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

Based on knowledge of early embryo development,where anterior neural ectoderm (ANE) development is regulated by native inhibitors of bone morphogenic protein (BMP) and Nodal/Activin signaling,most published protocols of human embryonic stem cell differentiation to ANE have demonstrated a crucial role for Smad signaling in neural induction. The drawbacks of such protocols include the use of an embryoid body culture step and use of polypeptide secreted factors that are both expensive and,when considering clinical applications,have significant challenges in terms of good manufacturing practices compliancy. The use of small molecules to direct differentiation of pluripotent stem cells toward a specified lineage represents a powerful approach to generate specific cell types for further understanding of biological function,for understanding disease processes,for use in drug discovery,and finally for use in regenerative medicine. We therefore aimed to find controlled and reproducible animal-component-free differentiation conditions that would use only small molecules. Here,we demonstrate that pluripotent stem cells can be reproducibly and efficiently differentiated to PAX6(+) (a marker of neuroectoderm) and OCT4(-) (a marker of pluripotent stem cells) cells with the use of potent small inhibitors of the BMP and Activin/Nodal pathways,and in animal-component-free conditions,replacing the frequently used Noggin and SB431542. We also show by transcript analysis,both at the population level and for the first time at the single-cell level,that differentiated cells express genes characteristic for the development of ANE,in particular for the development of the future forebrain. View Publication

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis,but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm,precardiac or presomitic mesoderm within the first 24 h of differentiation,respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members,antagonist of Nodal signalling LEFTY1 and CER1,are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive,functional role of the BCD,we show its utility as a method to control lineage-specific differentiation. Furthermore,these findings have profound consequences for inter-experimental comparability,reproducibility,bioprocess optimization and scale-up. View Publication

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. View Publication

Recent studies have suggested that REX1 (reduced expression 1) plays an important role in pluripotency,proliferation,and differentiation. However,the molecular mechanisms involved in REX1-dependent regulation of diverse cellular processes remain unclear. To elucidate the regulatory functions of REX1 in human embryonic stem cells (hESCs),comparative proteomic analysis was performed on REX1 RNAi specifically silenced hESCs. Analysis of the proteome via nano-LC-MS/MS identified 140 differentially expressed proteins (DEPs) displaying a textgreater2-fold difference in expression level between control and REX1 knockdown (KD) hESCs,which were then compared with transcriptome data and validated by quantitative real-time RT-PCR and Western blotting. These DEPs were analyzed by GO,pathway,and functional clustering analyses to determine the molecular functions of the proteins and pathways regulated by REX1. The REX1 KD-mediated DEPs mapped to major biological processes involved in the regulation of ribosome-mediated translation and mitochondrial function. Functional network analysis revealed a highly interconnected network among these DEPs and indicated that these interconnected proteins are predominantly involved in translation and the regulation of mitochondrial organization. These findings regarding REX1-mediated regulatory network have revealed the contributions of REX1 to maintaining the status of hESCs and have improved our understanding of the molecular events that underlie the fundamental properties of hESCs. View Publication

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) $$$$ and $$$$ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells. View Publication

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is a crucial step in cell therapy. In the present study,we demonstrate that functional CD34(+) progenitor cells can be efficiently produced from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) by combined modulation of 2 signaling pathways. A higher proportion of CD34(+) cells (∼ 20%) could be derived from hPSCs by inhibition of mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling and activation of bone morphogenic protein-4 (BMP4) signaling. hPSC-derived CD34(+) progenitor cells further developed to endothelial and smooth muscle cells with functionality. Moreover,they contributed directly to neovasculogenesis in ischemic mouse hind limbs,thereby resulting in improved blood perfusion and limb salvage. Our results suggest that combined modulation of signaling pathways may be an efficient means of differentiating hPSCs into functional CD34(+) progenitor cells. View Publication

Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system,along with their G-coupled cannabinoid receptors (CB�? and CB₂) and the enzymes involved in their biosynthesis and degradation. However,their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here,we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol),whereas CB₂ receptors are expressed in human and murine HSPCs. On ligand stimulation with CB₂ agonists,CB₂ receptors induced chemotaxis,migration,and enhanced colony formation of bone marrow cells,which were mediated via ERK,PI3-kinase,and Gαi-Rac1 pathways. In vivo,the CB₂ agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition,granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB₂ antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together,these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB₂/CB₂ agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus,CB₂ agonists may be therapeutically applied in clinical conditions,such as bone marrow transplantation. View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover,safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple,nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe,efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research,disease modeling,and regenerative medicine. ?? 2010 Elsevier Inc. View Publication

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication