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

It has been recently reported that the regulatory circuitry formed by OCT4,miR-302,and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C,a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs,directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression,decreased BMP signaling,and enhanced TGF$\$ JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown,but not the control,cells within 3 days,accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together,our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF. View Publication

Stem cells,especially human embryonic stem cells (hESCs),are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol,EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs,we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs,particularly those associated with molecular pathways for metabolic processes,oxidative stress,and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOH's effect than their differentiated counterparts,with methylation on the promoter regions of chromosomes 2,16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes,which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis. ?? 2014 Published by Elsevier B.V. View Publication

BACKGROUND Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs). METHODS Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm,anteriorized to a foregut fate,and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs,which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position. RESULTS Activin-A treatment,followed by transforming growth factor-$\$,induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2),SRY (Sex Determining Region Y)-Box 17 (SOX17),and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Cells expressing Nkx2.1 were identified at 40% to 60% efficiency. Within whole-lung scaffolds and under perfusion culture,cells further upregulated Nkx2.1 expression. After orthotopic transplantation,grafts were perfused and ventilated by host vasculature and airways. CONCLUSIONS Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation. View Publication

As the prognosis of invasive aspergillosis remains unacceptably poor in patients undergoing hematopoietic stem cell transplantation (HSCT),there is a growing interest in the adoptive transfer of antifungal effector cells,such as Natural Killer (NK) cells. Because immunosuppressive agents are required in most HSCT recipients,knowledge of the impact of these compounds on the antifungal activity of NK cells is a prerequisite for clinical trials. We,therefore,assessed the effect of methylprednisolone (mPRED),cyclosporin A (CsA) and mycophenolic acid (MPA) at different concentrations on proliferation,apoptosis/necrosis,and the direct and indirect anti-Aspergillus activity of human NK cells. Methylprednisolone decreased proliferation and increased apoptosis of NK cells in a significant manner. After seven days,a reduction of viable NK cells was seen for all three immunosuppressants,which was significant for MPA only. Cyclosporin A significantly inhibited the direct hyphal damage by NK cells in a dose-dependent manner. None of the immunosuppressive compounds had a major impact on the measured levels of interferon-$\gamma$,granulocyte-macrophage colony-stimulating factor and RANTES (regulated on activation,normal T cell expressed and secreted; CCL5). Our data demonstrate that commonly used immunosuppressive compounds have distinct effects on proliferation,viability and antifungal activity of human NK cells,which should be considered in designing studies on the use of NK cells for adoptive antifungal immunotherapy. View Publication

In this study,Chen et al. describe two independent mechanisms that control ?-catenin levels in neuroglial cells and drive their proliferation. The work provides mechanistic insight into the impact of MEK activation resulting from the biallelic loss of NF1 or BRAF rearrangement in pediatric gliomas. The two major genomic alterations in pediatric pilocytic astrocytoma (PA) are NF1 loss and KIAA1549:BRAF rearrangement. Although these molecular changes result in increased MEK activity and tumor growth,it is not clear exactly how MEK controls human neuroglial cell proliferation. Leveraging human-induced pluripotent stem cells harboring these PA-associated alterations,we used a combination of genetic and pharmacological approaches to demonstrate that MEK-regulated cell growth is mediated by ?-catenin through independent mechanisms involving IRX2 control of CTNNB1 transcription and NPTX1 stabilization of ?-catenin protein levels. These results provide new mechanistic insights into MEK regulation of human brain cell function. View Publication

Neurodevelopmental studies employing animal models encounter challenges due to interspecies differences and ethical concerns. Maturing neurons of human origin,undergoing several developmental stages,present a powerful alternative. In this study,human embryonic stem cell (H9 cell line) was differentiated into neural stem cells and subsequently matured into neurons over 30 days. Ion AmpliSeq™ was used for transcriptomic characterization of human stem cell-derived neurons at multiple time points. Data analysis revealed a progressive increase of markers associated with neuronal development and astrocyte markers,indicating the establishment of a co-culture accommodating both glial and neurons. Transcriptomic and pathway enrichment analysis also revealed a more pronounced GABAergic phenotype in the neurons,signifying their specialization toward this cell type. The findings confirm the robustness of these cells across different passages and demonstrate detailed progression through stages of development. The model is intended for neurodevelopmental applications and can be adapted to investigate how genetic modifications or exposure to chemicals,pharmaceuticals,and other environmental factors influence neurons and glial maturation. View Publication

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes,but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study,we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs,enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro,as well as heterotopic bone formation in vivo. Similarly,treating hMSC with Phalloidin,which is known to stabilize polymerized actin filaments,increased hMSCs viability and OB differentiation. Conversely,Cytocholasin D,an inhibitor of actin polymerization,reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level,preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover,depolymerizing actin reduced FAK,p38 and JNK activation during OB differentiation of hMSCs,while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation. View Publication

Aurora kinases play an important role in chromosome alignment,segregation,and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases,and ZM447439,a potent inhibitor of Aurora kinases,effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152,a highly selective inhibitor of Aurora B kinase,on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60,NB4,MOLM13),ALL line (PALL-2),biphenotypic leukemia (MV4-11),acute eosinophilic leukemia (EOL-1),and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM,as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis,as measured by cell-cycle analysis and annexin V staining,respectively. Of note,AZD1152 synergistically enhanced the antiproliferative activity of vincristine,a tubulin depolymerizing agent,and daunorubicin,a topoisomerase II inhibitor,against the MOLM13 and PALL-2 cells in vitro. Furthermore,AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together,AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation. View Publication