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

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value,thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity,only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2,PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb,heart and embryonic development related transcription factors and biological processes. Moreover,this study uncovered novel possible mechanisms,such as the inhibition of RANBP1,that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1,GSTA2),that protect the cell from secondary oxidative stress. As a proof of principle,we demonstrated that a combination of transcriptomics and proteomics,along with consistent differentiation of hESCs,enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. View Publication

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians� View Publication

Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However,precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs,and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase,which blocks entry into mitosis by phosphorylating CDK1 at Y15,was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal,but not ectodermal,lineages. Functionally,WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775. View Publication

It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ˜50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor,UBTF,from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency,hESCs were treated with the Pol I inhibitor,CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers,reduces the expression of pluripotency markers,and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene,and corresponding reduction in transcription,represent early regulatory events during the directed differentiation of pluripotent stem cells. View Publication

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB,it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study,we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly,fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation,suggesting that although BCR and FcγRIIB can both recruit SHIP,this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation,but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP,whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk,as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP,but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling. View Publication

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells. The low efficiency with which iPS cells are derived hinders studies on the molecular mechanism of reprogramming,and integration of viral transgenes,in particular the oncogenes c-Myc and Klf4,may handicap this method for human therapeutic applications. Here we report that valproic acid (VPA),a histone deacetylase inhibitor,enables reprogramming of primary human fibroblasts with only two factors,Oct4 and Sox2,without the need for the oncogenes c-Myc or Klf4. The two factor-induced human iPS cells resemble human ES cells in pluripotency,global gene expression profiles and epigenetic states. These results support the possibility of reprogramming through purely chemical means,which would make therapeutic use of reprogrammed cells safer and more practical. View Publication

In mouse,a subset of dendritic cells (DCs) known as CD8alpha+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However,translation into clinical protocols has been hampered by the failure to identify CD8alpha+ DCs in humans. Here,we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8alpha+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8alpha+ DCs,human DNGR-1+ BDCA3hi DCs express Necl2,CD207,BATF3,IRF8,and TLR3,but not CD11b,IRF4,TLR7,or (unlike CD8alpha+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8,but not of TLR7,and produce interleukin (IL)-12 when given innate and T cell-derived signals. Notably,DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy. View Publication

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine,pharmacological drug screening,and toxicity testing. However,full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study,we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 10(6) hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A,Wnt3a,and sodium butyrate to the culture medium. For further maturation,hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP),a marker for DE,was significantly (p textless 0.05) higher in 2D cultures,while secretion of albumin,a typical characteristic for mature hepatocytes,was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2,CYP2B6,and CYP3A4 in both groups,although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p textless 0.05) higher in 3D bioreactors compared with 2D cultures,which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin,cytokeratin 18 (CK18),and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition,cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures. View Publication

Human embryonic stem cell (hESC) line chHES-468 was derived from abnormal blastocyst donated by polycystic kidney syndrome (PKD) patient after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-468 cell line carried a heterozygous mutation,c.1052610527delAG,of PKD1. Characteristic tests proved that the chHES-468 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. View Publication

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity,and prepared them in formations of single chain,single chain with joint segment,dual chain with joint segment,and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain,which has a storage modulus of 25 kPa,supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. View Publication

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka,who first generated iPSCs by retroviral transduction of four reprogramming factors,several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However,the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study,three different strategies,based on retroviral vectors,episomal vectors,and Sendai virus vectors,were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells,including the expression of alkaline phosphatase and stemness maker genes,and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion,the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs. View Publication

We have developed models of HIV latency using microglia derived from adult human patient brain cortex and transformed with the SV40 T large and hTERT antigens. Latent clones infected by HIV reporter viruses display high levels of spontaneous HIV reactivation in culture. BrainPhys,a medium highly representative of the CNS extracellular environment,containing low glucose and 1{\%} FBS,reduced,but did not prevent,HIV reactivation. We hypothesized that spontaneous HIV reactivation in culture was due to the expression of pro-inflammatory genes,such as TNF-alpha$,taking place in the absence of the natural inhibitory signals from astrocytes and neurons. Indeed,expression and secretion of TNF-alpha$ is strongly reduced in HIV-latently infected microglia compared to the subset of cells that have undergone spontaneous HIV reactivation. Whereas inhibitors of NF-kappa$B or of macrophage activation only had a short-term silencing effect,addition of dexamethasone (DEXA),a glucocorticoid receptor (GR) agonist and mediator of anti-inflammation,silenced the HIV provirus in a long-term,and shRNA-mediated knock-down of GR activated HIV. DEXA also decreased secretion of a number of cytokines,including TNF-alpha$. Chromatin immunoprecipitation analysis revealed that DEXA strongly increased GR occupancy at the HIV promoter,and reduced histone 3 acetylated levels. Moreover,TNF-alpha$ expression inhibitors in combination with DEXA induced further HIV silencing and increased the histone 3 lysine 27 tri-methylated epigenetic mark of repression at the HIV promoter region. We conclude that GR is a critical repressor of HIV transcription in microglia,and a novel potential pharmacological target to restrict HIV expression in the CNS. View Publication