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

Genome-wide association studies (GWASs) have reported many single nucleotide polymorphisms (SNPs) associated with psychiatric disorders,but knowledge is lacking regarding molecular mechanisms. Here we show that risk alleles spanning multiple genes across the 10q24.32 schizophrenia-related locus are associated in the human brain selectively with an increase in the expression of both BLOC-1 related complex subunit 7 (BORCS7) and a previously uncharacterized,human-specific arsenite methyltransferase (AS3MT) isoform (AS3MT(d2d3)),which lacks arsenite methyltransferase activity and is more abundant in individuals with schizophrenia than in controls. Conditional-expression analysis suggests that BORCS7 and AS3MT(d2d3) signals are largely independent. GWAS risk SNPs across this region are linked with a variable number tandem repeat (VNTR) polymorphism in the first exon of AS3MT that is associated with the expression of AS3MT(d2d3) in samples from both Caucasians and African Americans. The VNTR genotype predicts promoter activity in luciferase assays,as well as DNA methylation within the AS3MT gene. Both AS3MT(d2d3) and BORCS7 are expressed in adult human neurons and astrocytes,and they are upregulated during human stem cell differentiation toward neuronal fates. Our results provide a molecular explanation for the prominent 10q24.32 locus association,including a novel and evolutionarily recent protein that is involved in early brain development and confers risk for psychiatric illness. View Publication

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development,several issues regarding their pluripotency,differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study,we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that,upon spontaneous differentiation,iPS-MBMC present high amounts of terminal $\$-galactopyranoside residues,pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation,prompting an ectoderm commitment. Exposed $\$-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface,which could modulate intercellular or intracellular interactions. Together,our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and,therefore,the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation. View Publication

mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here,we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels,particularly when oxidative phosphorylation is impaired,such as in neurons treated with mitochondrial inhibitors,or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly,in mitochondrially defective neurons,but not neuroprogenitor cells,ribosomal S6 and S6 kinase phosphorylation increased over time,despite activation of AMPK,which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis,a major energy-consuming process,may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration. View Publication

In mammals,the continuous production of hematopoietic cells (HCs) is sustained by a small number of hematopoietic stem cells (HSCs) residing in the bone marrow. Early HSC activity arises in the aorta-gonad mesonephros region,within cells localized to the ventral floor of the major blood vessels,suggesting that the first HSCs may be derived from cells capable of giving rise to the hematopoietic system and to the endothelial cells of the vasculature. TIE1 (TIE) and TIE2 (TEK) are related receptor tyrosine kinases with an embryonic expression pattern in endothelial cells,their precursors,and HCs,suggestive of a role in the divergence and function of both lineages. Indeed,gene targeting approaches have shown that TIE1,TIE2,and ligands for TIE2,the angiopoietins,are essential for vascular development and maintenance. To explore possible roles for these receptors in HCs,we have examined the ability of embryonic cells lacking both TIE1 and TIE2 to contribute to developmental and adult hematopoiesis by generating chimeric animals between normal embryonic cells and cells lacking these receptors. We show here that TIE receptors are not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo and fetus; surprisingly,however,these receptors are specifically required during postnatal bone marrow hematopoiesis. View Publication

Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs),including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),large numbers of PSC-derived cell products are in demand for applications in drug screening,disease modeling,and especially cell therapy. In stem cell-based therapy,tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO,0.86 $$m) for MRI analysis. The protocol described PSC expansion and differentiation into NPs,and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation. View Publication

Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease,such as Parkinson's disease (PD),in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis,release,and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2,henceforth referred to as GIRK2),representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (textless10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites,and in the soma. Finally,neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models. View Publication

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally,myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally,stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs,with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells. View Publication

Human bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes,chondrocytes,and adipocytes in vivo and in vitro. The aim of this study was to investigate the in vitro differentiation of MSCs into cells of the endothelial lineage. MSCs were generated out of mononuclear bone marrow cells from healthy donors separated by density gradient centrifugation. Cells were characterized by flow cytometry using a panel of monoclonal antibodies and were tested for their potential to differentiate along different mesenchymal lineages. Isolated MSCs were positive for the markers CD105,CD73,CD166,CD90,and CD44 and negative for typical hematopoietic and endothelial markers. They were able to differentiate into adipocytes and osteocytes after cultivation in respective media. Differentiation into endothelial-like cells was induced by cultivation of confluent cells in the presence of 2% fetal calf serum and 50 ng/ml vascular endothelial growth factor. Laser scanning cytometry analysis of the confluent cells in situ showed a strong increase of expression of endothelial-specific markers like KDR and FLT-1,and immunofluorescence analysis showed typical expression of the von Willebrand factor. The functional behavior of the differentiated cells was tested with an in vitro angiogenesis test kit where cells formed characteristic capillary-like structures. We could show the differentiation of expanded adult human MSCs into cells with phenotypic and functional features of endothelial cells. These predifferentiated cells provide new options for engineering of artificial tissues based on autologous MSCs and vascularized engineered tissues. View Publication