搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species,the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest,followed by an immunodepletion step using microbeads coated with CD11b,CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion,a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1),CD90,CD105 and CD73 cell surface markers,but negative for the hematopoietic surface markers CD14,CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic,osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. View Publication

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The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 microM fumonisin B1,a fungal inhibitor of mammalian ceramide synthase,inhibited incorporation of [3H]galactose/glucosamine and [14C]-serine into complex sphingolipids of cultured cerebellar neurons. Under this condition,the expression of Purkinje cell-enriched sphingolipids,including GD1 alpha,9-O-acetylated LD1 and GD3,and sphingomyelin,was significantly decreased. After 2 weeks' exposure to fumonisin B1,dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag,but their branches began to degenerate. In some cells,formation of elongated dendrite trees was severely impaired. However,treatment with fumonisin B1 also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells,morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B1. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed,these effects of fumonisin B1 were reversed,but not completely,by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino dcaproyl]sphingosine (C6-NBD-ceramide),a synthetic derivative of ceramide. Thus,we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells. View Publication

Neurogenesis occurs continuously in two brain regions of adult mammals,underpinned by a pool of resident neural stem cells (NSCs) that can differentiate into all neural cell types. To advance our understanding of NSC function and to develop therapeutic and diagnostic approaches,it is important to accurately identify and enrich for NSCs. There are no definitive markers for the identification and enrichment of NSCs present in the mouse brain. Recently,a fluorescent rosamine dye,CDy1,has been identified as a label for pluripotency in cultured human embryonic and induced pluripotent stem cells. As similar cellular characteristics may enable the uptake and retention of CDy1 by other stem cell populations,we hypothesized that this dye may also enrich for primary NSCs from the mouse brain. Because the subventricular zone (SVZ) and the hippocampus represent brain regions that are highly enriched for NSCs in adult mammals,we sampled cells from these areas to test this hypothesis. These experiments revealed that CDy1 staining indeed allows for enrichment and selection of all neurosphere-forming cells from both the SVZ and the hippocampus. We next examined the effectiveness of CDy1 to select for NSCs derived from the SVZ of aged animals,where the total pool of NSCs present is significantly lower than in young animals. We found that CDy1 effectively labels the NSCs in adult and aged animals as assessed by the neurosphere assay and reflects the numbers of NSCs present in aged animals. CDy1,therefore,appears to be a novel marker for enrichment of NSCs in primary brain tissue preparations. View Publication

Stem cell-derived ?-cells (SC-BCs) represent a potential source for curing diabetes. To date,in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation,indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here,we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards ?-cells in vitro. To this aim,a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most ?-cell markers,but did not improve ?-cell identity. In conclusion,the study describes the production of a PanMa hydrogel,which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs. View Publication

Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here,we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture. Whole-cell patch-clamp demonstrated subthreshold synaptic inputs and action potential firing of neurons. Spontaneous intracellular calcium signals turned into synchronous large-scale oscillations upon combined disinhibition of NMDA receptors and blocking of GABA A receptors. Brief metabolic inhibition to mimic transient energy restriction in the ischemic brain induced reversible intracellular calcium loading of cBOS. Moreover,metabolic inhibition induced a reversible decline in neuronal ATP as revealed by ATeam1.03 YEMK . Overall,cBOS provide a powerful platform to assess morphological and functional aspects of human neural cells in intact minimal networks and to address the pathways that drive cellular damage during brain ischemia. Subject areas: Neuroscience,Cellular neuroscience,Stem cells research View Publication

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study,we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4,hSox2,hKlf4,and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci,a hallmark of DM1,were detected in DM1 iPSCs,neural stem cells (NSCs),and terminally differentiated neurons and astrocytes. In conclusion,we have successfully established disease-specific human DM1 iPSC lines,NSCs,and neuronal lineages with pathognomonic intranuclear RNA foci,which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development. View Publication

BACKGROUND: The testing of cord blood (CB) progenitor and stem cell units for transplantation suitability involves enumeration of total nucleated cells before freezing. CD34+ cell counts may also be a means of determining suitability. Studies have been conducted to evaluate how specific storage conditions influence cell counts. STUDY DESIGN AND METHODS: CB units were processed by hydroxyethyl starch volume reduction. Cryopreserved-thawed samples were diluted 1:3 without washing. CD34+ cells were measured with three commercially available assay methods. In specific studies,apoptosis-indicating reagents were included. CB units were analyzed for nucleated cells,aldehyde dehydrogenase-containing cells,and progenitor colonies. RESULTS: CD34+ cell levels and nucleated cells were retained during storage in test tubes at 1 to 6 degrees C for 3 days. Cryopreserved-thawed samples showed a reduction in CD34+ cells relative to prefreeze levels with the largest decrease with the Stem-Kit (Beckman Coulter) restricted gating procedure. Prefreeze samples contained minimal numbers of presumed apoptotic cells detected with 7-aminoactinomycin D or SYTO16,but after cryopreservation-thawing there was an increase. Nucleated cell levels determined with a hematology analyzer or flow cytometry were reduced after thawing. Cryopreservation-thawing reduced the percentage of CD34+ cells positive for the presence of aldehyde dehydrogenase and the number of progenitor colonies. These differences were significant. CONCLUSION: These studies indicate that CD34+ cell counts were maintained when CB samples were stored at 1 to 6 degrees C in test tubes for 3 days. Cryopreservation-thawing resulted in changes in a number of parameters including the percentage of CD34+ cells that were aldehyde dehydrogenase(+) and the number of 7-aminoactinomycin D(+) cells and SYTO16(low) cells. View Publication

Hematopoietic stem cells (HSCs) react to various stress conditions. However,it is unclear whether and how HSCs respond to severe anemia. Here,we demonstrate that upon induction of acute anemia,HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia,lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs,transcription levels of lipid metabolism-related genes,such as very low-density lipoprotein receptor ( Vldlr ),are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential,whereas HSCs in Apoe knockout mice do not respond to anemia induction. Vldlr high HSCs show higher erythroid potential,which is enhanced after acute anemia induction. Vldlr high HSCs are epigenetically distinct because of their low chromatin accessibility,and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions. Subject terms: Haematopoietic stem cells,Fat metabolism,Chromatin,Anaemia View Publication

We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition,anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling,leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6),a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. View Publication