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

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population,and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular,disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors,aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits,we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as globulomers" View Publication

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation,whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans,or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs,suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci,characteristic of the inactive X. Moreover,analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs. View Publication

Transcription factors NANOG,OCT4,and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however,their expression profiles during early differentiation of hES cells are unclear. In this study,we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG,OCT4,and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells,characterized by specific expression patterns of NANOG,OCT4,and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time,indicating a gradual mode of developmental transition in these cells. Notably,distinct regulation of SOX2 during early differentiation events was detected,highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation. View Publication

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival,self-renewal,and differentiation. Thus,hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel,i.e. on standard substrates,without affecting hPSC morphology,growth rate or the ability to differentiate into multiple lineages both invitro and invivo. Importantly,QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically,the interaction of QHREDGS with ??1-integrins increased expression of integrin-linked kinase (ILK),increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2),and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance. ?? 2014 Elsevier Ltd. View Publication

The in vitro differentiation of human induced pluripotent stem cells (hiPSC) to generate specific types of cells is inefficient,and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC,we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK) suicide gene at the endogenous OCT4 (POU5F1) locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only,we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV) prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus,we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN) and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences,flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed,three contained the HSV1-TK transgene at the OCT4 locus,but they were not sensitive to GCV. The other six clones were GCV-sensitive,but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days,indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed. View Publication

STUDY HYPOTHESIS Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? STUDY FINDING We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture,often accompanied by erosion of XCI-specific methylation,and a frequent loss of random XCI in the cultures. WHAT IS KNOWN ALREADY Variable XCI patterns have been reported in female hPSC,not only between different hPSC lines,but also between sub-passages of the same cell line,however the reasons for this variability remain unknown. Moreover,while non-random XCI-linked DNA methylation patterns have been previously reported,their origin and extent have not been investigated. STUDY DESIGN,SAMPLES/MATERIALS,METHODS We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines,during long-term culture and after differentiation,by gene expression analysis,histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence,XIST expression by real-time PCR,and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR,and more in depth by massive parallel bisulphite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE All hPSC lines showed XCI,but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation,it did not result in X chromosome reactivation. Moreover,lines without strong erosion of methylation frequently displayed non-random DNA methylation,which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore,we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally,differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. LIMITATIONS,REASONS FOR CAUTION All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. WIDER IMPLICATIONS OF THE FINDINGS Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC,more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen,grant 1502512N),Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel,on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest. View Publication

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses,cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated,adult donors and found frequent cross-group BCRs,both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab,encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response. View Publication

Freshly isolated,uncultured,autologous adipose derived regenerative cells (ADRCs) have emerged as a promising tool for regenerative cell therapy. The Transpose RT system (InGeneron,Inc.,Houston,TX,USA) is a system for isolating ADRCs from adipose tissue,commercially available in Europe as a CE-marked medical device and under clinical evaluation in the United States. This system makes use of the proprietary,enzymatic Matrase Reagent for isolating cells. The present study addressed the question whether the use of Matrase Reagent influences cell yield,cell viability,live cell yield,biological characteristics,physiological functions or structural properties of the ADRCs in final cell suspension. Identical samples of subcutaneous adipose tissue from 12 subjects undergoing elective lipoplasty were processed either with or without the use of Matrase Reagent. Then,characteristics of the ADRCs in the respective final cell suspensions were evaluated. Compared to non-enzymatic isolation,enzymatic isolation resulted in approximately twelve times higher mean cell yield (i.e.,numbers of viable cells/ml lipoaspirate) and approximately 16 times more colony forming units. Despite these differences,cells isolated from lipoaspirate both with and without the use of Matrase Reagent were independently able to differentiate into cells of all three germ layers. This indicates that biological characteristics,physiological functions or structural properties relevant for the intended use were not altered or induced using Matrase Reagent. A comprehensive literature review demonstrated that isolation of ADRCs from lipoaspirate using the Transpose RT system and the Matrase Reagent results in the highest viable cell yield among published data regarding isolation of ADRCs from lipoaspirate. View Publication

Different innate immune cell types are known to release extracellular traps (ETs) in response to invasive pathogens,including parasites. These ETs function to trap,immobilize,and eventually kill pathogens. In line with this,monocytes and macrophages have been shown to release ETs,known as monocyte/macrophage extracellular traps (METs). Toxoplasma gondii (T. gondii) is an apicomplexan zoonotic parasite that infects humans and homeothermic animals. While most studies have focused on prolonged exposure of immune cells to T. gondii,this study characterized the early innate immune reaction of mononuclear phagocytes to vital T. gondii tachyzoites. Methods: Primary human and bovine monocytes,monocytic THP-1 cells,and THP-1 cell-derived macrophages (M0-,M1-,and M2-like) were exposed to T. gondii tachyzoites for 4 h. Scanning electron microscopy (SEM),transmission electron microscopy (TEM),immunofluorescencemicroscopy,and confocal microscopy were used to visualize cell activation and the presence of METs. Additionally,the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-6,and expression of Toll-like receptor (TLR) 2 and TLR4 were analyzed. Results and discussion: Microscopic analysis illustrated the activation of all cell types tested within 4 h of exposure to T. gondii tachyzoites. Numerous tachyzoites were found intracellularly in THP-1 cell-derived M1-like macrophages. Furthermore,the co-localization of extracellular DNA (extDNA) and histones in extracellular web-like fibers proved classical characteristics of extruded T. gondii-induced METs,although this was a rare event. In primary human monocytes,an increased release of IL-1β and IL-6 was observed following exposure to T. gondii tachyzoites. When co-stimulated with lipopolysaccharide (LPS),primary human monocytes showed an enhanced release of IL-1β and IL-6 in response to T. gondii. In contrast to monocytic THP-1 cells,THP-1 cell-derived M1-like macrophages released IL-1β in response to T. gondii tachyzoite exposure. When additionally stimulated by LPS,all THP-1 cell-derived macrophages showed an enhanced release of IL-1β,and monocytic THP-1 cells an increased release of IL-6 in response to T. gondii tachyzoites. This study provides insights into the early innate immune response of human and bovine mononuclear phagocytes to T. gondii. While cytokine secretion was prominent,MET formation was rare in the early response (i.e. < 4 h of exposure) to T. gondii tachyzoites. View Publication

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca(2+) level ([Ca(2+)] i ) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus,the presence of active GABA-A receptors,associated with phenotype determination via Ca(2+)-signalling was demonstrated in differentiating human DA neurons. View Publication

INTRODUCTION: Ischemic stroke is a leading cause of death and disability,but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study,we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.backslashnbackslashnMETHODS: Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.backslashnbackslashnRESULTS: After 11 days of neural induction by using the small-molecule protocol,over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude,repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.backslashnbackslashnCONCLUSIONS: Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo,enhance regenerative activities,and improve sensory recovery after ischemic stroke. View Publication

Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ,patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs),a recently emerged novel genome editing tool,to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon,TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD,which represents a significant advance toward hiPSC-based cell and gene therapies. View Publication