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

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma,an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis,bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2,respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors,and transduced cells were cultured in a semisolid medium permissive for the development of erythroid,myeloid,and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro,in contrast to Tax2-transduced cells,which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected,suggesting that Tax1 inhibited the maturation of relatively early,uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis,lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells,an activity that is not displayed by Tax2,and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo,the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2. View Publication

We used a panel of human and mouse fibroblasts with various abilities for supporting the prolonged growth of human embryonic stem cells (hESCs) to elucidate growth factors required for hESC survival,proliferation,and maintenance of the undifferentiated and pluripotent state (self-renewal). We found that supportive feeder cells secrete growth factors required for both hESC survival/proliferation and blocking hESC spontaneous differentiation to achieve self-renewal. The antidifferentiation soluble factor is neither leukemia inhibitory factor nor Wnt,based on blocking experiments using their antagonists. Because Wnt/beta-catenin signaling has been implicated in cell-fate determination and stem cell expansion,we further examined the effects of blocking or adding recombinant Wnt proteins on undifferentiated hESCs. In the absence of feeder cell-derived factors,hESCs cultured under a feeder-free condition survived/proliferated poorly and gradually differentiated. Adding recombinant Wnt3a stimulated hESC proliferation but also differentiation. After 4-5 days of Wnt3a treatment,hESCs that survived maintained the undifferentiated phenotype but few could form undifferentiated hESC colonies subsequently. Using a functional reporter assay,we found that the beta-catenin-mediated transcriptional activation in the canonical Wnt pathway was minimal in undifferentiated hESCs,but greatly upregulated during differentiation induced by the Wnt treatment and several other methods. Thus,Wnt/beta-catenin activation does not suffice to maintain the undifferentiated and pluripotent state of hESCs. We propose a new model for the role of Wnt/beta-catenin signaling in undifferentiated hESCs. View Publication

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is,however,challenged by a limited supply of appropriate cells. Therefore,we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study,we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine,a well-known demethylating agent,and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1,1,or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR,RT-PCR,immunofluorescence,and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes,we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion,these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes. View Publication

Abnormal activation of B lymphocytes is a feature commonly seen in human immunodeficiency virus type 1 (HIV-1)-infected persons. However,the mechanism(s) responsible for this dysfunction is still poorly understood. Having recently shown that CD40L,the ligand for CD40,is inserted within emerging HIV-1 particles,we hypothesized that the contact between virus-anchored host CD40L and CD40 on the surface of B lymphocytes might result in the activation of this cell type. We report here that CD40L-bearing viruses,but not isogenic virions lacking host-derived CD40L,can induce immunoglobulin G and interleukin-6 production. Furthermore,such viral entities were found to induce B-cell homotypic adhesion. These effects were paralleled at the intracellular level by the nuclear translocation of the ubiquitous transcription factor NF-kappaB. The presence of host-derived CD40L within virions resulted in an increased virus attachment to B cells and a more-efficient B-cell-mediated transfer of HIV-1 to autologous CD4(+) T lymphocytes. All the above processes were independent of the virus-encoded envelope glycoproteins. Altogether,the data gathered from this series of investigations suggest that the incorporation of host-encoded CD40L in HIV-1 is likely to play a role in the B-cell abnormalities that are seen in infected individuals. View Publication

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations,which is lacking in the field,is also crucial. Here,we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine,a small molecule that activates the SHH pathway,could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+,Irx3+,and Pax7-),with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus,the directed neural differentiation system with small molecules,even without further purification,will facilitate basic and translational studies using human motoneurons at a minimal cost. View Publication

Luminal and myoepithelial cells have been separated from normal adult human breast epithelium using fluorescence activated cell sorting. Their isolation was based on the exclusive expression of two surface antigens,epithelial membrane antigen (EMA) and the common acute lymphoblastic leukaemia antigen (CALLA/CD10/neutral endopeptidase 24.11). Sorted luminal and myoepithelial cells displayed distinctively different morphologies when maintained in monolayer culture,differences which were enhanced by the addition of hydrocortisone,insulin and cholera toxin to the culture medium. The EMA-positive cells formed an attenuated monolayer with indistinct cell boundaries while CALLA-positive cells,by contrast,formed tightly packed arrays of refractile cells. The distribution of the cell type-specific markers cytokeratin 18 (luminal cells) and smooth muscle alpha-actin (myoepithelial cells) indicated that the sorted populations were approximately 98% pure. However,a significant minority (approximately 15%) of sorted luminal cells consistently expressed the basal-cell marker cytokeratin 14 in culture. A marked difference was noted in the proliferative behaviour of the two types of sorted cells,with myoepithelial cells dividing rapidly in response to the humoural additives,in contrast to the luminal cells which proliferated slowly. Both types of sorted cells could be cloned in the presence of feeder layers of mouse fibroblasts. Clones of luminal and myoepithelial cells were also distinctive; all spread" luminal clones were similar in appearance to each other� View Publication

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse,but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed textless10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However,the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses textless20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells,mES cells lacking H2AX,a histone protein involved in the DNA damage response,were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining,H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs),an increase in dsb rejoining rate,and a decrease in Ku70/80. Unlike mouse ES,human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells,they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM,and they add to the growing list of differences in the way rodent and human cells deal with DNA damage. View Publication

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity,we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo,activated and memory B cells expressed lower levels of CD1d compared with resting,naive,and marginal zone-like B cells. In vitro,CD1d was downregulated by all forms of B cell activation,leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone,whereas activation via the BCR significantly upregulated CD1c,particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes,an effect that was reversed by RARα agonists. However,BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells,in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity. View Publication

Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated,turning MSCs into variety of cell lineages of mesodermal,ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial,these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently,using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function,as well as with specific signalling pathways that promote neural fate commitment,we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). However,the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early-passage hMSCs,comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early- and late-passage hMSCs. The results demonstrated that early-passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs,accompanied by morphological changes into neural-like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early-passage hMSCs could explain their higher plasticity and pliability for neural induction. Copyright textcopyright 2012 John Wiley & Sons,Ltd. View Publication

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol,passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization,centrifugation or drug treatment. It also allows for higher throughput,requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation,colony expansion,cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion,and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages,this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium. View Publication

Hepatocytes play a central and crucial role in cholesterol and lipid homeostasis,and their proper function is of key importance for cardiovascular health. In particular,hepatocytes (especially periportal hepatocytes) endogenously synthesize large amounts of cholesterol and secrete it into circulating blood via apolipoprotein particles. Cholesterol-secreting hepatocytes are also the clinically-relevant cells targeted by statin treatment in vivo. The study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that do not recapitulate those key aspects of normal human hepatocyte function that result from genetic variation of individuals within a population. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells can provide a cell culture model for the study of cholesterol homeostasis,dyslipidemias,the action of statins and other pharmaceuticals important for cardiovascular health. We have analyzed expression of core components for cholesterol homeostasis in untreated human iPS cells and in response to pravastatin. Here we show the production of differentiated cells resembling periportal hepatocytes from human pluripotent stem cells. These cells express a broad range of apolipoproteins required for secretion and elimination of serum cholesterol,actively secrete cholesterol into the medium,and respond functionally to statin treatment by reduced cholesterol secretion. Our research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to human cholesterol homeostasis at both cellular and molecular levels. Importantly,it permits for the first time to also functionally assess the impact of genetic polymorphisms on cholesterol homeostasis. Finally,the system will also be useful for mechanistic studies of heritable dyslipidemias,drug discovery,and investigation of modes of action of cholesterol-modulatory drugs. View Publication

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however,although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes,most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here,the authors report a 3D cellular microenvironment plate (3D-CEP),which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG),which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally,global gene expression analyses are used to elucidate small variations among different test environments. Interestingly,the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine. View Publication