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

The CD34 molecule belongs to the mucin membrane molecule family and is expressed on virtually all normal haematopoietic progenitor cells (HPC). Due to its heavy glycosylation,several different epitopes exist on the molecule. Based on the sensitivity of the glycosylated molecule to degradation with a glycoprotease from Pasteurella haemolytica and neuraminidase,three classes of epitopes have been identified. The class I and II epitopes are probably related to the glycosylated part of the molecule while class III epitopes are core protein related. It has been known for some time that CD34 class I epitopes are absent on CD34 molecules expressed on high endothelial venules. Here we review recent observations that expression of both class I and II epitopes,but not class III epitopes,is impaired on mature myeloid CD34-pos. HPC while no diverse class epitope expression was observed on immature HPC. In addition,cells from patients with CD34-pos. acute myeloid leukaemia of FAB classification M4-M5,i.e.,leukaemic blast cells of relatively mature morphologic phenotype,also express less class I and II epitopes than class III epitopes. It therefore seems that HPC maturation and class I and II epitope deprivation are concomitant events and that CD34 class I and II epitopes are lost prior to downregulation of the CD34 molecule per se. The biological significance of this observation is discussed as well as the need to carefully select CD34-specific monoclonal antibodies for research and clinical purposes. View Publication

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process,we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold,and 1% of the Myc-overexpressing cells,but none of the control cells,retained self-renewal capacity even under differentiation-inducing conditions. A Myc mutant (MycV394D) deficient in binding to Miz-1,did not increase the percentage of self-renewing cells but was able to stimulate proliferation of NPCs as efficiently as wild-type Myc,indicating that these two cellular phenomena are regulated by at least partially different pathways. Our results suggest that Myc,through Miz-1,enhances self-renewal of NPCs and influences the way progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. View Publication

The conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces,which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable,continuous propagation of two hESC lines on microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks,thus opening the prospect of propagation in controlled bioreactors. ?? 2009 Elsevier B.V. All rights reserved. View Publication

Experimentation with the progenitor/stem cells in adult prostate epithelium can be inconvenient due to a tight time line from tissue acquisition to cell isolation and to downstream experiments. To circumvent this inconvenience,we developed a simple technical procedure for culturing epithelial cells derived from human prostate tissue. In this study,benign prostate tissue was enzymatically digested and fractionated into epithelium and stroma,which were then cultured in the medium designed for prostate epithelial and stromal cells,respectively. The cultured cells were analyzed by immunocytochemical staining and flow cytometry. Prostate tissue-regenerating capacity of cultured cells in vitro was determined by co-culturing epithelial and stromal cells in dihydrotestosterone-containing RPMI. Cell lineages in formed acini-like structures were determined by immunohistochemistry. The culture of epithelial cells mainly consisted of basal cells. A minor population was negative for known lineage markers and positive for CD133. The culture also contained cells with high activity of aldehyde dehydrogenase. After co-culturing with stromal cells,the epithelial cells were able to form acini-like structures containing multiple cell lineages. Thus,the established culture of prostate epithelial cells provides an alternative source for studying progenitor/stem cells of prostate epithelium. View Publication

The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand,FasL. Although Fas is highly expressed in cancer cells,insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here,we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor,fasudil,that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL),fasudil promoted cancer cell apoptosis,but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis. View Publication

The generation of insulin-producing pancreatic $\beta$ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However,insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide $\beta$ cells. Here,we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive $\beta$ cells from hPSC in vitro. These stem-cell-derived $\beta$ cells (SC-$\beta$) express markers found in mature $\beta$ cells,flux Ca(2+) in response to glucose,package insulin into secretory granules,and secrete quantities of insulin comparable to adult $\beta$ cells in response to multiple sequential glucose challenges in vitro. Furthermore,these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner,and transplantation of these cells ameliorates hyperglycemia in diabetic mice. View Publication

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover,Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands,such as HIV and CpG respectively,turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions,and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection,but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here,we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α,TNF-α,IFN-γ and IL-12,and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations,the addition of NK cells did not promote the release of these mediators,suggesting that once efficiently triggered by the virus,pDCs could not integrate new activating signals delivered by NK cells. However,high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly,we identified the alarmin HMGB1,released at pDC-NK cell synapse,as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover,HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1,HMGB1-specific antibodies,sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether,these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells,and they suggest a novel mechanism of innate control of HIV-1 infection. View Publication

To investigate intestinal health and its potential disruptors in vitro,representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However,the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently,the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge,IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model,while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately,comparison to in vivo transcriptomics data showed limited similarities,which could be explained by essential differences in the study setups. Altogether,hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-74802-w. View Publication