an In Vitro Study

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP- BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration

Effects of nilotinib on regulatory T cells: the dose matters

<p>Abstract</p> <p>Background</p> <p>Nilotinib is a tyrosine kinase inhibitor with high target specificity. Here, we characterized the effects of nilotinib for the first time on CD4⁺CD25⁺regulatory T cells (Tregs) which regulate anti-tumor/leukemia immune responses.</p> <p>Design and Methods</p> <p>Carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2-deoxy -uridine (BrdU) were used to assess the proliferation and cell cycle distribution of Tregs. The expression of the transcription factor forkhead box P3 (FoxP3) and the glucocorticoid-induced tumor necrosis factor receptor (GITR) were measured by flow cytometry. Western blotting analysis was used to detect the effects of nilotinib on the signal transduction cascade of T-cell receptor (TCR) in Tregs.</p> <p>Results</p> <p>Nilotinib inhibited the proliferation and suppressive capacity of Tregs in a dose-dependent manner. However, the production of cytokines secreted by Tregs and CD4⁺CD25^-T cells was only inhibited at high concentrations of nilotinib exceeding the mean therapeutic serum concentrations of the drug in patients. Only high doses of nilotinib arrested both Tregs and CD4⁺CD25^-T cells in the G₀/G₁phase and down-regulated the expression of FoxP3 and GITR. In western blotting analysis, nilotinib did not show significant inhibitory effects on TCR signaling events in Tregs and CD4⁺CD25^-T cells.</p> <p>Conclusions</p> <p>These findings indicate that nilotinib does not hamper the function of Tregs at clinical relevant doses, while long-term administration of nilotinib still needs to be investigated.</p

Labeling of mesenchymal stromal cells with iron oxide-poly(l-lactide) nanoparticles for magnetic resonance imaging: uptake, persistence, effects on cellular function and magnetic resonance imaging properties

Background aims. Mesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the noninterference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI. Methods.We studied the effects of iron oxide-poly(L-lactide) nanoparticles in MSC with flow cytom-etry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant® proliferation testing, colony-forming unit-fibroblast (CFU-F) assays, flow chamber adhesion testing, immuno-logic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats. Results. It could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks. Conclusions. These particles qualify for studying MSC homing and trafficking via MRI

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data

Background Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. Methods Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone. Results Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. Conclusions Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.publishedVersio

CD90 is dispensable for white and beige/brown adipocyte differentiation

Brown adipose tissue (BAT) is a thermogenic organ in rodents and humans. In mice, the transplantation of BAT has been successfully used to combat obesity and its comorbidities. While such beneficial properties of BAT are now evident, the developmental and cellular origins of brown, beige, and white adipocytes have remained only poorly understood, especially in humans. We recently discovered that CD90 is highly expressed in stromal cells isolated from human white adipose tissue (WAT) compared to BAT. Here, we studied whether CD90 interferes with brown or white adipogenesis or white adipocyte beiging. We applied flow cytometric sorting of human adipose tissue stromal cells (ASCs), a CRISPR/Cas9 knockout strategy in the human Simpson-Golabi-Behmel syndrome (SGBS) adipocyte model system, as well as a siRNA approach in human approaches supports the hypothesis that CD90 affects brown or white adipogenesis or white adipocyte beiging in humans. Taken together, our findings call the conclusions drawn from previous studies, which claimed a central role of CD90 in adipocyte differentiation, into question

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876

Paroxysmal nocturnal hemoglobinuria (PNH): higher sensitivity and validity in diagnosis and serial monitoring by flow cytometric analysis of reticulocytes

Flow cytometric analysis of GPI-anchored proteins (GPI-AP) is the gold standard for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Due to therapy options and the relevance of GPI-deficient clones for prognosis in aplastic anaemia detection of PNH is gaining importance. However, no generally accepted standard has been established. This study analysed the usefulness of a flow cytometric panel with CD58, CD59 on reticulocytes and erythrocytes, CD24/CD66b and CD16, FLAER on granulocytes and CD14, and CD48 on monocytes. Actual cut-off (mean + 2 SD) for GPI-deficient cells was established in healthy blood donors. We studied 1,296 flow cytometric results of 803 patients. Serial monitoring was analysed during a median follow-up of 1,039 days in 155 patients. Of all, 22% and 48% of 155 follow-up patients. showed significant GPI-AP-deficiency at time of initial analyses. During follow-up in 9%, a new PNH diagnosis, and in 28%, a significant change of size or lineage involvement was demonstrated. Highly significant correlations for GPI-AP deficiency were found within one cell lineage (r2 = 0.61–0.95, p < 0.0001) and between the different cell lineages (r2 = 0.49–0.88, p < 0.0001). Especially for detection of small GPI-deficient populations, reticulocytes and monocytes proved to be sensitive diagnostic tools. Our data showed superiority of reticulocyte analyses compared with erythrocyte analyses due to transfusion and hemolysis independency especially in cases with small GPI-deficient populations. In conclusion, a screening panel of at least two different GPI-AP markers on granulocytes, erythrocytes, and reticulocytes provides a simple and rapid method to detect even small GPI-deficient populations. Among the markers in our panel, CD58 and CD59 on reticulocytes, CD24/66b, and eventually FLAER on granulocytes as well as CD14 on monocytes were most effective for flow cytometric diagnosis of GPI deficiency

CD95-independent, caspase- and mitochondria-dependent apoptosis induction, apoptosis independent proliferation

1. Inhaltsverzeichnis 2. Summary/Zusammenfassung 3. Einleitung 4. Zielsetzung 5. Materialien 6. Methoden 7. Ergebnisse 8. Diskussion 9. Abkürzungen und Synonyme 10. Appendix 11. Literatur 12. Erklärung zu eigenen Publikationen 13. Weitere eigene Publikationen 14. Danksagung 15. LebenslaufDreiwertige anorganische Arsenverbindungen wie Arsentrioxid oder Arsensulfid werden seit über 2400 Jahren in der Medizin verwandt. Nachdem Arsentrioxid durch die Entdeckung der Röntgenstrahlung und die Entwicklung potenter Zytostatika bei der Therapie akuter myeloischer Leukämien immer mehr verdrängt worden war, fanden organische und anorganische dreiwertige Arsenverbindungen bis Ende des 20. Jahrhunderts bei der Therapie der refraktären oder rezidivierten akuten Promyelozytenleukämie (APL) Verwendung. Bis vor kurzem wurde vermutet, daß Arsentrioxid seine therapeutischen Effekte nur in APL- Zellen entwickeln könne, da die Arsentrioxid-Wirkung an die Anwesenheit des Fusionsproteins PML-RARalpha gekoppelt sei. In dieser Arbeit wurde daher die Wirkung von Arsentrioxid auf Apoptose, Differenzierung und Proliferation an 22 malignen lymphatischen und myeloischen Zellinien unterschiedlichen Differenzierungsstadiums und Herkunftsgewebes untersucht. Aufgrund der unterschiedlichen Sensitivität dieser Zellinien gegenüber Arsentrioxid-Konzentrationen, welche auch bei der APL-Therapie im Plasma von Patienten zu erzielen sind, konnten drei Sensitivitätsgruppen für Zellinien definiert werden: Zellinien mit Sensitivität gegenüber 0,1 µM (Gruppe A), 1 µM (Gruppe B) und 5 µM (Gruppe C) Arsentrioxid. Arsentrioxid- Behandlung von Zellen führte zu einer Aktivierung der Caspasen-Kaskade, zum Zusammenbruch des mitochondrialen Membranpotentials und zur Bildung von ROS (reactive oxygen species). Die Mitochondrien sind als primärer Wirkungsort des Arsentrioxids anzusehen, da Caspasen-Inhibitoren zwar einen Rückgang des Prozentsatzes apoptotischer Zellen bewirkten und die Aktivierung von Caspasen hemmten, aber nur einen geringen Einfluß auf die Bildung von ROS und den Funktionsverlust der Mitochondrien hatten. NO-Radikale und erhöhte p53-Expression traten hingegen nur bei einigen Zellinien nach Arsentrioxid- Behandlung auf. Im Rahmen der Arsentrioxid-induzierten Apoptose konnte weder eine CD95-Induktion gemessen noch die Arsentrioxid-induzierte Apoptose durch Blockierung des CD95-Rezeptors gehemmt werden. Neben der Apoptose-Induktion zeigte Arsentrioxid eine Apoptose-unabhängige Proliferationsinhibition. Die Proliferationsinhibition trat bereits bei einer Arsentrioxid-Konzentration auf, welche entweder keine Apoptose auslöste oder aber Apoptose nur in einem kleinen Anteil der Gesamtpopulation induzierte. Aufgrund dieser Arbeit wurde eine Systematik eingeführt, anhand derer Zellinien gemäß ihrer Sensitivität gegenüber Arsentrioxid-induzierter Apoptose klassifiziert werden können. Ein für zahlreiche hämatopoetische Zellinien allgemeingültiger Mechanismus der Apoptose-Induktion wurde beschrieben: die Caspasen-unabhängige Mitochondrieninaktivierung. Es wurde ferner gezeigt, daß es im Rahmen der Arsentrioxid-Behandlung hämatopoetischer Zellinien zu einer Apoptose-unabhängigen Proliferationsinhibition kommen kann.Trivalent anorganic arsenic compounds have been used in medicine for more than 2400 years. However, with the discovery of radiotherapy and high potential cytostatic drugs in the 20th century, arsenic(III)-oxide disappeared as one of the cancer standard therapies. Because of its effects in acute promyelocytic leikemia (APL), the therapeutic use of arsenic(III)-oxide was thought to be restricted to cells in which the fusion protein PML-RARalpha is present. In this work, the effects of arsenic(III)-oxide on apoptosis and differentiation was analyzed in 22 cell. These cell lines represented various stages of lympho-haematopoietic differentiation. According to their sensitivity towards apoptosis induction a classification system was introduced: induction of apoptosis with concentrations 0,1 µM, 1 µM or 5 µM arsenic(III)-oxide was classified as sensitivity group A, group B or group C, respectively. The potential mechanisms responsible for apoptosis induction were analysed in representative cell lines of all three sensitivity groups. Arsenic(III)-oxide induced activity of caspases, breakdown of the mitochondrial membrane potential and synthesis of reactive oxygen species (ROS), whereas an increase of NO-radicals and p53-protein level could be measured only for some cell lines. Mitochondria can be regarded as the primary target of action for arsenic(III)-oxide, as caspases specific inhibitors could reduce the percentage of apoptotic (i. e. Annexin V-FITC and 7-amino-actinomycin (7?AAD)-positive) cells and caspase activity (measured by FITC-VAD-FMK- binding) but had no effect on the breakdown of mitochondrial membrane potential. The treatment of cell lines with arsenic(III)-oxide had no regulatory effect on CD95, and no inhibition of arsenic(III)-oxide-induced apoptosis could be detected by blocking the CD95 pathway. Beside the effect of apoptosis induction, treatment of cell lines with arsenic(III)-oxide also lead to an apoptosis independent inhibition of proliferation. Proliferation inhibition could already be observed for concentrations of arsenic(III)-oxide that themselves were not able to induce apoptosis or induced apoptosis only in a low percentage of the cell population. A classification system of sensitivity for haematopoietic cell lines to arsenic(III)-oxide was introduced by this work. This system is based on the sensitivity of cell lines towards arsenic(III)-oxide induced apoptosis. This work clearly shows that a common mechanism is responsible for arsenic(III)-oxide effects on a variety of haematopoeitic cell lines: the caspase-independent breakdown of mitochondrial membrane potential. Furthermore, this work clearly shows that an apoptosis-independent inhibition of cell proliferation can occur during arsenic(III)-oxide treatment of haematopoietic cell lines