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

Human embryonic stem cells (hESCs) offer exciting potential in regenerative medicine for the treatment of a host of diseases including cancer,Alzheimer's and Parkinson's disease. They also provide insight into human development and disease and can be used as models for drug discovery and toxicity analyses. The key properties of hESCs that make them so promising for medical use are that they have the ability to self-renew indefinitely in culture and they are pluripotent,which means that they can differentiate into any of more than 200 human cell types. Since proteins are the effectors of cellular processes,it is important to investigate hESC expression at the protein level as well as at the transcript level. In addition,post-translational modifications,such as phosphorylation,may influence the activity of pivotal proteins in hESCs,and this information can only be determined by studying the proteome. In this review,we summarize the results obtained from several proteomics analyses of hESCs that have been reported in the last few years. View Publication

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets,epidermal Langerhans cells (LCs) and dermal DCs,which can be distinguished by the expression of C-type lectin receptors,Langerin and DC-SIGN,respectively. Although peripheral blood monocytes differentiate into these distinct subsets,monocyte-derived LCs (moLCs) induced by coculture with GM-CSF,IL-4,and TGF-$\beta$1 coexpress both Langerin and DC-SIGN,suggesting that the environmental cues remain unclear. In this study,we show that LC differentiation is TGF-$\beta$1 dependent and that cofactors such as IL-4 and TNF-$\alpha$ promote TGF-$\beta$1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-$\alpha$-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs,dexamethasone-treated moLCs express CD1a,whereas monocyte-derived DCs (moDCs) express CD1b,CD1c,and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and $\alpha$-galactosylceramide,respectively. Strikingly,CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis,these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin. View Publication

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens,but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells,we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs,40{\%} of antigen-specific SED B cells bind antigen within 2 h,whereas unspecifc cells do not,indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice,but is unperturbed in mice depleted of classical dendritic cells (DC). Thus,we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses,and should be taken into account when developing mucosal vaccines. View Publication

The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However,comparing the two assays,cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells,p = 0.0001). Overall,60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells� View Publication

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article,we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro,respectively. Furthermore,our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells,along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover,we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion,our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system. View Publication

Greater than 75% of all hematologic malignancies derive from germinal center (GC) or post-GC B cells,suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID),GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair,homologous recombination,base excision repair,and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells,reflecting activation of a process we have termed somatic hyperrepair (SHR). Using an in vitro system,we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators,SHR induction strictly required signals provided by contact with activated CD4+ T cells,and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression,as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process,our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells. View Publication

miRNA,short non-coding RNA,are rapidly emerging as important regulators in cell homeostasis,as well as potential players in cellular degeneration. The latter has led to interest in them as both biomarkers and as potential therapeutics. Retinal ganglion cells (RGC),whose axons connect the eye to the brain,are central nervous system cells of great interest,yet their study is largely restricted to animals due to the difficulty in obtaining healthy human RGC. Using a CRISPR/Cas9-based reporter embryonic stem cell line,human RGC were generated and their miRNA profile characterized using NanoString miRNA assays. We identified a variety of retinal specific miRNA upregulated in ESC-derived RGC,with half of the most abundant miRNA also detectable in purified rat RGC. Several miRNA were however identified to be unique to RGC from human. The findings show which miRNA are abundant in RGC and the limited congruence with animal derived RGC. These data could be used to understand miRNA’s role in RGC function,as well as potential biomarkers or therapies in retinal diseases involving RGC degeneration. View Publication

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases,regenerative medicine,and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs,which involves step-by-step differentiation of hiPSCs into definitive endoderm,anterior foregut endoderm,NKX2.1+ lung progenitors,and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs,expressing basal cell markers (NGFR,KRT5,and TP63),could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs,potentially applicable for gene therapy research. View Publication

Two groups recently reported the in vitro differentiation of human embryonic stem cells into insulin-secreting cells,achieving an elusive goal for regenerative medicine. Herein we provide a perspective regarding these developments,compare phenotypes of the insulin-containing cells to human $\beta$ cells,and discuss implications for type 1 diabetes research and clinical care. View Publication

Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups,previously introduced by nitrogen atmospheric pressure nonequilibrium plasma,are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1,a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and,in the swollen state,are translucent,soft,and pliable,yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant,since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds,the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions. View Publication