• High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

      Hemmen, Katherina; Reinl, Tobias; Buttler, Kerstin; Behler, Friederike; Dieken, Hauke; Jänsch, Lothar; Wilting, Jörg; Weich, Herbert A; Department of Gene Regulation, HZI, Build. D, Inhoffenstr. 7, 38124, Braunschweig, Germany. katharina.hemmen@ewetel.net (2011-05)
      Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
    • Integrated strategy for the production of therapeutic retroviral vectors.

      Carrondo, Manuel; Panet, Amos; Wirth, Dagmar; Coroadinha, Ana Sofia; Cruz, Pedro; Falk, Haya; Schucht, Roland; Dupont, Francis; Geny-Fiamma, Cécile; Merten, Otto-Wilhelm; et al. (2011-03)
      The broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.
    • Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis.

      Oberhardt, Matthew A; Puchałka, Jacek; Martins dos Santos, Vítor A P; Papin, Jason A; Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America. (2011-03)
      In the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.