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

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells,which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered,the rank order of potency is oleoyl-LPA textgreater arachidonoyl-LPA textgreater linolenoyl-LPA textgreater linoleoyl-LPA textgreater stearoyl-LPA = palmitoyl-LPA textgreater myristoyl-LPA. The shorter-chain species,lauroyl- and decanoyl-LPA,show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses textgreater 15 microM. Similarly,the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate,a putative messenger with some structural similarities to LPA,elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however,cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that,although many features of the LPA structure are important for optimal activity,the phosphate group is most critical,suggesting that this moiety is directly involved in receptor activation. View Publication

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone,doxorubicin,and daunorubicin,reduces daunorubicin accumulation and retention,and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells. View Publication

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here,we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes,including tal1/scl,gata1,gata2,hoxB4,and lhx2,into CM ESCs. By immunochemical and morphological analyses,we demonstrated that overexpression of tal1/scl,but not the remaining genes,dramatically increased hematopoiesis of CM ESCs,resulting in multiple blood-cell lineages. Furthermore,flow cytometric analysis demonstrated that CD34,a hematopoietic stem/progenitor cell marker,was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells. View Publication

Human pluripotent stem cells (hPSCs) are proving to be a valuable source of endothelial cells (ECs),pericytes,and vascular smooth muscle cells (vSMCs). Although an increasing number of phenotypic markers are becoming available to determine the phenotypes of these cells in vitro,the ability to integrate and form functional vessels in the host organism,typically mouse,remains critical for the assessment of EC functional competence. However,current mouse models require relatively large numbers of cells that might be difficult to derive simultaneously from multiple hPSCs lines. Therefore,there is an urgent need for new functional assays that are robust and can be performed with small numbers of cells. Here we describe a novel zebrafish xenograft model to test functionality of hPSC-derived ECs. The assay can be performed in 10 days and requires only ˜100-400 human cells per embryo. Thus,the zebrafish xenograft model can be useful for the accurate and rapid assessment of functionality of hPSC-derived ECs in a lower vertebrate model that is widely viewed by regulatory authorities as a more acceptable alternative to adult mice. View Publication

Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However,the generation methods reported so far vary greatly in quality and efficiency. Here,we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs� View Publication

The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs,heterogeneity,and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs,but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques,such as coculture,physical manipulation,sorting,or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First,epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days,iPSCs showed upregulation of mesodermal genes (MSX2,NCAM,HOXA2) and downregulation of pluripotency genes (OCT4,LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes,reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype,expressed high levels of vimentin and N-cadherin,and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs,whereas adipogenic differentiation was limited,as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture,providing a robust,clinically applicable,and efficient system for generating MSCs from human iPSCs. View Publication

West Nile virus (WNV) infection is a mosquito-borne zoonosis with increasing prevalence in the United States. WNV infection begins in the skin,and the virus replicates initially in keratinocytes and dendritic cells (DCs). In the skin and cutaneous lymph nodes,infected DCs are likely to interact with invariant natural killer T cells (iNKTs). Bidirectional interactions between DCs and iNKTs amplify the innate immune response to viral infections,thus controlling viral load and regulating adaptive immunity. iNKTs are stimulated by CD1d-bound lipid antigens or activated indirectly by inflammatory cytokines. We exposed human monocyte-derived DCs to WNV Kunjin and determined their ability to activate isolated blood iNKTs. DCs became infected as judged by synthesis of viral mRNA and Envelope and NS-1 proteins,but did not undergo significant apoptosis. Infected DCs up-regulated the co-stimulatory molecules CD86 and CD40,but showed decreased expression of CD1d. WNV infection induced DC secretion of type I interferon (IFN),but no or minimal interleukin (IL)-12,IL-23,IL-18 or IL-10. Unexpectedly,we found that the WNV-infected DCs stimulated human iNKTs to up-regulate CD69 and produce low amounts of IL-10,but not proinflammatory cytokines such as IFN-γ or tumour necrosis factor (TNF)-α. Both CD1d and IFNAR blockade partially abrogated this iNKT response,suggesting involvement of a T cell receptor (TCR)-CD1d interaction and type I interferon receptor (IFNAR) signalling. Thus,WNV infection interferes with DC-iNKT interactions by preventing the production of proinflammatory cytokines. iNKTs may be a source of IL-10 observed in human flavivirus infections and initiate an anti-inflammatory innate response that limits adaptive immunity and immune pathology upon WNV infection. View Publication

The MUC1 transmembrane mucin is expressed on the surface of activated human T cells; however,the physiologic signals responsible for the regulation of MUC1 in T cells are not known. The present studies demonstrate that IL-7,but not IL-2 or IL-4,markedly induces MUC1 expression on CD3+ T cells. MUC1 was also up-regulated by IL-15,but to a lesser extent than that found with IL-7. The results show that IL-7 up-regulates MUC1 on CD4+,CD8+,CD25+,CD69+,naive CD45RA+,and memory CD45RO+ T cells. In concert with induction of MUC1 expression by IL-7,activated dendritic cells (DC) that produce IL-7 up-regulate MUC1 on allogeneic CD3+ T cells. DC also induce MUC1 expression on autologous CD3+ T cells in the presence of recall Ag. Moreover,DC-induced MUC1 expression on T cells is blocked by a neutralizing anti-IL-7 Ab. The results also demonstrate that DC induce polarization of MUC1 on T cells at sites opposing the DC-T cell synapse. These findings indicate that DC-mediated activation of Ag-specific T cells is associated with induction and polarization of MUC1 expression by an IL-7-dependent mechanism. View Publication

Recently,many hurdles and limitations for production of clinically applicable iPSC derivatives have been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood. Here we describe the generation of transgene-free hiPS cells from cord blood derived CD34+ cells,reprogrammed using CytoTune™ Sendai reprogramming vectors. CD34+ cell isolation,cultivation,reprogramming and establishment of resulting hiPSC lines were performed under the exclusive usage of animal-derived component-free (ADCF) materials and components. View Publication

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein,hiPSCs were differentiated into alpha-smooth muscle actin ($$-SMA) and calponin-positive VSMCs,which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted,which contained cells largely positive for $$-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover,when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts,they remained unruptured and patent with active vascular remodeling,and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs,and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease. View Publication

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized,the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here,using a short fragment of laminin 411 (LM411-E8),an ECM predominantly expressed in the vascular endothelial basement membrane,we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (textgreater95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate. View Publication

We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post-mitotic,HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8-10 weeks for the production of functional mature motoneurons. In comparison with other methods,our protocol does not use feeder cells,has a minimum dependence on proteins (purmorphamine replacing SHH),has controllable adherent selection and is adaptable for scalable suspension culture. View Publication