REBIRTH Cluster of Excellence

Hannover, Germany

REBIRTH Cluster of Excellence

Hannover, Germany
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Kellner M.,Hannover Medical School | Heidrich M.,Laser Zentrum Hannover e.V. | Beigel R.,Hannover Medical School | Lorbeer R.-A.,Laser Zentrum Hannover e.V. | And 11 more authors.
Journal of Applied Physiology | Year: 2012

The current study focuses on the use of scanning laser optical tomography (SLOT) in imaging of the mouse lung ex vivo. SLOT is a highly efficient fluorescence microscopy technique allowing rapid scanning of samples of a size of several millimeters, thus enabling volumetric visualization by using intrinsic contrast mechanisms of previously fixed lung lobes. Here, we demonstrate the imaging of airways, blood vessels, and parenchyma from whole, optically cleared mouse lung lobes with a resolution down to the level of single alveoli using absorption and autofluorescence scan modes. The internal structure of the lung can then be analyzed nondestructively and quantitatively in three-dimensional datasets in any preferred planar orientation. Moreover, the procedure preserves the microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, the current study has shown that SLOT is a valuable technique to study the internal structure of the mouse lung. Copyright © 2012 the American Physiological Society.


Limbourg A.,Research Group Regenerative Agents | Limbourg A.,Integrated Research Center Transplantation Tx | Schnabel S.,Research Group Regenerative Agents | Lozanovski V.J.,Research Group Regenerative Agents | And 14 more authors.
Cell Regeneration | Year: 2014

The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4+ cells in postnatal mice. While the prevalence of GFP+ cells in testis rapidly declined with age, the skin-resident GFP+ population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP+ epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming. © 2014 Limbourg et al.; licensee BioMed Central Ltd.


Schmeckebier S.,Hannover Medical School | Schmeckebier S.,German Center for Lung Research | Mauritz C.,Hannover Medical School | Mauritz C.,German Center for Lung Research | And 26 more authors.
Tissue Engineering - Part A | Year: 2013

Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application. © Copyright 2013, Mary Ann Liebert, Inc. 2013.


PubMed | Research Group Regenerative Agents, Hannover Medical School, Hanover Veterinary School, REBIRTH Cluster of Excellence and 2 more.
Type: Journal Article | Journal: Cell regeneration (London, England) | Year: 2014

The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4 (+) cells in postnatal mice. While the prevalence of GFP(+) cells in testis rapidly declined with age, the skin-resident GFP(+) population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP(+) epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming.


Heidrich M.,Laser Zentrum Hannover e.V. | Kellner M.,Hannover Medical School | Beigel R.,Hannover Medical School | Lorbeer R.-A.,Laser Zentrum Hannover e.V. | And 12 more authors.
Progress in Biomedical Optics and Imaging - Proceedings of SPIE | Year: 2013

New optical techniques have the potential to fill the gap between radiological and microscopic approaches to assess the lung's internal structure. Since its quantitative assessment requires unbiased sampling and measurement principles, imaging of the whole lung with sufficient resolution for visualizing details is important. To address this request, we applied scanning laser optical tomography (SLOT) for the three dimensional imaging of mouse lung ex vivo. SLOT is a highly efficient flourescence and transmission microscopy technique allowing for 3D imaging of specimen of sizes up to several millimeters. Previously fixed lung lobes and whole lungs were optically cleared and subsequently imaged with SLOT while making use of intrinsic contrast mechanisms like absorption and autofluorescence. Imaging of airways, blood vessels and parenchyma is demonstrated. Volumetric SLOT datasets of the lung's internal structure can be analyzed in any preferred planar orientation. Moreover, the sample preparation preserves microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, SLOT is a useful technique to visualize and survey the internal structure of mouse lung at different scales and with various contrast mechanisms. Potential applications of SLOT in lung research are e.g. quantitative phenotype analysis of mouse models of human lung disease in combination with stereological methods. © 2013 SPIE.


Grothausmann R.,Hannover Medical School | Kellner M.,Hannover Medical School | Kellner M.,Biomedical Research in Endstage and Obstructive Lung Disease Hanover BREATH | Heidrich M.,Laser Zentrum Hannover e.V. | And 10 more authors.
Computational and Mathematical Methods in Medicine | Year: 2015

In lungs the number of conducting airway generations as well as bifurcation patterns varies across species and shows specific characteristics relating to illnesses or gene variations. A method to characterize the topology of the mouse airway tree using scanning laser optical tomography (SLOT) tomograms is presented in this paper. It is used to test discrimination between two types of mice based on detected differences in their conducting airway pattern. Based on segmentations of the airways in these tomograms, the main spanning tree of the volume skeleton is computed. The resulting graph structure is used to distinguish between wild type and surfactant protein (SP-D) deficient knock-out mice. © 2015 Roman Grothausmann et al.


Becker S.,Hannover Medical School | Steinmeyer J.,Hannover Medical School | Avsar M.,Hannover Medical School | Hoffler K.,Hannover Medical School | And 10 more authors.
Transplant International | Year: 2016

Normothermic ex vivo lung perfusion (EVLP) has developed as a powerful technique to evaluate particularly marginal donor lungs prior to transplantation. In this study, acellular and cellular perfusate compositions were compared in an identical experimental setting as no consensus has been reached on a preferred technique yet. Porcine lungs underwent EVLP for 12 h on the basis of an acellular or a cellular perfusate composition after 24 h of cold ischaemia as defined organ stress. During perfusion, haemodynamic and respiratory parameters were monitored. After EVLP, the lung condition was assessed by light and transmission electron microscopy. Aerodynamic parameters did not show significant differences between groups and remained within the in vivo range during EVLP. Mean oxygenation indices were 491 ± 39 in the acellular group and 513 ± 53 in the cellular group. Groups only differed significantly in terms of higher pulmonary artery pressure and vascular resistance in the cellular group. Lung histology and ultrastructure were largely well preserved after prolonged EVLP and showed only minor structural alterations which were similarly present in both groups. Prolonged acellular and cellular EVLP for 12 h are both feasible with lungs prechallenged by ischaemic organ stress. Physiological and ultrastructural analysis showed no superiority of either acellular or cellular perfusate composition. © 2015 Steunstichting ESOT.


Warlich E.,Miltenyi Biotec GmbH | Warlich E.,Hannover Medical School | Schambach A.,Hannover Medical School | Schambach A.,Harvard University | And 7 more authors.
PLoS ONE | Year: 2014

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues. © 2014 Warlich et al.


PubMed | Hannover Medical School and REBIRTH Cluster of Excellence
Type: Comparative Study | Journal: Transplant international : official journal of the European Society for Organ Transplantation | Year: 2016

Normothermic ex vivo lung perfusion (EVLP) has developed as a powerful technique to evaluate particularly marginal donor lungs prior to transplantation. In this study, acellular and cellular perfusate compositions were compared in an identical experimental setting as no consensus has been reached on a preferred technique yet. Porcine lungs underwent EVLP for 12 h on the basis of an acellular or a cellular perfusate composition after 24 h of cold ischaemia as defined organ stress. During perfusion, haemodynamic and respiratory parameters were monitored. After EVLP, the lung condition was assessed by light and transmission electron microscopy. Aerodynamic parameters did not show significant differences between groups and remained within the in vivo range during EVLP. Mean oxygenation indices were 491 39 in the acellular group and 513 53 in the cellular group. Groups only differed significantly in terms of higher pulmonary artery pressure and vascular resistance in the cellular group. Lung histology and ultrastructure were largely well preserved after prolonged EVLP and showed only minor structural alterations which were similarly present in both groups. Prolonged acellular and cellular EVLP for 12 h are both feasible with lungs prechallenged by ischaemic organ stress. Physiological and ultrastructural analysis showed no superiority of either acellular or cellular perfusate composition.

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