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

Leukocyte recruitment to inflammation sites progresses in a multistep cascade. Chemokines regulate multiple steps of the cascade,including arrest,transmigration,and chemotaxis. The most important chemokine receptor in mouse neutrophils is CXCR2,which couples through G$\alpha$i2- and G$\alpha$i3-containing heterotrimeric G proteins. Neutrophils arrest in response to CXCR2 stimulation. This is defective in G$\alpha$i2-deficient neutrophils. In this study,we show that G$\alpha$i3-deficient neutrophils showed reduced transmigration but normal arrest in mice. We also tested G$\alpha$i2- or G$\alpha$i3-deficient neutrophils in a CXCL1 gradient generated by a microfluidic device. G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils showed significantly reduced migration and directionality. This was confirmed in a model of sterile inflammation in vivo. G$\alpha$i2-,but not G$\alpha$i3-,deficient neutrophils showed decreased Ca(2+) flux in response to CXCR2 stimulation. Conversely,G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils exhibited reduced AKT phosphorylation upon CXCR2 stimulation. We conclude that G$\alpha$i2 controls arrest and G$\alpha$i3 controls transmigration and chemotaxis in response to chemokine stimulation of neutrophils. View Publication

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro,and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously,we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this NIA Mouse ESC Bank we generated and characterized 48 additional mouse ESC lines,in which single TFs in each line could be induced in a doxycycline-controllable manner. Together,with the previous ESC lines,the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g.,neural lineages by Myt1 Isl1,and St18; mesodermal lineages by Pitx1,Pitx2,Barhl2,and Lmx1a; white blood cells by Myb,Etv2,and Tbx6,and ovary by Pitx1,Pitx2,and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. View Publication

SUMMARY Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here,we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways,such as Hippo-LATS-YAP/TAZ signaling,enriched in these cells. Next,we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular,morphologic,and functional phenotype reminiscent of human AT1 cells,including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s. In brief Kotton and colleagues generate human alveolar epithelial type I cells (AT1s) from induced pluripotent stem cells (iPSCs). The resulting cells can be grown as 3D organoids or in 2D air-liquid interface cultures,displaying many of the molecular,morphologic,and functional phenotypes of primary AT1s. Graphical abstract View Publication

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone,muscle,endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin,neurofilament,A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4,SCF,Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase,GalC,Olig2 and the late-stage marker MOSP are observed,as is the Schwann cell marker PMP22. In summary,Lin(neg) UCB cells,when appropriately cultured,are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation. View Publication

A simple,scalable,and reproducible technology that allows direct formation of large numbers of homogeneous and synchronized embryoid bodies (EBs) of defined sizes from dissociated human induced pluripotent stem cells (hiPSCs) was developed. Non-cell-adhesive hydrogels were used to create round-bottom microwells to host dissociated hiPSCs. No Rho-associated kinase inhibitor (ROCK-i),or centrifugation was needed and the side effects of ROCK-i can be avoided. The key requirement for the successful EB formation in addition to the non-cell-adhesive round-bottom microwells is the input cell density per microwell. Too few or too many cells loaded into the microwells will compromise the EB formation process. In parallel,we have tested our microwell-based system for homogeneous hEB formation from dissociated human embryonic stem cells (hESCs). Successful production of homogeneous hEBs from dissociated hESCs in the absence of ROCK-i and centrifugation was achieved within an optimal range of input cell density per microwell. Both the hiPSC- and hESC-derived hEBs expressed key proteins characteristic of all the three developmental germ layers,confirming their EB identity. This novel EB production technology may represent a versatile platform for the production of homogeneous EBs from dissociated human pluripotent stem cells (hPSCs). View Publication

The Met receptor tyrosine kinase has been shown to be overexpressed or mutated in a variety of solid tumors and has,therefore,been identified as a good candidate for molecularly targeted therapy. Activation of the Met tyrosine kinase by the TPR gene was originally described in vitro through carcinogen-induced rearrangement. The TPR-MET fusion protein contains constitutively elevated Met tyrosine kinase activity and constitutes an ideal model to study the transforming activity of the Met kinase. We found,when introduced into an interleukin 3-dependent cell line,TPR-MET induces factor independence and constitutive tyrosine phosphorylation of several cellular proteins. One major tyrosine phosphorylated protein was identified as the TPR-MET oncoprotein itself. Inhibition of the Met kinase activity by the novel small molecule drug SU11274 [(3Z)-N-(3-chlorophenyl)-3-([3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl]methylene)-N-methyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide] led to time- and dose-dependent reduced cell growth. The inhibitor did not affect other tyrosine kinase oncoproteins,including BCR-ABL,TEL-JAK2,TEL-PDGFbetaR,or TEL-ABL. The Met inhibitor induced G(1) cell cycle arrest and apoptosis with increased Annexin V staining and caspase 3 activity. The autophosphorylation of the Met kinase was reduced on sites that have been shown previously to be important for activation of pathways involved in cell growth and survival,especially the phosphatidylinositol-3'-kinase and the Ras pathway. In particular,we found that the inhibitor blocked phosphorylation of AKT,GSK-3beta,and the pro-apoptotic transcription factor FKHR. The characterization of SU11274 as an effective inhibitor of Met tyrosine kinase activity illustrates the potential of targeting for Met therapeutic use in cancers associated with activated forms of this kinase. View Publication

Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific,robust,and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of textgreateror=30 AP(+) cells that coexpress OCT4,NANOG,SSEA3,SSEA4,TRA-1-60,and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition,including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly,this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple,reliable,broadly applicable,and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation. View Publication

A rapid and efficient method to stimulate bone regeneration would be useful in orthopaedic stem cell therapies. Rolipram is an inhibitor of phosphodiesterase 4 (PDE4),which mediates cyclic adenosine monophosphate (cAMP) degradation. Systemic injection of rolipram enhances osteogenesis induced by bone morphogenetic protein 2 (BMP-2) in mice. However,there is little data on the precise mechanism,by which the PDE4 inhibitor regulates osteoblast gene expression. In this study,we investigated the combined ability of BMP-2 and cilomilast,a second-generation PDE4 inhibitor,to enhance the osteoblastic differentiation of mesenchymal stem cells (MSCs). The alkaline phosphatase (ALP) activity of MSCs treated with PDE4 inhibitor (cilomilast or rolipram),BMP-2,and/or H89 was compared with the ALP activity of MSCs differentiated only by osteogenic medium (OM). Moreover,expression of Runx2,osterix,and osteocalcin was quantified using real-time polymerase chain reaction (RT-PCR). It was found that cilomilast enhances the osteoblastic differentiation of MSCs equally well as rolipram in primary cultured MSCs. Moreover,according to the H89 inhibition experiments,Smad pathway was found to be an important signal transduction pathway in mediating the osteogenic effect of BMP-2,and this effect is intensified by an increase in cAMP levels induced by PDE4 inhibitor. View Publication

The development of xeno-free,chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard,various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal,i.e.,proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly,when surface chemistry of the substrates was uniformly controlled by collagen conjugation,the stiffness of substrate was inversely related to the sphericity,a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture,implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall,we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal. View Publication

Breast tumors contain a small population of tumor initiating stem-like cells,termed breast cancer stem cells (BCSCs). These cells,which are refractory to chemotherapy and radiotherapy,are thought to persist following treatment and drive tumor recurrence. We examined whether BCSCs are similarly resistant to hyperthermic therapy,and whether nanoparticles could be used to overcome this resistance. Using a model of triple-negative breast cancer stem cells,we show that BCSCs are markedly resistant to traditional hyperthermia and become enriched in the surviving cell population following treatment. In contrast,BCSCs are sensitive to nanotube-mediated thermal treatment and lose their long-term proliferative capacity after nanotube-mediated thermal therapy. Moreover,use of this therapy in vivo promotes complete tumor regression and long-term survival of mice bearing cancer stem cell-driven breast tumors. Mechanistically,nanotube thermal therapy promotes rapid membrane permeabilization and necrosis of BCSCs. These data suggest that nanotube-mediated thermal treatment can simultaneously eliminate both the differentiated cells that constitute the bulk of a tumor and the BCSCs that drive tumor growth and recurrence. View Publication