搜索结果: 'gmp t cell activator'

A growth factor that is mitogenic for vascular endothelial cells,with an ED50 of approximately 1 ng/ml,has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line. The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction. This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts,properties that further distinguish it from other well-characterized vascular endothelial cell mitogens. In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor,which have no secretory leader sequences and might only be released by leakage from damaged cells,the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway. This secretable growth factor could,therefore,be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with blood-vessel growth and maintenance. View Publication

mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl- cotransporter NKCC1 (SLC12A2),a known regulator of cell volume,is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity,as well as activation of Akt and Erk,leading to activation of mTORC1 in cells,colonic organoids,and mouse colon. Moreover,NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1,therefore,suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly,by linking ion transport and cell volume regulation to mTORC1 function,NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation. View Publication

This study focuses on improving the identification of chicken primordial germ cells (PGCs),which are vital for genetic transmission and biotechnological applications. Traditional markers like SSEA1 and CVH have limitations—SSEA1 lacks specificity,and CVH is intracellular. A monoclonal antibody was generated by injecting chicken PGCs into mice,producing one that specifically binds to PGCs and decreases with cell differentiation. Mass spectrometry identified its target as the MYH9 protein. The resulting αMYH9 antibody effectively labels PGCs at various developmental stages,offering a valuable tool for isolating viable PGCs and advancing avian genetics,agriculture,and biotechnology. View Publication

BACKGROUND Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC),particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies,and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct,the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS CSC populations in HPV16-positive and HPV-negative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH),an in vitro tumorsphere formation assay,and an in vivo limiting cell dilution in nonobese diabetic/severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1,a CSC marker,to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC. RESULTS HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining,further extending the association between HPV16 and CSCs. CONCLUSIONS The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore,the reliance on the CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression. View Publication

The cell cycle (CC) underpins diverse cell processes like cell differentiation,cell expansion,and tumorigenesis but current single-cell (sc) strategies study CC as: coarse phases,rely on transcriptomic signatures,use imaging modalities limited to adherent cells,or lack high-throughput multiplexing. To solve this,we develop an expanded,Mass Cytometry (MC) approach with 48 CC-related molecules that deeply phenotypes the diversity of scCC states. Using Cytometry by Time of Flight,we quantify scCC states across suspension and adherent cell lines,and stimulated primary human T cells. Our approach captures the diversity of scCC states,including atypical CC states beyond canonical definitions. Pharmacologically-induced CC arrest reveals that perturbations exacerbate noncanonical states and induce previously unobserved states. Notably,primary cells escaping CC inhibition demonstrated aberrant CC states compared to untreated cells. Our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems with simultaneous multiplexing and integration with MC platforms. Subject terms: Assay systems,Proteomics,Cell biology,Immunology,Systems biology View Publication

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However,the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells,which play a critical role in the development of the autoimmune response,has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs,whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression,induced via STAT3 phosphorylation,mediates IL-1beta and IL-23 down-regulation,while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc),IL-17A,and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc,IL-17A,and IL-23R,but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3,respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS. View Publication

There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases,safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection,processing,testing,banking,packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) that prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore,to avoid any potential cryoprotectant related complications,reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs),animal protein free freezing solutions,cryoprotectants,freezing & thawing protocols,viability assays,packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed. View Publication

The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting,we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC(+)) and epithelial-specific antigen (ESA(+)) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC(-)/ESA(+)). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins,claudin-1 and occludin,and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures,the MUC(+)/ESA(+) epithelial cell line was luminal epithelial restricted in its differentiation repertoire,the suprabasal-derived MUC(-)/ESA(+) epithelial cell line was able to generate itself as well as MUC(+)/ESA(+) epithelial cells and Thy-1(+)/alpha-smooth muscle actin(+) (ASMA(+)) myoepithelial cells. The MUC(-)/ESA(+) epithelial cell line further differed from the MUC(+)/ESA(+) epithelial cell line by the expression of keratin K19,a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane,the MUC(+)/ESA(+) epithelial cell line formed acinus-like spheres. In contrast,the MUC(-)/ESA(+) epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus,MUC(-)/ESA(+) epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast. View Publication

Transforming growth factor (TGF)-beta superfamily proteins play a key role in the regulation of human embryonic stem cells (hESCs). Those of the TGFbeta/activin/nodal branch seem to support self-renewal and pluripotency,whereas those of the bone morphogenic protein (BMP) branch induce differentiation. In contrast to this generalization,we found that hESC remained undifferentiated after knockdown of SMAD4 with inducible short hairpin RNA interference,although the knockdown inhibited TGFbeta signaling and rendered the cells nonresponsive to BMP-induced differentiation. Moreover,the rapid differentiation of hESC after pharmacological inhibition of TGFbeta/activin/nodal receptor signaling was restricted after SMAD4 knockdown. These results suggest that TGFbeta/activin/nodal signaling supports the undifferentiated phenotype of hESC by suppressing BMP activity. During long-term culture,SMAD4 knockdown cell populations became less stable and more permissive to neural induction,a situation that was rescued by re-establishment of SMAD4 expression. These results suggest that SMAD4 is not required for maintenance of the undifferentiated state of hESC,but rather to stabilize that state. View Publication

BACKGROUND Patient-derived induced pluripotent stem cells (iPSCs) are an innovative source as an in vitro model for neurological diseases. Recent studies have demonstrated the differentiation of brain microvascular endothelial cells (BMECs) from various stem cell sources,including iPSC lines. However,the impact of the culturing conditions used to maintain such stem cell pluripotency on their ability to differentiate into BMECs remains undocumented. In this study,we investigated the effect of different sources of Matrigel and stem cell maintenance medium on BMEC differentiation efficiency. METHODS The IMR90-c4 iPSC line was maintained on mTeSR1 or in essential-8 (E-8) medium on growth factor-reduced (GFR) Matrigel from three different manufacturers. Cells were differentiated into BMECs following published protocols. The phenotype of BMEC monolayers was assessed by immunocytochemistry. Barrier function was assessed by transendothelial electrical resistance (TEER) and permeability to sodium fluorescein,whereas the presence of drug efflux pumps was assessed by uptake assay using fluorescent substrates. RESULTS Stem cell maintenance medium had little effect on the yield and barrier phenotype of IMR90-derived BMECs. The source of GFR-Matrigel used for the differentiation process significantly impacted the ability of IMR90-derived BMECs to form tight monolayers,as measured by TEER and fluorescein permeability. However,the Matrigel source had minimal effect on BMEC phenotype and drug efflux pump activity. CONCLUSION This study supports the ability to differentiate BMECs from iPSCs grown in mTeSR1 or E-8 medium and also suggests that the origin of GFR-Matrigel has a marked inpact on BMEC barrier properties. View Publication