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Search Results: 1 - 10 of 3544 matches for " Alexandra Stolzing "
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Differentiation of mouse bone marrow derived stem cells toward microglia-like cells
Arnd Hinze, Alexandra Stolzing
BMC Cell Biology , 2011, DOI: 10.1186/1471-2121-12-35
Abstract: We assessed in vitro-derived microglia differentiation by marker expression (CD11b/CD45, F4/80), but also for the first time for functional performance (phagocytosis, oxidative burst) and in situ migration into living brain tissue. Integration, survival and migration were assessed in organotypic brain slices.The cells differentiated from mouse BM show function, markers and morphology of primary microglia and migrate into living brain tissue. Flt3L displays a negative effect on differentiation while GM-CSF enhances differentiation.We conclude that in vitro-derived microglia are the phenotypic and functional equivalents to primary microglia and could be used in cell therapy.Microglias constitute about 10% of the cell population of the brain and represent the most important first immune defense of the CNS. They are phagocytic, cytotoxic, antigen-presenting cells which promote brain tissue repair after injury [1]. Primary microglia differ from other blood macrophages in the expression levels of markers like CD11b/CD45low/high [2], CD68 low/high [3] and substance P levels [4]. Because of the overlap in markers there is an ongoing discussion about the distinction between dendritic cells, macrophages and microglia. The regulation of marker levels and activity has led to the proposition that microglia could be immature or resting macrophages [5]. However, there is a lack of correlation between marker expression and actual functional capacity, which is the most important hallmark for therapeutic use. Microglia in the brain normally display a quiescent state in which phagocytosis, immune response and migration are down-regulated and the microglia show a ramified morphology with long processes [6]. Microglia react to inflammation by switching to an activated state and taking on an amoeboid morphology [7]. They migrate towards sites of injury and lesion and extracellular debris such as amyloid-β plaques [8]. An important function of microglia is the "oxidative burst" - a sudden
Microglia differentiation using a culture system for the expansion of mice non-adherent bone marrow stem cells
Arnd Hinze, Alexandra Stolzing
Journal of Inflammation , 2012, DOI: 1476-9255-9-12
Abstract: Non-adherent bone marrow derived stem cells (NA-BMC) are derived by selective adhesion (‘preplating’) and are non adhesive adult stem cells. We investigated the changes in bone marrow cell populations by this repeated selective adhesion and compared the potential of the derived cells to differentiate towards microglia. Cells were differentiated with astrocyte conditioned medium (ACM) and granulocyte-monocyte colony stimulating factor (GM-CSF).NA-BMC cultures show a steep raise in the fraction of stem cells during the cultivation time and the differentiation potential is of the same quality as established protocols. Around 70% of the cells are microglia defined as being positive for CD11b/CD45 and show phagocytosis activity and oxidative bursts.The non-adherent cell system has the advantage that is produces stem cell progenitors during expansion and provides good microglial differentiation.
Influence of Murine Mesenchymal Stem Cells on Proliferation, Phenotype, Vitality, and Cytotoxicity of Murine Cytokine-Induced Killer Cells in Coculture
Martin Bach, Christoph Schimmelpfennig, Alexandra Stolzing
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088115
Abstract: Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.
Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning
Anastasia Efimenko, Ekaterina Starostina, Natalia Kalinina, Alexandra Stolzing
Journal of Translational Medicine , 2011, DOI: 10.1186/1479-5876-9-10
Abstract: ADSC were harvested from young (1-3 month), adult (12 month) and aged (18-24 month) mice and cultured under normoxic (20%) and hypoxic (1%) conditions for 48 h. Differences in proliferation, apoptosis and telomere length were assessed in addition to angiogenic properties of ADSC.Proliferation potential and telomere length were decreased in aged ADSC compared to young ADSC. Frequency of apoptotic cells was higher in aged ADSC. Gene expression of pro-angiogenic factors including vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and hepatic growth factor (HGF) were down-regulated with age, which could be restored by hypoxia. Transforming growth factor (TGF-β) increased in the old ADSC but was reduced by hypoxia.Expression of anti-angiogenic factors including thrombospondin-1 (TBS1) and plasminogen activator inhibitor-1 (PAI-1) did increase in old ADSC, but could be reduced by hypoxic stimulation. Endostatin (ENDS) was the highest in aged ADSC and was also down-regulated by hypoxia. We noted higher gene expression of proteases system factors like urokinase-type plasminogen activator receptor (uPAR), matrix metalloproteinases (MMP2 and MMP9) and PAI-1 in aged ADSC compared to young ADSC, but they decreased in old ADSC. Tube formation on matrigel was higher in the presence of conditioned medium from young ADSC in comparison to aged ADSC.ADSC isolated from older animals show changes, including impaired proliferation and angiogenic stimulation. Angiogenic gene expression can be partially be improved by hypoxic preconditioning, however the effect is age-dependent. This supports the hypothesis that autologous ADSC from aged subjects might have an impaired therapeutic potential.Mesenchymal stem cells (MSC) have therapeutic potential in bone marrow transplantation [1,2], tissue engineering [3], and cell therapy [4]. Adipose-derived stem cells (ADSC) are relatively easy to obtain from adipose tissue and are more frequent than MSC in bone marrow [5]. They
Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide
Naaldijk Yahaira,Staude Marek,Fedorova Viktoriya,Stolzing Alexandra
BMC Biotechnology , 2012, DOI: 10.1186/1472-6750-12-49
Abstract: Background Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed. Results The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a ‘straight freeze’ protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods. Conclusion A 5% DMSO / 5% HES solution cryopreservation solution using a ‘straight freeze’ approach can be recommended for rat MSC.
The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins
Katrin Paulsen, Svantje Tauber, Johanna Timm, Nadine Goelz, Claudia Dumrese, Alexandra Stolzing, Ralf Hass, Oliver Ullrich
Cell Communication and Signaling , 2011, DOI: 10.1186/1478-811x-9-33
Abstract: Cannabinoids have been used as medicinal plant extracts for a long time. Already 4000 years ago, the Chinese emperor successfully treated diseases associated with increased immune reaction and inflammation using plant extracts [1]. The fact that the signaling pathways of the cannabinoid system are conserved throughout various species suggests an evolutionary benefit [2,3]. Cannabinoid signals can be mediated by different receptors, the first cannabinoid receptor CB1 was discovered in 1990 in the brain. A few years later a second receptor CB2 was cloned from immune cells [4,5]. The activation of CB1 and CB2 results in different cellular responses: (i) inhibition of adenylyl cyclase and the cAMP/protein kinase A (PKA)-dependent pathway by inhibitory G-proteins (Gi) which leads to a reduced production of cAMP [6] while recent research suggests that these receptors can also stimulate cAMP production by directly stimulating G-proteins (Gs) [7] (ii) stimulation of mitogen-activated protein kinase (MAPK) cascade, especially the extracellular signal kinase (ERK) [8] and the p38 MAPK cascade [9]. Whereas the CB1 receptor is mainly, but not exclusively, expressed on neurons, the CB2 receptor is primarily present in immune cells [8,10]. Other receptors in the cannabinoid signal system include the vanilloid receptor type 1 (TRPV1) [11] and the G protein-coupled receptor 55, also known as GPR55 [12,13]. Interestingly, many cannabinoid-related compounds have little if any affinity for either of the two known cannabinoid receptors CB1 or CB2 - suggesting that other unidentified receptors might be involved. The endogenous ligands for the endocannabinoid system are anandamide (AEA), 2-Arachidonylglycerol (2-AG), noladin ether and virodhamine, but fast degradation of these substances by the specific monoglyceride lipase (in the case of 2-AG), serine hydrolase and fatty acid amide hydrolase (for AEA and 2-AG) limits the usage of these ligands for the study of the signaling pathways [1
Reprogramming of Human Huntington Fibroblasts Using mRNA
Antje Arnold,Yahaira M. Naaldijk,Claire Fabian,Henry Wirth,Hans Binder,Guido Nikkhah,Lyle Armstrong,Alexandra Stolzing
ISRN Cell Biology , 2012, DOI: 10.5402/2012/124878
Abstract: The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting. 1. Introduction The feasibility of reprogramming somatic cells to induced pluripotent stem cells (iPS) [1–4] has led to the possibility of developing disease-specific iPS cells for improved disease modeling in vitro [5–7] and potential use in clinical applications [8, 9]. Since the initial generation of iPS cells from mouse embryonic fibroblast (MEF) cells [1], there have been numerous refinements of the method. The potential therapeutic application of initial iPS cell lines was hampered by the fact that applied methods of iPS cell derivation modified the host genome through the integration of DNA sequences [3, 10–15]. Kim and colleagues [16] showed that it is possible to reprogram human foreskin fibroblasts through exposure to membrane-permeable recombinant proteins of the pluripotency factors Oct-4, Sox-2, Klf-4, and c-Myc. The factors were fused to a 9-arginine sequence to establish the ability of cell penetration. HEK 293 cells were transfected with plasmids for producing the described proteins. The whole HEK 293 cell extract was used for reprogramming. The method was refined by Zhou et al. [17] for MEF cells using recombinant cell-penetrating proteins. It has been shown that the modified mRNA-mediated delivery of reprogramming factors based on nucleofection is an efficient and nontoxic alternative approach to cell modification [18] which has recently facilitated the derivation of iPS cell lines [19–21]. Here, we investigate in vitro transcribed mRNA transfection as a method for producing iPS cells that does not bear any risk with respect
Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment
Stephan Fricke,Manuela Ackermann,Alexandra Stolzing,Christoph Schimmelpfennig,Nadja Hilger,Jutta Jahns,Guido Hildebrandt,Frank Emmrich,Peter Ruschpler,Claudia P?sel,Manja Kamprad,Ulrich Sack
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0006157
Abstract: Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4?/?, HLA-DR3+ mice.
Effect of Age and Diabetes on the Response of Mesenchymal Progenitor Cells to Fibrin Matrices
A. Stolzing,H. Colley,A. Scutt
International Journal of Biomaterials , 2011, DOI: 10.1155/2011/378034
Abstract: Mesenchymal stem cells are showing increasing promise in applications such as tissue engineering and cell therapy. MSC are low in number in bone marrow, and therefore in vitro expansion is often necessary. In vivo, stem cells often reside within a niche acting to protect the cells. These niches are composed of niche cells, stem cells, and extracellular matrix. When blood vessels are damaged, a fibrin clot forms as part of the wound healing response. The clot constitutes a form of stem cell niche as it appears to maintain the stem cell phenotype while supporting MSC proliferation and differentiation during healing. This is particularly appropriate as fibrin is increasingly being suggested as a scaffold meaning that fibrin-based tissue engineering may to some extent recapitulate wound healing. Here, we describe how fibrin modulates the clonogenic capacity of MSC derived from young/old human donors and normal/diabetic rats. Fibrin was prepared using different concentrations to modulate the stiffness of the substrate. MSC were expanded on these scaffolds and analysed. MSC showed an increased self-renewal on soft surfaces. Old and diabetic cells lost the ability to react to these signals and can no longer adapt to the changed environment.
Modeling Spatial Opportunity Structures and Youths’ Transitions from School to Training  [PDF]
Alexandra Wicht, Alexandra Nonnenmacher
Open Journal of Statistics (OJS) , 2017, DOI: 10.4236/ojs.2017.76071
Abstract: This paper examines the significance of spatial externalities for youths’ school-to-training transitions in Germany. For this purpose, it is necessary to address the methodological question of how an individual’s spatial context has to be operationalized with respect to both its extent and the problem of spatial autocorrelation. Our analyses show that the “zone of influence” comprises of the whole of Germany, not only close-by districts, and that these effects differ between structurally weak and strong regions. Consequently, assuming that only close proximity affects individual outcomes may disregard relevant contextual influences, and for spatial models that require an a priori definition of the weights for spatial units, it may be erroneous to make a decision based on this assumption. Concerning spatial autocorrelation, we found that neglecting local spatial autocorrelation at the context level causes considerable bias to the estimates, especially for districts that are close to the home district.
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