright, Meina Neumann-Schaal, Dietmar Schomburg

Date Published: 29th May 2017

Publication Type: Not specified

Protein abundance of AKT and ERK pathway components governs cell type-specific regulation of proliferation.

Abstract (Expand)

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, … is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro-proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type-specific proliferation. First, cell type-specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate-limiting for faster cycling cells while slower cell cycles are controlled at the G1-S progression. The integrated mathematical model of Epo-driven proliferation explains cell type-specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti-proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

Authors: L. Adlung, S. Kar, M. C. Wagner, B. She, S. Chakraborty, J. Bao, S. Lattermann, M. Boerries, H. Busch, P. Wuchter, A. D. Ho, J. Timmer, M. Schilling, T. Hofer, U. Klingmuller

Date Published: 24th Jan 2017

Publication Type: Journal

A systems biology approach reveals major metabolic changes in the thermoacidophilic archaeon Sulfolobus solfataricus in response to the carbon source L-fucose versus D-glucose

Abstract (Expand)

Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known … for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, we analyse growth on different carbon sources using an integrated systems biology approach. The comparison of growth on L-fucose and D-glucose allows first insights into the genome-wide changes in response to the two carbon sources and revealed a new pathway for L-fucose degradation in S. solfataricus. During growth on L-fucose we observed major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3-hydroxypropionate/4-hydroxybutyrate cycle. Within the newly discovered pathway for L-fucose degradation the following key reactions were identified: (i) L-fucose oxidation to L-fuconate via a dehydrogenase, (ii) dehydration to 2-keto-3-deoxy-L-fuconate via dehydratase, (iii) 2-keto-3-deoxy-L-fuconate cleavage to pyruvate and L-lactaldehyde via aldolase and (iv) L-lactaldehyde conversion to L-lactate via aldehyde dehydrogenase. This pathway as well as L-fucose transport shows interesting overlaps to the D-arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species. This article is protected by copyright. All rights reserved.

Authors: J. Wolf, H. Stark, K. Fafenrot, A. Albersmeier, T. K. Pham, K. B. Muller, B. Meyer, L. Hoffmann, L. Shen, S. P. Albaum, T. Kouril, K. Schmidt-Hohagen, M. Neumann-Schaal, C. Brasen, J. Kalinowski, P. C. Wright, S. V. Albers, D. Schomburg, B. Siebers

Date Published: 10th Sep 2016

Publication Type: Not specified

L1 regularization facilitates detection of cell type-specific parameters in dynamical systems

Abstract (Expand)

MOTIVATION: A major goal of drug development is to selectively target certain cell types. Cellular decisions influenced by drugs are often dependent on the dynamic processing of information. Selective … responses can be achieved by differences between the involved cell types at levels of receptor, signaling, gene regulation or further downstream. Therefore, a systematic approach to detect and quantify cell type-specific parameters in dynamical systems becomes necessary. RESULTS: Here, we demonstrate that a combination of nonlinear modeling with L1 regularization is capable of detecting cell type-specific parameters. To adapt the least-squares numerical optimization routine to L1 regularization, sub-gradient strategies as well as truncation of proposed optimization steps were implemented. Likelihood-ratio tests were used to determine the optimal regularization strength resulting in a sparse solution in terms of a minimal number of cell type-specific parameters that is in agreement with the data. By applying our implementation to a realistic dynamical benchmark model of the DREAM6 challenge we were able to recover parameter differences with an accuracy of 78%. Within the subset of detected differences, 91% were in agreement with their true value. Furthermore, we found that the results could be improved using the profile likelihood. In conclusion, the approach constitutes a general method to infer an overarching model with a minimum number of individual parameters for the particular models. AVAILABILITY AND IMPLEMENTATION: A MATLAB implementation is provided within the freely available, open-source modeling environment Data2Dynamics. Source code for all examples is provided online at http://www.data2dynamics.org/ CONTACT: bernhard.steiert@fdm.uni-freiburg.de.

Authors: B. Steiert, J. Timmer, C. Kreutz

Date Published: 3rd Sep 2016

Publication Type: Not specified

Driving the Model to Its Limit: Profile Likelihood Based Model Reduction

Abstract (Expand)

In systems biology, one of the major tasks is to tailor model complexity to information content of the data. A useful model should describe the data and produce well-determined parameter estimates and … predictions. Too small of a model will not be able to describe the data whereas a model which is too large tends to overfit measurement errors and does not provide precise predictions. Typically, the model is modified and tuned to fit the data, which often results in an oversized model. To restore the balance between model complexity and available measurements, either new data has to be gathered or the model has to be reduced. In this manuscript, we present a data-based method for reducing non-linear models. The profile likelihood is utilised to assess parameter identifiability and designate likely candidates for reduction. Parameter dependencies are analysed along profiles, providing context-dependent suggestions for the type of reduction. We discriminate four distinct scenarios, each associated with a specific model reduction strategy. Iterating the presented procedure eventually results in an identifiable model, which is capable of generating precise and testable predictions. Source code for all toy examples is provided within the freely available, open-source modelling environment Data2Dynamics based on MATLAB available at http://www.data2dynamics.org/, as well as the R packages dMod/cOde available at https://github.com/dkaschek/. Moreover, the concept is generally applicable and can readily be used with any software capable of calculating the profile likelihood.

Authors: T. Maiwald, H. Hass, B. Steiert, J. Vanlier, R. Engesser, A. Raue, F. Kipkeew, H. H. Bock, D. Kaschek, C. Kreutz, J. Timmer

Date Published: 3rd Sep 2016

Publication Type: Not specified

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

Abstract (Expand)

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs … such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.

Authors: R. Merkle, B. Steiert, F. Salopiata, S. Depner, A. Raue, N. Iwamoto, M. Schelker, H. Hass, M. Wasch, M. E. Bohm, O. Mucke, D. B. Lipka, C. Plass, W. D. Lehmann, C. Kreutz, J. Timmer, M. Schilling, U. Klingmuller

Date Published: 6th Aug 2016

Publication Type: Journal

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

Abstract (Expand)

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good … manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion.

Authors: P. Wuchter, M. Vetter, R. Saffrich, A. Diehlmann, K. Bieback, A. D. Ho, P. Horn

Date Published: 26th Feb 2016

Publication Type: Journal

Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells.

Abstract (Expand)

In previous studies human mesenchymal stromal cells (MSCs) maintained the "stemness" of human hematopoietic progenitor cells (HPCs) through direct cell-cell contact in two-dimensional co-culture … systems. We establish a three-dimensional (3D) co-culture system based on a custom-made chip, the 3(D)-KITChip, as an in vitro model system of the human hematopoietic stem cell niche. This array of up to 625 microcavities, with 300 mum size in each orientation, was inserted into a microfluidic bioreactor. The microcavities of the 3(D)-KITChip were inoculated with human bone marrow MSCs together with umbilical cord blood HPCs. MSCs used the microcavities as a scaffold to build a complex 3D mesh. HPCs were distributed three-dimensionally inside this MSC network and formed ss-catenin- and N-cadherin-based intercellular junctions to the surrounding MSCs. Using RT(2)-PCR and western blots, we demonstrate that a proportion of HPCs maintained the expression of CD34 throughout a culture period of 14 days. In colony-forming unit assays, the hematopoietic stem cell plasticity remained similar after 14 days of bioreactor co-culture, whereas monolayer co-cultures showed increasing signs of HPC differentiation and loss of stemness. These data support the notion that the 3D microenvironment created within the microcavity array preserves vital stem cell functions of HPCs more efficiently than conventional co-culture systems.

Authors: P. Wuchter, R. Saffrich, S. Giselbrecht, C. Nies, H. Lorig, S. Kolb, A. D. Ho, E. Gottwald

Date Published: 3rd Feb 2016

Publication Type: Journal

Context-specific flow through the MEK/ERK module produces cell- and ligand-specific patterns of ERK single and double phosphorylation.

Abstract (Expand)

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of … the MAPK kinase MEK and its targets extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK.

Authors: N. Iwamoto, L. A. D'Alessandro, S. Depner, B. Hahn, B. A. Kramer, P. Lucarelli, A. Vlasov, M. Stepath, M. E. Bohm, D. Deharde, G. Damm, D. Seehofer, W. D. Lehmann, U. Klingmuller, M. Schilling

Date Published: 2nd Feb 2016

Publication Type: Journal

Data2Dynamics: a modeling environment tailored to parameter estimation in dynamical systems.

Abstract (Expand)

UNLABELLED: Modeling of dynamical systems using ordinary differential equations is a popular approach in the field of systems biology. Two of the most critical steps in this approach are to construct … dynamical models of biochemical reaction networks for large datasets and complex experimental conditions and to perform efficient and reliable parameter estimation for model fitting. We present a modeling environment for MATLAB that pioneers these challenges. The numerically expensive parts of the calculations such as the solving of the differential equations and of the associated sensitivity system are parallelized and automatically compiled into efficient C code. A variety of parameter estimation algorithms as well as frequentist and Bayesian methods for uncertainty analysis have been implemented and used on a range of applications that lead to publications. AVAILABILITY AND IMPLEMENTATION: The Data2Dynamics modeling environment is MATLAB based, open source and freely available at http://www.data2dynamics.org. CONTACT: andreas.raue@fdm.uni-freiburg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Authors: A. Raue, B. Steiert, M. Schelker, C. Kreutz, T. Maiwald, H. Hass, J. Vanlier, C. Tonsing, L. Adlung, R. Engesser, W. Mader, T. Heinemann, J. Hasenauer, M. Schilling, T. Hofer, E. Klipp, F. Theis, U. Klingmuller, B. Schoberl, J. Timmer

Date Published: 1st Nov 2015

Publication Type: Journal

Disentangling the Complexity of HGF Signaling by Combining Qualitative and Quantitative Modeling.

Abstract (Expand)

Signaling pathways are characterized by crosstalk, feedback and feedforward mechanisms giving rise to highly complex and cell-context specific signaling networks. Dissecting the underlying relations … is crucial to predict the impact of targeted perturbations. However, a major challenge in identifying cell-context specific signaling networks is the enormous number of potentially possible interactions. Here, we report a novel hybrid mathematical modeling strategy to systematically unravel hepatocyte growth factor (HGF) stimulated phosphoinositide-3-kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling, which critically contribute to liver regeneration. By combining time-resolved quantitative experimental data generated in primary mouse hepatocytes with interaction graph and ordinary differential equation modeling, we identify and experimentally validate a network structure that represents the experimental data best and indicates specific crosstalk mechanisms. Whereas the identified network is robust against single perturbations, combinatorial inhibition strategies are predicted that result in strong reduction of Akt and ERK activation. Thus, by capitalizing on the advantages of the two modeling approaches, we reduce the high combinatorial complexity and identify cell-context specific signaling networks.

Authors: L. A. D'Alessandro, R. Samaga, T. Maiwald, S. H. Rho, S. Bonefas, A. Raue, N. Iwamoto, A. Kienast, K. Waldow, R. Meyer, M. Schilling, J. Timmer, S. Klamt, U. Klingmuller

Date Published: 24th Apr 2015

Publication Type: Journal

Identification of isoform-specific dynamics in phosphorylation-dependent STAT5 dimerization by quantitative mass spectrometry and mathematical modeling.

Abstract (Expand)

STAT5A and STAT5B are important transcription factors that dimerize and transduce activation signals of cytokine receptors directly to the nucleus. A typical cytokine that mediates STAT5 activation is … erythropoietin (Epo). Differential functions of STAT5A and STAT5B have been reported. However, the extent to which phosphorylated STAT5A and STAT5B (pSTAT5A, pSTAT5B) form homo- or heterodimers is not understood, nor is how this might influence the signal transmission to the nucleus. To study this, we designed a concept to investigate the isoform-specific dimerization behavior of pSTAT5A and pSTAT5B that comprises isoform-specific immunoprecipitation (IP), measurement of the degree of phosphorylation, and isoform ratio determination between STAT5A and STAT5B. For the main analytical method, we employed quantitative label-free and -based mass spectrometry. For the cellular model system, we used Epo receptor (EpoR)-expressing BaF3 cells (BaF3-EpoR) stimulated with Epo. Three hypotheses of dimer formation between pSTAT5A and pSTAT5B were used to explain the analytical results by a static mathematical model: formation of (i) homodimers only, (ii) heterodimers only, and (iii) random formation of homo- and heterodimers. The best agreement between experimental data and model simulations was found for the last case. Dynamics of cytoplasmic STAT5 dimerization could be explained by distinct nuclear import rates and individual nuclear retention for homo- and heterodimers of phosphorylated STAT5.

Authors: M. E. Boehm, L. Adlung, M. Schilling, S. Roth, U. Klingmuller, W. D. Lehmann

Date Published: 5th Dec 2014

Publication Type: Journal

Mesenchymal stromal cells inhibit proliferation of virus-specific CD8(+) T cells.

Abstract (Expand)

Mesenchymal stromal cells (MSCs) possess broad immunomodulatory capacities that are currently investigated for potential clinical application in treating autoimmune disorders. Third-party MSCs suppress … alloantigen-induced proliferation of peripheral blood mononuclear cells providing the rationale for clinical use in graft-versus-host disease (GvHD). We confirmed that MSCs strongly inhibited proliferation of CD8(+) T cells in a mixed lymphocyte reaction. However, MSCs also suppressed proliferation of T cells specifically recognizing cytomegalovirus (CMV) and influenza virus. Inhibition was dose dependent, but independent of the culture medium. MSCs inhibited proliferation of specific CD8(+) T cells and the release of IFN-gamma by specific CD8(+) T cells for immunodominant HLA-A2- and HLA-B7- restricted antigen epitopes derived from CMV phosphoprotein 65 and influenza matrix protein. This is in contrast to a recently reported scenario where MSCs exert differential effects on alloantigen and virus-specific T cells potentially having an impact on surveillance and prophylaxis of patients treated by MSCs.

Authors: G. Malcherek, N. Jin, A. G. Huckelhoven, J. Mani, L. Wang, U. Gern, A. Diehlmann, P. Wuchter, A. Schmitt, B. Chen, A. D. Ho, M. Schmitt

Date Published: 18th Sep 2014

Publication Type: Journal

Cause and cure of sloppiness in ordinary differential equation models

Abstract (Expand)

Data-based mathematical modeling of biochemical reaction networks, e.g., by nonlinear ordinary differential equation (ODE) models, has been successfully applied. In this context, parameter estimation … and uncertainty analysis is a major task in order to assess the quality of the description of the system by the model. Recently, a broadened eigenvalue spectrum of the Hessian matrix of the objective function covering orders of magnitudes was observed and has been termed as sloppiness. In this work, we investigate the origin of sloppiness from structures in the sensitivity matrix arising from the properties of the model topology and the experimental design. Furthermore, we present strategies using optimal experimental design methods in order to circumvent the sloppiness issue and present nonsloppy designs for a benchmark model.

Authors: Christian Tönsing, Jens Timmer, Clemens Kreutz

Date Published: 1st Aug 2014

Publication Type: Not specified

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

Abstract (Expand)

BACKGROUND AIMS: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different … MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Authors: P. Wuchter, K. Bieback, H. Schrezenmeier, M. Bornhauser, L. P. Muller, H. Bonig, W. Wagner, R. Meisel, P. Pavel, T. Tonn, P. Lang, I. Muller, M. Renner, G. Malcherek, R. Saffrich, E. C. Buss, P. Horn, M. Rojewski, A. Schmitt, A. D. Ho, R. Sanzenbacher, M. Schmitt

Date Published: 27th May 2014

Publication Type: Journal

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells.

Abstract (Expand)

The interaction between the stromal cell-derived factor-1 alpha (SDF-1alpha, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The … CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1alpha axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1alpha levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1alpha significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1alpha interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1alpha gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1alpha axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.

Authors: P. Wuchter, C. Leinweber, R. Saffrich, M. Hanke, V. Eckstein, A. D. Ho, M. Grunze, A. Rosenhahn

Date Published: 17th Dec 2013

Publication Type: Journal

Differences between healthy hematopoietic progenitors and leukemia cells with respect to CD44 mediated rolling versus adherence behavior on hyaluronic acid coated surfaces.

Abstract (Expand)

We previously demonstrated that leukemia cell lines expressing CD44 and hematopoietic progenitor cells (HPC) from umbilical cord blood (CB) showed rolling on hyaluronic acid (HA)-coated surfaces under … physiological shear stress. In the present study, we quantitatively assessed the interaction of HPC derived from CB, mobilized peripheral blood (mPB) and bone marrow (BM) from healthy donors, as well as primary leukemia blasts from PB and BM of patients with acute myeloid leukemia (AML) with HA. We have demonstrated that HPC derived from healthy donors showed relative homogeneous rolling and adhesion to HA. In contrast, highly diverse behavioral patterns were found for leukemia blasts under identical conditions. The monoclonal CD44 antibody (clone BU52) abrogated the shear stress-induced rolling of HPC and leukemia blasts, confirming the significance of CD44 in this context. On the other hand, the immobile adhesion of leukemia blasts to the HA-coated surface was, in some cases, not or incompletely inhibited by BU52. The latter property was associated with non-responsiveness to induction chemotherapy and subsequently poor clinical outcome.

Authors: M. Hanke, I. Hoffmann, C. Christophis, M. Schubert, V. T. Hoang, A. Zepeda-Moreno, N. Baran, V. Eckstein, P. Wuchter, A. Rosenhahn, A. D. Ho

Date Published: 26th Nov 2013

Publication Type: Journal

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor.

Abstract (Expand)

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C … chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1alpha (SDF-1alpha) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro. METHODS: We monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor. RESULTS: Co-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34(+) cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations-and conversely their hematopoiesis supportive functions-may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor. CONCLUSIONS: These results support the notion that the CXCR4/SDF-1alpha axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.

Authors: A. Ludwig, R. Saffrich, V. Eckstein, T. Bruckner, W. Wagner, A. D. Ho, P. Wuchter

Date Published: 15th Oct 2013

Publication Type: Journal

[Mesenchymal stroma cells (MSCs) for the treatment of rheumatic disease].

Abstract

Not specified

Authors: M. Schmitt, L. P. Muller, G. Keysser, H. M. Lorenz, A. D. Ho, P. Wuchter

Date Published: 6th Sep 2013

Publication Type: Journal

Excemplify: a flexible template based solution, parsing and managing data in spreadsheets for experimentalists.

Abstract (Expand)

In systems biology, quantitative experimental data is the basis of building mathematical models. In most of the cases, they are stored in Excel files and hosted locally. To have a public database for … collecting, retrieving and citing experimental raw data as well as experimental conditions is important for both experimentalists and modelers. However, the great effort needed in the data handling procedure and in the data submission procedure becomes the crucial limitation for experimentalists to contribute to a database, thereby impeding the database to deliver its benefit. Moreover, manual copy and paste operations which are commonly used in those procedures increase the chance of making mistakes. Excemplify, a web-based application, proposes a flexible and adaptable template-based solution to solve these problems. Comparing to the normal template based uploading approach, which is supported by some public databases, rather than predefining a format that is potentiall impractical, Excemplify allows users to create their own experiment-specific content templates in different experiment stages and to build corresponding knowledge bases for parsing. Utilizing the embedded knowledge of used templates, Excemplify is able to parse experimental data from the initial setup stage and generate following stages spreadsheets automatically. The proposed solution standardizes the flows of data traveling according to the standard procedures of applying the experiment, cuts down the amount of manual effort and reduces the chance of mistakes caused by manual data handling. In addition, it maintains the context of meta-data from the initial preparation manuscript and improves the data consistency. It interoperates and complements RightField and SEEK as well.

Authors: L. Shi, L. Jong, U. Wittig, P. Lucarelli, M. Stepath, S. Mueller, L. A. D'Alessandro, U. Klingmuller, W. Muller

Date Published: 3rd Apr 2013

Publication Type: Journal

Covering a broad dynamic range: information processing at the erythropoietin receptor.

Abstract (Expand)

Cell surface receptors convert extracellular cues into receptor activation, thereby triggering intracellular signaling networks and controlling cellular decisions. A major unresolved issue is the … identification of receptor properties that critically determine processing of ligand-encoded information. We show by mathematical modeling of quantitative data and experimental validation that rapid ligand depletion and replenishment of the cell surface receptor are characteristic features of the erythropoietin (Epo) receptor (EpoR). The amount of Epo-EpoR complexes and EpoR activation integrated over time corresponds linearly to ligand input; this process is carried out over a broad range of ligand concentrations. This relation depends solely on EpoR turnover independent of ligand binding, which suggests an essential role of large intracellular receptor pools. These receptor properties enable the system to cope with basal and acute demand in the hematopoietic system.

Authors: V. Becker, M. Schilling, J. Bachmann, U. Baumann, A. Raue, T. Maiwald, J. Timmer, U. Klingmuller

Date Published: 11th Jun 2010

Publication Type: Journal

Computational processing and error reduction strategies for standardized quantitative data in biological networks.

Abstract (Expand)

High-quality quantitative data generated under standardized conditions is critical for understanding dynamic cellular processes. We report strategies for error reduction, and algorithms for automated … data processing and for establishing the widely used techniques of immunoprecipitation and immunoblotting as highly precise methods for the quantification of protein levels and modifications. To determine the stoichiometry of cellular components and to ensure comparability of experiments, relative signals are converted to absolute values. A major source for errors in blotting techniques are inhomogeneities of the gel and the transfer procedure leading to correlated errors. These correlations are prevented by randomized gel loading, which significantly reduces standard deviations. Further error reduction is achieved by using housekeeping proteins as normalizers or by adding purified proteins in immunoprecipitations as calibrators in combination with criteria-based normalization. Additionally, we developed a computational tool for automated normalization, validation and integration of data derived from multiple immunoblots. In this way, large sets of quantitative data for dynamic pathway modeling can be generated, enabling the identification of systems properties and the prediction of targets for efficient intervention.

Authors: M. Schilling, T. Maiwald, S. Bohl, M. Kollmann, C. Kreutz, J. Timmer, U. Klingmuller

Date Published: 13th Dec 2005

Publication Type: Journal

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