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

NK cells express different TLRs,such as TLR3,TLR7,and TLR9,but little is known about their role in NK cell stimulation. In this study,we used specific agonists (poly(I:C),loxoribine,and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12,IL-15,or IFN-alpha,and investigated the secretion of IFN-gamma,cytotoxicity,and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine,in conjunction with IL-12,but not IL-15,triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also,secretion of IFN-gamma was dependent on MEK1/ERK,p38 MAPK,p70(S6) kinase,and NF-kappaB,but not on calcineurin. IFN-alpha induced a similar effect,but promoted lesser IFN-gamma secretion. However,cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged,as well as the expression of the NKG2D receptor. Excitingly,IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12,and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12,and this effect can be further enhanced by costimulation through NKG2D. Hence,integration of the signaling cascades that involve TLR3,TLR7,IL-12,and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions,which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer. View Publication

Human bone marrow (BM) cells contain low levels of the DNA repair protein,O6-alkylguanine-DNA alkyltransferase,which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor,interleukin-3,stem cell factor,the combination,or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition,O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU,resulting in a reduction in the dose of drug (termed the dose-modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since,unlike many other cell types,proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase,myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials. View Publication

OBJECTIVE: We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS: After single cell deposition onto 96-well plates,cells were cocultured for 10 days with the stem cell supporting cell line AFT024,fibronectin (FN),or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS: Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times,a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS: For the first time,we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC),this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential. View Publication

Type 1 interferon-producing cells (IPCs),also known as plasmacytoid dendritic cell (DC) precursors,represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection,autoimmune SLE,and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3),IL-7,stem cell factor (SCF),macrophage-colony-stimulating factor (M-CSF),and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture,they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L,inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 x 10(6) IPCs per 10(6) CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-alpha (IFN-alpha) and to differentiate into mature DCs. This culture system,combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF-mobilized donors,permits the generation of more than 10(9) IPCs from a single blood donor. View Publication

Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here,we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages,we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage,we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes,formed large numbers of multilineage and myeloid colonies,and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit,greater than 95% CD45(+) hematopoietic cells could be generated. In addition,we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification,whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development. View Publication

In human cytomegalovirus (HCMV)-seropositive patients,CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However,analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors,availability of cells,and low frequency of reactivation. In addition,multiple progenitor cell types express surface CD34,and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study,we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines,WA01 and WA09,to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however,these cells are a heterogeneous pool with donor-to-donor variation in functional,genetic,and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states,which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation. View Publication

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells,including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation,nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy. View Publication

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium,functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts,we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system,these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs,we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust,scalable,and consistent methodology. In the present study,we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set,we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality,with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed,demonstrating the value of this biologically relevant and reproducible technology. In addition,this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. View Publication

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential,however,is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here,we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG,we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes,and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane,suggesting that the ectodomain is not required for protein loading. Finally,exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain,confirming a role of the ectodomain in cell tropism. In summary,our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells. View Publication

Embryonic stem cells (ESCs) are an attractive source of cells for disease modeling in vitro and may eventually provide access to cells/tissues for the treatment of many degenerative diseases. However,applications of ESC-derived cell types are largely hindered by the lack of highly efficient methods for lineage-specific differentiation. Using a high-content screen,we have identified a small molecule,named stauprimide,that increases the efficiency of the directed differentiation of mouse and human ESCs in synergy with defined extracellular signaling cues. Affinity-based methods revealed that stauprimide interacts with NME2 and inhibits its nuclear localization. This,in turn,leads to downregulation of c-Myc,a key regulator of the pluripotent state. Thus,our findings identify a chemical tool that primes ESCs for efficient differentiation through a mechanism that affects c-Myc expression,and this study points to an important role for NME2 in ESC self-renewal. View Publication