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

Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here,we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did,however,require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-catenin protein levels in stem and progenitor cells. View Publication

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids,pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction,the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step,3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors,and they proliferate and expand over 1-3 months to give rise to intestinal tissue,complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date,this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro. View Publication

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet,acquiring donor cells is,in most instances,an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances,as the isolation of urinary cells is simple (30 ml of urine are sufficient),cost-effective and universal (can be applied to any age,gender and race). Moreover,the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential,and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases,including those affecting the kidney. View Publication

CD147 (alias emmprin or basigin),an integral plasma membrane glycoprotein and a member of the Ig superfamily,is widespread in normal tissues,but highly up-regulated in many types of malignant cancer cells. CD147 is multifunctional,with numerous binding partners. Recent studies suggest that complexes of CD147 with the hyaluronan receptor CD44 and associated transporters and receptor tyrosine kinases are enriched in the plasma membrane of cancer stem-like cells. Here,we show that subpopulations of tumor cell lines constitutively expressing high levels of cell-surface CD147 exhibit cancer stem-like cell properties; that is,they exhibit much greater invasiveness,anchorage-independent growth,spheroid formation,and drug resistance in vitro and higher tumorigenicity in vivo than those constitutively expressing low levels of cell-surface CD147. Primary CD147-rich cell subpopulations derived from mouse mammary adenocarcinomas also exhibit high levels of invasiveness and spheroid-forming capacity,whereas CD147-low cells do not. Moreover,localization at the plasma membrane of CD44,the EGF receptor,the ABCB1 and ABCG2 drug transporters,and the MCT4 monocarboxylate transporter is elevated in cells constitutively expressing high levels of cell-surface CD147. These results show that CD147 is associated with assembly of numerous pro-oncogenic proteins in the plasma membrane and may play a fundamental role in properties characteristic of cancer stem-like cells. View Publication

Under stress conditions such as infection or inflammation the body rapidly needs to generate new blood cells that are adapted to the challenge. Haematopoietic cytokines are known to increase output of specific mature cells by affecting survival,expansion and differentiation of lineage-committed progenitors,but it has been debated whether long-term haematopoietic stem cells (HSCs) are susceptible to direct lineage-specifying effects of cytokines. Although genetic changes in transcription factor balance can sensitize HSCs to cytokine instruction,the initiation of HSC commitment is generally thought to be triggered by stochastic fluctuation in cell-intrinsic regulators such as lineage-specific transcription factors,leaving cytokines to ensure survival and proliferation of the progeny cells. Here we show that macrophage colony-stimulating factor (M-CSF,also called CSF1),a myeloid cytokine released during infection and inflammation,can directly induce the myeloid master regulator PU.1 and instruct myeloid cell-fate change in mouse HSCs,independently of selective survival or proliferation. Video imaging and single-cell gene expression analysis revealed that stimulation of highly purified HSCs with M-CSF in culture resulted in activation of the PU.1 promoter and an increased number of PU.1(+) cells with myeloid gene signature and differentiation potential. In vivo,high systemic levels of M-CSF directly stimulated M-CSF-receptor-dependent activation of endogenous PU.1 protein in single HSCs and induced a PU.1-dependent myeloid differentiation preference. Our data demonstrate that lineage-specific cytokines can act directly on HSCs in vitro and in vivo to instruct a change of cell identity. This fundamentally changes the current view of how HSCs respond to environmental challenge and implicates stress-induced cytokines as direct instructors of HSC fate. View Publication

Vaccines are among the most effective public health tools for combating certain infectious diseases such as influenza. The role of the humoral immune system in vaccine-induced protection is widely appreciated; however,our understanding of how antibody specificities relate to B cell function remains limited due to the complexity of polyclonal antibody responses. To address this,we developed the Spec-seq framework,which allows for simultaneous monoclonal antibody (mAb) characterization and transcriptional profiling from the same single cell. Here,we present the first application of the Spec-seq framework,which we applied to human plasmablasts after influenza vaccination in order to characterize transcriptional differences governed by B cell receptor (BCR) isotype and vaccine reactivity. Our analysis did not find evidence of long-term transcriptional specialization between plasmablasts of different isotypes. However,we did find enhanced transcriptional similarity between clonally related B cells,as well as distinct transcriptional signatures ascribed by BCR vaccine recognition. These data suggest IgG and IgA vaccine-positive plasmablasts are largely similar,whereas IgA vaccine-negative cells appear to be transcriptionally distinct from conventional,terminally differentiated,antigen-induced peripheral blood plasmablasts. View Publication

Antibodies secreted into mucosal barriers serve to protect the host from a variety of pathogens,and are the basis for successful vaccines1. In type I mucosa (such as the intestinal tract),dimeric IgA secreted by local plasma cells is transported through polymeric immunoglobulin receptors2 and mediates robust protection against viruses3,4. However,owing to the paucity of polymeric immunoglobulin receptors and plasma cells,how and whether antibodies are delivered to the type II mucosa represented by the lumen of the lower female reproductive tract remains unclear. Here,using genital herpes infection in mice,we show that primary infection does not establish plasma cells in the lamina propria of the female reproductive tract. Instead,upon secondary challenge with herpes simplex virus 2,circulating memory B cells that enter the female reproductive tract serve as the source of rapid and robust antibody secretion into the lumen of this tract. CD4 tissue-resident memory T cells secrete interferon-gamma,which induces expression of chemokines,including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in a CXCR3-dependent manner,and secrete virus-specific IgG2b,IgG2c and IgA into the lumen. These results reveal that circulating memory B cells act as a rapidly inducible source of mucosal antibodies in the female reproductive tract. View Publication

Human-based body-on-a-chip" technology provides powerful platforms in developing models for drug evaluation and disease evaluations in phenotypic models. Induced pluripotent stem cells (iPSCs) are ideal cell sources for generating different cell types for these in vitro functional systems and recapitulation of the neuromuscular reflex arc would allow for the study of patient specific neuromuscular diseases. Regarding relevant afferent (intrafusal fibers sensory neurons) and efferent (extrafusal fibers motoneurons) cells in vitro differentiation of intrafusal fiber from human iPSCs has not been established. This work demonstrates a protocol for inducing an enrichment of intrafusal bag fibers from iPSCs using morphological analysis and immunocytochemistry. Phosphorylation of the ErbB2 receptors and S46 staining indicated a 3-fold increase of total intrafusal fibers further confirming the efficiency of the protocol. Integration of induced intrafusal fibers would enable more accurate reflex arc models and application of this protocol on patient iPSCs would allow for patient-specific disease modeling." View Publication

Gelatin coatings are effective in increasing the retention of MSCs injected into the heart and minimizing the damage from acute myocardial infarction (AMI),but early studies suffered from low fractions of the MSCs coated with gelatin. Biotinylation of the MSC surface is a critical first step in the gelatin coating process,and in this study,we evaluated the use of biotinylated cholesterol lipid insertion" anchors as a substitute for the covalent NHS-biotin anchors to the cell surface. Streptavidin-eosin molecules where eosin is our photoinitiator can then be bound to the cell surface through biotin-streptavidin affinity. The use of cholesterol anchors increased streptavidin density on the surface of MSCs further driving polymerization and allowing for an increased fraction of MSCs coated with gelatin (83{\%}) when compared to NHS-biotin (52{\%}). Additionally the cholesterol anchors increased the uniformity of the coating on the MSC surface and supported greater numbers of coated MSCs even when the streptavidin density was slightly lower than that of an NHS-biotin anchoring strategy. Critically this improvement in gelatin coating efficiency did not impact cytokine secretion and other critical MSC functions. Proper selection of the cholesterol anchor and the biotinylation conditions supports cellular function and densities of streptavidin on the MSC surface of up to {\~{}}105 streptavidin molecules/$\mu$m2 . In all these cholesterol anchors offer an effective path towards the formation of conformal coatings on the majority of MSCs to improve the retention of MSCs in the heart following AMI." View Publication

Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research,particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y),termed conditionally reprogrammed cell (CRC) technology,enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings,we then created six CF cell CRC populations,three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted,and cells were subcultured,without irradiated feeders plus Y,into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y,dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage,but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus,CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers. View Publication