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- ItemPhenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells([London] : Macmillan Publishers Limited, part of Springer Nature, 2015) Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, DenisXenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.
- ItemZebrafish In-Vivo Screening for Compounds Amplifying Hematopoietic Stem and Progenitor Cells: - Preclinical Validation in Human CD34+ Stem and Progenitor Cells(London : Nature Publishing Group, 2017) Arulmozhivarman, Guruchandar; Kräter, Martin; Wobus, Manja; Friedrichs, Jens; Bejestani, Elham Pishali; Müller, Katrin; Lambert, Katrin; Alexopoulou, Dimitra; Dahl, Andreas; Stöter, Martin; Bickle, Marc; Shayegi, Nona; Hampe, Jochen; Stölzel, Friedrich; Brand, Michael; von Bonin, Malte; Bornhäuser, MartinThe identification of small molecules that either increase the number and/or enhance the activity of human hematopoietic stem and progenitor cells (hHSPCs) during ex vivo expansion remains challenging. We used an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model and identified histone deacetylase inhibitors (HDACIs), particularly valproic acid (VPA), as significant enhancers of the number of phenotypic HSPCs, both in vivo and during ex vivo expansion. The long-term functionality of these expanded hHSPCs was verified in a xenotransplantation model with NSG mice. Interestingly, VPA increased CD34+ cell adhesion to primary mesenchymal stromal cells and reduced their in vitro chemokine-mediated migration capacity. In line with this, VPA-treated human CD34+ cells showed reduced homing and early engraftment in a xenograft transplant model, but retained their long-term engraftment potential in vivo, and maintained their differentiation ability both in vitro and in vivo. In summary, our data demonstrate that certain HDACIs lead to a net expansion of hHSPCs with retained long-term engraftment potential and could be further explored as candidate compounds to amplify ex-vivo engineered peripheral blood stem cells.
- ItemLimbal stromal cells derived from porcine tissue demonstrate mesenchymal characteristics in vitro(London : Nature Publishing Group, 2017) Fernández-Pérez, Julia; Binner, Marcus; Werner, Carsten; Bray, Laura J.Limbal stromal cells (LSCs) from the human ocular surface display mesenchymal stromal cell characteristics in vitro. In this study, we isolated cells from the porcine limbal stroma (pLSCs), characterised them, and evaluated their ability to support angiogenesis and the culture of porcine limbal epithelial stem cells (pLESCs). The isolated cells adhered to plastic and grew in monolayers in vitro using serum-supplemented or serum-free medium. The pLSCs demonstrated expression of CD29, and cross-reactivity with anti-human CD45, CD90, CD105, CD146, and HLA-ABC. However, expression of CD105, CD146 and HLA-ABC reduced when cultured in serum-free medium. PLSCs did not undergo adipogenic or osteogenic differentiation, but differentiated towards the chondrogenic lineage. Isolated cells were also co-cultured with human umbilical vein endothelial cells (HUVECs) in star-shaped Poly(ethylene glycol) (starPEG)-heparin hydrogels to assess their pericyte capacity which supported angiogenesis networks of HUVECs. PLSCs supported the three dimensional HUVEC network for 7 days. The isolated cells were further growth-arrested and evaluated as feeder cells for pLESC expansion on silk fibroin membranes, as a potential carrier material for transplantation. PLSCs supported the growth of pLESCs comparably to murine 3T3 cells. In conclusion, although pLSCs were not completely comparable to their human counterpart, they display several mesenchymal-like characteristics in vitro.
- ItemDefined Geldrop Cultures Maintain Neural Precursor Cells(London : Nature Publishing Group, 2018) Vogler, Steffen; Prokoph, Silvana; Freudenberg, Uwe; Binner, Marcus; Tsurkan, Mikhail; Werner, Carsten; Kempermann, GerdDistinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.
- ItemNon-thermal plasma-treated solution demonstrates antitumor activity against pancreatic cancer cells in vitro and in vivo([London] : Macmillan Publishers Limited, 2017) Liedtke, Kim Rouven; Bekeschus, Sander; Kaeding, André; Hackbarth, Christine; Kuehn, Jens-Peter; Heidecke, Claus-Dieter; von Bernstorff, Wolfram; von Woedtke, Thomas; Partecke, Lars IvoPancreatic cancer is associated with a high mortality rate. In advanced stage, patients often experience peritoneal carcinomatosis. Using a syngeneic murine pancreatic cancer cell tumor model, the effect of non-thermal plasma (NTP) on peritoneal metastatic lesions was studied. NTP generates reactive species of several kinds which have been proven to be of relevance in cancer. In vitro, exposure to both plasma and plasma-treated solution significantly decreased cell viability and proliferation of 6606PDA cancer cells, whereas mouse fibroblasts were less affected. Repeated intraperitoneal treatment of NTP-conditioned medium decreased tumor growth in vivo as determined by magnetic resonance imaging, leading to reduced tumor mass and improved median survival (61 vs 52 days; p < 0.024). Tumor nodes treated by NTP-conditioned medium demonstrated large areas of apoptosis with strongly inhibited cell proliferation. Contemporaneously, no systemic effects were found. Apoptosis was neither present in the liver nor in the gut. Also, the concentration of different cytokines in splenocytes or blood plasma as well as the distribution of various hematological parameters remained unchanged following treatment with NTP-conditioned medium. These results suggest an anticancer role of NTP-treated solutions with little to no systemic side effects being present, making NTP-treated solutions a potential complementary therapeutic option for advanced tumors.
- ItemCryogel-supported stem cell factory for customized sustained release of bispecific antibodies for cancer immunotherapy(London : Nature Publishing Group, 2017) Aliperta, Roberta; Welzel, Petra B.; Bergmann, Ralf; Freudenberg, Uwe; Berndt, Nicole; Feldmann, Anja; Arndt, Claudia; Koristka, Stefanie; Stanzione, Marcello; Cartellieri, Marc; Ehninger, Armin; Ehninger, Gerhard; Werner, Carsten; Pietzsch, Jens; Steinbach, Jörg; Bornhäuser, Martin; Bachmann, Michael P.Combining stem cells with biomaterial scaffolds provides a promising strategy for the development of drug delivery systems. Here we propose an innovative immunotherapeutic organoid by housing human mesenchymal stromal cells (MSCs), gene-modified for the secretion of an anti-CD33-anti-CD3 bispecific antibody (bsAb), in a small biocompatible star-shaped poly(ethylene glycol)-heparin cryogel scaffold as a transplantable and low invasive therapeutic machinery for the treatment of acute myeloid leukemia (AML). The macroporous biohybrid cryogel platform displays effectiveness in supporting proliferation and survival of bsAb-releasing-MSCs overtime in vitro and in vivo, avoiding cell loss and ensuring a constant release of sustained and detectable levels of bsAb capable of triggering T-cell-mediated anti-tumor responses and a rapid regression of CD33 + AML blasts. This therapeutic device results as a promising and safe alternative to the continuous administration of short-lived immunoagents and paves the way for effective bsAb-based therapeutic strategies for future tumor treatments.
- ItemMicrofluidic cell sorting: Towards improved biocompatibility of extracorporeal lung assist devices([London] : Macmillan Publishers Limited, part of Springer Nature, 2018) Bleilevens, Christian; Lölsberg, Jonas; Cinar, Arne; Knoben, Maren; Grottke, Oliver; Rossaint, Rolf; Wessling, MatthiasExtracorporeal lung assist technology is one of the last options in critical care medicine to treat patients suffering from severe oxygenation and decarboxylation disorders. Platelet activation along with the consequent thrombus formation is a potentially life-threatening complication of this technique. To avoid platelet-dependent clot formation, this study aims at developing a microfluidic cell sorting chip that can bypass platelets prior to the membrane oxygenator of the extracorporeal lung assist device. The cell sorting chips were produced by maskless dip-in laser lithography, followed by soft lithography replication using PDMS. Citrated porcine whole blood with a clinically relevant haematocrit of 17% was used for the cell sorting experiments involving three different blood flow rates. The joint effects of flow focusing and hydrodynamic lifting forces within the cell sorting chip resulted in a reduction of up to 57% of the baseline platelet count. This cell sorting strategy is suitable for the continuous and label-free separation of red blood cells and platelets and is potentially applicable for increasing the biocompatibility and lifetime of current extracorporeal lung assist devices.
- ItemEngineering biofunctional in vitro vessel models using a multilayer bioprinting technique([London] : Macmillan Publishers Limited, part of Springer Nature, 2018) Schöneberg, Jan; De Lorenzi, Federica; Theek, Benjamin; Blaeser, Andreas; Rommel, Dirk; Kuehne, Alexander J. C.; Kießling, Fabian; Fischer, HorstRecent advances in the field of bioprinting have led to the development of perfusable complex structures. However, most of the existing printed vascular channels lack the composition or key structural and physiological features of natural blood vessels or they make use of more easily printable but less biocompatible hydrogels. Here, we use a drop-on-demand bioprinting technique to generate in vitro blood vessel models, consisting of a continuous endothelium imitating the tunica intima, an elastic smooth muscle cell layer mimicking the tunica media, and a surrounding fibrous and collagenous matrix of fibroblasts mimicking the tunica adventitia. These vessel models with a wall thickness of up to 425 µm and a diameter of about 1 mm were dynamically cultivated in fluidic bioreactors for up to three weeks under physiological flow conditions. High cell viability (>83%) after printing and the expression of VE-Cadherin, smooth muscle actin, and collagen IV were observed throughout the cultivation period. It can be concluded that the proposed novel technique is suitable to achieve perfusable vessel models with a biofunctional multilayer wall composition. Such structures hold potential for the creation of more physiologically relevant in vitro disease models suitable especially as platforms for the pre-screening of drugs.
- ItemMagnetically Controllable Polymer Nanotubes from a Cyclized Crosslinker for Site-Specific Delivery of Doxorubicin([London] : Macmillan Publishers Limited, part of Springer Nature, 2015) Newland, Ben; Leupelt, Daniel; Zheng, Yu; Thomas, Laurent S.V.; Werner, Carsten; Steinhart, Martin; Wang, WenxinExternally controlled site specific drug delivery could potentially provide a means of reducing drug related side effects whilst maintaining, or perhaps increasing therapeutic efficiency. The aim of this work was to develop a nanoscale drug carrier, which could be loaded with an anti-cancer drug and be directed by an external magnetic field. Using a single, commercially available monomer and a simple one-pot reaction process, a polymer was synthesized and crosslinked within the pores of an anodized aluminum oxide template. These polymer nanotubes (PNT) could be functionalized with iron oxide nanoparticles for magnetic manipulation, without affecting the large internal pore, or inherent low toxicity. Using an external magnetic field the nanotubes could be regionally concentrated, leaving areas devoid of nanotubes. Lastly, doxorubicin could be loaded to the PNTs, causing increased toxicity towards neuroblastoma cells, rendering a platform technology now ready for adaptation with different nanoparticles, degradable pre-polymers and various therapeutics.
- ItemCytochrome C oxidase Inhibition and Cold Plasma-derived Oxidants Synergize in Melanoma Cell Death Induction([London] : Macmillan Publishers Limited, part of Springer Nature, 2018-8-24) Gandhirajan, Rajesh Kumar; Rödder, Katrin; Bodnar, Yana; Pasqual-Melo, Gabriella; Emmert, Steffen; Griguer, Corinne E.; Weltmann, Klaus-Dieter; Bekeschus, SanderDespite striking advances in the treatment of metastasized melanoma, the disease is often still fatal. Attention is therefore paid towards combinational regimens. Oxidants endogenously produced in mitochondria are currently targeted in pre-clinical and clinical studies. Cytotoxic synergism of mitochondrial cytochrome c oxidase (CcO) inhibition in conjunction with addition of exogenous oxidants in 2D and 3D melanoma cell culture models were examined. Murine (B16) and human SK-MEL-28 melanoma cells exposed to low-dose CcO inhibitors (potassium cyanide or sodium azide) or exogenous oxidants alone were non-toxic. However, we identified a potent cytotoxic synergism upon CcO inhibition and plasma-derived oxidants that led to rapid onset of caspase-independent melanoma cell death. This was mediated by mitochondrial dysfunction induced by superoxide elevation and ATP depletion. This observation was validated by siRNA-mediated knockdown of COX4I1 in SK-MEL-28 cells with cytotoxicity in the presence of exogenous oxidants. Similar effects were obtained with ADDA 5, a recently identified specific inhibitor of CcO activity showing low toxicity in vivo. Human keratinocytes were not affected by this combinational treatment, suggesting selective effects on melanoma cells. Hence, targeting mitochondrial CcO activity in conjunction with exogenous pro oxidant therapies may constitute a new and effective melanoma treatment modality.