RESUMO
A/B toxins such as cholera toxin, Pseudomonas exotoxin and killer toxin K28 contain a KDEL-like amino acid motif at one of their subunits which ensures retrograde toxin transport through the secretory pathway of a target cell. As key step in host cell invasion, each toxin binds to distinct plasma membrane receptors that are utilized for cell entry. Despite intensive efforts, some of these receptors are still unknown. Here we identify the yeast H/KDEL receptor Erd2p as membrane receptor of K28, a viral A/B toxin carrying an HDEL motif at its cell binding ß-subunit. While initial toxin binding to the yeast cell wall is unaffected in cells lacking Erd2p, binding to spheroplasts and in vivo toxicity strongly depend on the presence of Erd2p. Consistently, Erd2p is not restricted to membranes of the early secretory pathway but extends to the plasma membrane where it binds and internalizes HDEL-cargo such as K28 toxin, GFP(HDEL) and Kar2p. Since human KDEL receptors are fully functional in yeast and restore toxin sensitivity in the absence of endogenous Erd2p, toxin uptake by H/KDEL receptors at the cell surface might likewise contribute to the intoxication efficiency of A/B toxins carrying a KDEL-motif at their cytotoxic A-subunit(s).
Assuntos
Fatores Matadores de Levedura/metabolismo , Receptores de Peptídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas , Proteínas de Choque Térmico HSP70 , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , EsferoplastosRESUMO
The estimated frequency of MSCs in BM is about 0.001-0.01% of total nucleated cells. Most commonly, one applied therapeutic cell dose is about 1-5 million MSCs/kg body weight, necessitating a reliable, fast, and safe expansion system. The limited availability of MSCs demands for an extensive ex vivo amplification step to accumulate sufficient cell numbers. Human platelet lysate (PL) has proven to be a safe and feasible alternative to animal-derived serum as supplement for MSC cultivation. We have investigated the functionally closed automated cell culture hollow fiber bioreactor Quantum cell expansion system as an alternative novel tool to conventional tissue flasks for efficient clinical-scale MSC isolation and expansion from bone marrow using PL. Cells expanded in the Quantum system fulfilled MSC criteria as shown by flow cytometry and adipogenic, chondrogenic, and osteogenic differentiation capacity. Cell surface expression of a variety of chemokine receptors, adhesion molecules, and additional MSC markers was monitored for several passages by flow cytometry. The levels of critical media components like glucose and lactate were analyzed. PDGF-AA, PDGF-AB/BB, bFGF, TGF-ß1, sICAM-1, sVCAM-1, RANTES, GRO, VEGF, sCD40L, and IL-6 were assessed using a LUMINEX platform. Originally optimized for the use of fetal calf serum (FCS) as supplement and fibronectin as coating reagent, we succeeded to obtain an average of more than 100×10(6) of MSCs from as little as 18.8-28.6 ml of BM aspirate using PL. We obtained similar yields of MSCs/µl BM in the FCS-containing and the xenogen-free expansion system. The Quantum system reliably produces a cellular therapeutic dose in a functionally closed system that requires minimal manipulation. Both isolation and expansion are possible using FCS or PL as supplement. Coating of the hollow fibers of the bioreactor is mandatory when loading MSCs. Fibronectin, PL, and human plasma may serve as coating reagents.
Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/normas , Separação Celular/instrumentação , Células-Tronco Mesenquimais/citologia , Biomarcadores/metabolismo , Reatores Biológicos , Plaquetas/química , Plaquetas/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Fibronectinas/química , Fibronectinas/metabolismo , Citometria de Fluxo , Humanos , Cariotipagem , Células-Tronco Mesenquimais/metabolismo , FenótipoRESUMO
BACKGROUND: Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. METHODS AND FINDINGS: We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. CONCLUSIONS: The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Adolescente , Adulto , Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Quimiocinas/metabolismo , Meios de Cultura/farmacologia , Citocinas/metabolismo , Citometria de Fluxo/métodos , Glucose/metabolismo , Humanos , Integrinas/metabolismo , Cariotipagem/métodos , Ácido Láctico/metabolismo , Reação em Cadeia da Polimerase/métodos , Células-Tronco , Engenharia Tecidual/métodosRESUMO
BACKGROUND AIMS: The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). METHODS: Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. RESULTS: PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-ß1 (TGF-ß1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-ß1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-ß1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. CONCLUSIONS: PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.
Assuntos
Plaquetas/metabolismo , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Células-Tronco Mesenquimais/citologia , Proteínas Proto-Oncogênicas c-sis/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Animais , Becaplermina , Remoção de Componentes Sanguíneos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismoRESUMO
We have investigated the uptake of cationic polystyrene nanoparticles by mesenchymal stem cells (MSCs) using confocal fluorescence microscopy and flow cytometry. Two types of nanoparticles of about 100 nm diameter with similar zeta potentials were employed in this study, plain polystyrene (PS) nanoparticles and amino-functionalized polystyrene (NPS) nanoparticles, each carrying about 6000 amino groups on the surface. To assess the relative importance of specific endocytosis mechanisms, uptake was observed in the presence of the drugs dynasore and chlorpromazine. NPS nanoparticles were rapidly internalized and accumulated to a much higher level in MSCs than PS nanoparticles, predominantly via the main clathrin-mediated pathway. PS nanoparticles were internalized mainly via clathrin-independent endocytosis. The pronounced difference in the internalization behavior of PS and NPS nanoparticles points to specific interactions of the amino groups on the nanoparticle surface with the endocytosis machinery of the cells.
Assuntos
Aminas/química , Células-Tronco Mesenquimais/química , Nanopartículas , Poliestirenos/química , Células Cultivadas , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia ConfocalRESUMO
The functionalization of well-defined PU nanocapsules with an aqueous core prepared by performing a polyaddition at the interface of inverse (water-in-oil) miniemulsion droplets is demonstrated. The miniemulsion technique involving the nanoreactor concept allows one to obtain an encapsulation efficiency as high as 90% within the nanocapsules. A pH independent fluorescent dye is used as a model system for the aqueous core. By varying the molar ratio of the diol to the diisocyanate at a fixed surfactant concentration, the shell thickness of the nanocapsules can be finely tuned. The carboxy- and amino-functionalized surface of the nanocapsules can be tailored by an in-situ carboxymethylation reaction and by physical adsorption of a cationic polyelectrolyte, i.e. PAEMA or PEI. The increased uptake of amino-functionalized fluorescent nanocapsules by HeLa cells clearly demonstrates the potential of the functionalized nanocapsules to be successfully exploited as biocarriers.
Assuntos
Portadores de Fármacos/química , Emulsões/química , Nanocápsulas/química , Poliuretanos/química , Adsorção , Condutividade Elétrica , Fluorescência , Células HeLa , Humanos , Microscopia Confocal , Nanocápsulas/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
The endocytotic mechanisms involved in the uptake of charged polystyrene nanoparticles into HeLa cells were investigated. Uptake experiments were done in the presence or absence of drugs known to inhibit various factors in endocytosis. Independent of the particle charge, endocytosis is highly dependent on dynamin, F-actin, and tyrosine-specific protein kinases, which suggests a dynamin-dependent and lipid raft-dependent mechanism. However, cholesterol depletion did not hinder particle uptake. Regarding positively charged particles, macropinocytosis, the microtubule network, and cyclooxygenases are also involved. The clathrin-dependent pathway plays a minor role.