Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO

Hannover, Germany

Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO

Hannover, Germany
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Ackermann M.,Hannover Medical School | Ackermann M.,Hannover Medical School | Kuhn A.,Hannover Medical School | Kuhn A.,Hannover Medical School | And 10 more authors.
Transfusion Medicine and Hemotherapy | Year: 2017

Background: Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), have the capacity to differentiate towards all three germ layers and have been highlighted as an attractive cell source for the field of regenerative medicine. Thus, stable expression of therapeutic transgenes in iPSCs, as well as thereof derived progeny of hematopoietic lineage, may lay the foundation for innovative cell replacement therapies. Methods: We have utilized human iPSC lines genetically modified by lentiviral vector technology or targeted integration of reporter genes to evaluate transgene expression during hematopoietic specification and differentiation towards macrophages. Results: Use of lentiviral vectors equipped with an ubiquitous chromatin opening element (CBX3-UCOE) as well as zinc finger nuclease-mediated targeting of an expression cassette into the human adeno-Associated virus integration site 1 (AAVS1) safe harbor resulted in stable transgene expression in iPSCs. When iPSCs were differentiated along the myeloid pathway into macrophages, both strategies yielded sustained transgene expression during the hematopoietic specification process including mature CD14+ and CD11b+ macrophages. Conclusion: Combination of human iPSC technology with either lentiviral vector technology or designer nuclease-based genome editing allows for the generation of transgenic iPSC-derived macrophages with stable transgene expression which may be useful for novel cell and gene replacement therapies. © 2017 S. Karger GmbH, Freiburg.


Deriu M.A.,University of Applied Sciences and Arts Southern Switzerland | Grasso G.,University of Applied Sciences and Arts Southern Switzerland | Massai D.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Danani A.,University of Applied Sciences and Arts Southern Switzerland
Proteins: Structure, Function and Bioinformatics | Year: 2016

Ataxin-1 is the protein responsible for the Spinocerebellar ataxia type 1, an incurable neurodegenerative disease caused by polyglutamine expansion. The AXH domain plays a pivotal role in physiological functions of Ataxin-1. In Spinocerebellar ataxia 1, the AXH domain is involved in the misfolding and aggregation pathway. Here molecular modeling is applied to investigate the protein-protein interactions contributing to the AXH dimer stability. Particular attention is focused on: (i) the characterization of AXH monomer-monomer interface; (ii) the molecular description of the AXH monomer-monomer interaction dynamics. Technically, an approach based on functional mode analysis, here applied to replica exchange molecular dynamics trajectories, was employed. The findings of this study are consistent with previous experimental results and elucidate the pivotal role of the I580 residue in mediating the AXH monomer-monomer interaction dynamics. © 2016 Wiley Periodicals, Inc.


Katsirntaki K.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Katsirntaki K.,Hannover Medical School | Katsirntaki K.,Biomedical Research in Endstage and Obstructive Lung Disease Hanover BREATH | Mauritz C.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | And 29 more authors.
Tissue Engineering - Part A | Year: 2015

Respiratory progenitors can be efficiently generated from pluripotent stem cells (PSCs). However, further targeted differentiation into bronchoalveolar sublineages is still in its infancy, and distinct specifying effects of key differentiation factors are not well explored. Focusing on airway epithelial Clara cell generation, we analyzed the effect of the glucocorticoid dexamethasone plus cAMP-elevating agents (DCI) on the differentiation of murine embryonic and induced pluripotent stem cells (iPSCs) into bronchoalveolar epithelial lineages, and whether keratinocyte growth factor (KGF) might further influence lineage decisions. We demonstrate that DCI strongly induce expression of the Clara cell marker Clara cell secretory protein (CCSP). While KGF synergistically supports the inducing effect of DCI on alveolar markers with increased expression of surfactant protein (SP)-C and SP-B, an inhibitory effect on CCSP expression was shown. In contrast, neither KGF nor DCI seem to have an inducing effect on ciliated cell markers. Furthermore, the use of iPSCs from transgenic mice with CCSP promoter-dependent lacZ expression or a knockin of a YFP reporter cassette in the CCSP locus enabled detection of derivatives with Clara cell typical features. Collectively, DCI was shown to support bronchoalveolar specification of mouse PSCs, in particular Clara-like cells, and KGF to inhibit bronchial epithelial differentiation. The targeted in vitro generation of Clara cells with their important function in airway protection and regeneration will enable the evaluation of innovative cellular therapies in animal models of lung diseases. © Mary Ann Liebert, Inc. 2015.


Kuetemeyer K.,Laser Zentrum Hannover e.V. | Kensah G.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Heidrich M.,Laser Zentrum Hannover e.V. | Meyer H.,Laser Zentrum Hannover e.V. | And 3 more authors.
Optics Express | Year: 2011

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies. © 2011 Optical Society of America.


Olmer R.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Olmer R.,Hannover Medical School | Lange A.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Lange A.,Hannover Medical School | And 8 more authors.
Tissue Engineering - Part C: Methods | Year: 2012

Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end, we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption, but in parallel functional integration of all probes mandatory for process monitoring, that is, for pO 2 and pH, experiments were performed in 100mL culture volume in a "mini reactor platform" consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×108 hiPSCs/100mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained, underlining practical utility of this new process. The presented data provide key steps toward scalable mass expansion of human iPS and ES cells thereby enabling translation of stem cell research to (pre)clinical application in relevant large animal models and valuable in vitro assays for drug development and validation as well. © Copyright 2012, Mary Ann Liebert, Inc.


Vukadinovic-Nikolic Z.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Andree B.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Dorfman S.E.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | Pflaum M.,Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO | And 11 more authors.
Tissue Engineering - Part A | Year: 2014

The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for the repair of ischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The main drawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus of this study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patch by combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineering an artificial tissue of 11 cm2 in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) in SIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were co-isolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize pre-existing vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CC contracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast to undirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology with well-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shaped and random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrode array measurements. A dense network of CD31+/eNOS+ cells was detected as permeating the whole construct. Superficial supplementation of RHE-A cells to SIS-CC led to the migration of these cells through the CC, resulting in the re-population of pre-existing vessel structures within the decelluarized SIS. By infusion of RHE-A cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well as distribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contact with RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaM represent a promising step toward engineering a functional perfusion system, enabling the engineering of vascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair. © 2014 Mary Ann Liebert, Inc.


PubMed | Hannover Medical School, Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO and Helmholtz Center for Infection Research
Type: | Journal: Molecular medicine (Cambridge, Mass.) | Year: 2016

Platelet (PLT) transfusion is indispensable to maintain homeostasis in thrombocytopenic patients. However, PLT transfusion refractoriness is a common life-threatening condition observed in multi-transfused patients. The most frequent immune cause for PLT transfusion refractoriness is the presence of alloantibodies specific for human leukocyte antigen (HLA) class I epitopes. Here, we have silenced the expression of HLA class I to generate a stable HLA-universal induced pluripotent stem cell (iPSC) line that can be used as a renewable cell source for the generation of low immunogenic cell products. The expression of HLA class I was silenced by up to 82% and remained stable during iPSC cultivation. In this study, we have focused on the generation of megakaryocytes (MK) and PLTs from a HLA-universal iPSC source under feeder-free and xeno-free conditions. On day 19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after


PubMed | Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO
Type: Journal Article | Journal: Tissue engineering. Part C, Methods | Year: 2012

Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end, we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption, but in parallel functional integration of all probes mandatory for process monitoring, that is, for pO and pH, experiments were performed in 100mL culture volume in a mini reactor platform consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 210 hiPSCs/100mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained, underlining practical utility of this new process. The presented data provide key steps toward scalable mass expansion of human iPS and ES cells thereby enabling translation of stem cell research to (pre)clinical application in relevant large animal models and valuable in vitro assays for drug development and validation as well.

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