A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product

Introduction: Mesenchymal stromal/stem cells (MSCs) derived from fat tissue are an encouraging tool for regenerative medicine. They share properties similar to the bone marrow-derived MSCs, but the amount of MSCs per gram of fat tissue is 500x higher. The fat tissue can easily be digested by collagenase, releasing a heterogeneous cell fraction called stromal vascular fraction (SVF) which contains a variable amount of stromal/stem cells. In Europe, cell products like the SVF derived from fat tissue are considered advanced therapy medicinal product (ATMPs). As a consequence, the manufacturing process has to be approved via GMP-compliant process validation. The problem of the process validation for SVF is the heterogeneity of this fraction. Methods: Here, we modified existing purification strategies by adding an additional plastic adherence incubation of maximal 20 hours after SVF isolation. The resulting cell fraction was characterized and compared to SVF as well as cultivated adipose-derived stromal/stem cells (ASCs) with respect to viability and cell yield, the expression of surface markers, differentiation potential and cytokine expression. Results: Short-term incubation significantly reduced the heterogeneity of the resulting cell fraction compared to SVF. The cells were able to differentiate into adipocytes, chondrocytes, and osteoblasts. More importantly, they expressed trophic proteins which have been previously associated with the beneficial effects of MSCs. Furthermore, GMP compliance of the production process described herein was acknowledged by the national regulatory agencies (DE_BB_01_GMP_2017_1018). Conclusion: Addition of a short purification-step after the SVF isolation is a cheap and fast strategy to isolate a homogeneous uncultivated GMP-compliant cell fraction of ASCs

Structure, function and regulation of the tight junction protein Tricellulin

Die Abdichtung des Interzellularspalts durch die Tight Junctions ist für Epithelien und Endothelien von zentraler Bedeutung, um als effektive Barriere den parazellulären Transport zwischen den Zellen hindurch zu kontrollieren. Als Hauptkomponenten der Tight Junctions wurden Occludin und die Familie der Claudine beschrieben. In einer kürzlich veröffentlichten Studie konnte Tricellulin als weiteres integrales Membranprotein identifiziert werden, das hauptsächlich an Kontaktstellen von drei benachbarten Zellen lokalisiert ist. Das spezielle Interesse dieser Arbeit lag darin, Tricellulin funktionell zu charakterisieren. Dabei konnte durch immunfluoreszenzmikroskopische Untersuchungen gezeigt werden, dass die C-terminale cytoplasmatische Domäne von Tricellulin für die Membranlokalisation wichtig ist. Der N-Terminus ist an der bevorzugten Lokalisation in trizellulären Tight Junctions beteiligt. In funktionellen Untersuchungen an stabil transfizierten MDCK C11-Klonen konnte im Rahmen dieser Arbeit nachgewiesen werden, dass die cytoplasmatischen Domänen von Tricellulin jedoch auf den transepithelialen Widerstand sowie die Permeabilitätseigenschaften eines Epithels keinen Einfluss haben. Diese Parameter werden vermutlich durch die extrazellulären Loops reguliert. Mit weiterführenden Analysen konnte anhand von Co-Immunpräzipitationsexperimenten gezeigt werden, dass Tricellulin homomere Komplexe ausbildet. Ferner lassen die Ergebnisse dieser Arbeit vermuten, dass diese Tricellulin-Tricellulin- Assoziation durch die Transmembrandomänen oder die extrazellulären Domänen des Proteins vermittelt wird. Zudem wurde nachgewiesen, dass Tricellulin und Occludin heteromere Proteinkomplexe ausbilden. Diese Interaktion erfolgt ebenfalls über die Transmembrandomänen oder die extrazellulären Loops. Die physiologische Bedeutung dieser Komplexe ist bisher jedoch noch nicht im Detail geklärt. Untersuchungen zu posttranslationalen Modifikationen von Tricellulin ergaben, dass sich Tricellulin und Occludin hinsichtlich der Kinasen, die beide Proteine modifizieren, unterscheiden. So wird der C-Terminus von Occludin durch CK1 und CK2 phosphoryliert, wohingegen für den C-Terminus von Tricellulin keine Phosphorylierung nachgewiesen werden konnte. Es konnten jedoch Hinweise für eine Phosphorylierung von Tricellulin durch PKA erarbeitet werden. Als weitere posttranslationale Modifikation konnte eine Ubiquitinierung von Tricellulin nachgewiesen werden. Die beobachtete Mono- Ubiquitinierung ist möglicherweise an der Regulation der Endocytose von Tricellulin beteiligt, die durch Zelloberflächen-Biotinylierungsexperimente nachgewiesen werden konnte.Sealing the intercellular space by tight junctions is essential for endothelial and epithelial cell-sheets for controlling paracellular transport. As the main components of tight junctions occludin and the family of the claudins were identified. A recently published study presented tricellulin as a new integral membrane protein located predominantly at contact-sites between three neighbouring cells. The special interest of this work was the functional characterization of tricellulin. Immunofluorescence microscopic analyses depicted a role of the C-terminal cytoplasmic domain for cell surface localisation of tricellulin. In contrast the N-terminus is involved in the preferred assembly in tricellular tight junctions. Functional analyses with stable transfected MDCK C11-clones revealed, that the cytoplasmic domains of tricellulin do not affect the paracellular resistence or the barrier properties of an epithelial cell-layer. These parameter are presumably regulated by the extracellular loops. Co-immunoprecipitation experiments indicated the existence of homomeric Tricellulin complexes. Furthermore, the results of this study suggest that the tricellulin-tricellulin association is mediated by the transmembrane domains or the extracellular loops of the protein. Furthermore, it was demonstrated, that tricellulin and occludin accumulate in heteromeric complexes. This interaction similary appears to be mediated by the transmembrane domains or the extracellular loops of both proteins. So far, the physiological role of these complexes is not known in detail. Assays concerning posttranscriptional modifications displayed, that tricellulin and occludin are phosphorylated by different kinases. The C-terminus of Occludin is modified by CK1 and CK2 whereas these kinases do not phosphorylate the C-terminus of Tricellulin. However, there is evidence for a phosphorylation of tricellulin by PKA. Moreover modification of tricellulin by ubiquitin was shown in this work for the first time. The observed mono- ubiquitination is potentially involved in the regulation of endocytotic internalisation of tricellulin. Endocytosis of tricellulin was confirmed by cell-surface-biotinylation-experiments

Identification of a SIRT1 mutation in a family with type 1 Diabetes

Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes

The database of the PREDICTS (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems) project

The PREDICTS project—Projecting Responses of Ecological Diversity In Changing Terrestrial Systems (www.predicts.org.uk)—has collated from published studies a large, reasonably representative database of comparable samples of biodiversity from multiple sites that differ in the nature or intensity of human impacts relating to land use. We have used this evidence base to develop global and regional statistical models of how local biodiversity responds to these measures. We describe and make freely available this 2016 release of the database, containing more than 3.2 million records sampled at over 26,000 locations and representing over 47,000 species. We outline how the database can help in answering a range of questions in ecology and conservation biology. To our knowledge, this is the largest and most geographically and taxonomically representative database of spatial comparisons of biodiversity that has been collated to date; it will be useful to researchers and international efforts wishing to model and understand the global status of biodiversity