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

Although prolonged transgene expression in progenitor cells might be desirable for modified cell therapy,the viral promoter-based expression vector tends to promote transgene expression only for a limited period. Here,we examined the ability of cellular promoters from elongation factor-1alpha (EF-1alpha) and ubiquitin C to drive gene expression in hematopoietic TF-1 and mesenchymal progenitor cells. We compared the expression levels and duration of a model gene,interleukin-2,generated by the cellular promoters to those by the cytomegalovirus (CMV) promoter. The EF-1alpha and ubiquitin C promoters drove prolonged gene expression in hematopoietic TF-1 and mesenchymal progenitor cells,whereas the CMV promoter did not. At day 7 after transfection in TF-1 cells,the mRNA expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 118- and 56-fold higher,respectively,than those driven by the CMV promoter. Similarly,in mesenchymal progenitor cells,the expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 98- and 20-fold higher,respectively,than that driven by the CMV promoter-encoding plasmid. Moreover,the ubiquitin C promoter directed higher levels of green fluorescence protein expression in mesenchymal progenitor cells than did the CMV promoter. These results indicate that the use of cellular promoters such as those for EF-1alpha and ubiquitin C might direct prolonged gene expression in hematopoietic and mesenchymal progenitor cells. View Publication

ETS2 and ERG are transcription factors,encoded on human chromosome 21 (Hsa21),that have been implicated in human cancer. People with Down syndrome (DS),who are trisomic for Hsa21,are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1,which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis,we expressed ETS2,ERG,and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2,ERG,and FLI-1 facilitated the expansion of megakaryocytes from wild-type,Gata1-knockdown,and Gata1s knockin progenitors,but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41(+) cells generated from Gata1s-knockin progenitors,their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin,but not wild-type,fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway,commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis. View Publication

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA),we studied the HLA-DR expression on CD45dimCD34+CD38+ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[-]) leukocytes. HLA-DR-lacking (DR[-]) cells accounted for 13.0-57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(-) leukocytes. The incubation of sorted DR(-) HSPCs in the presence of IFN-$\gamma$ for 72??h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g.,CD48,CD74,and CD86) in DR(-) cells,which was not evident in HLA-class I(-) HSPCs. DR(-) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(-) HSPCs. These findings suggest that CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(-) HSPCs in a distinct way from CD8+ T cells recognizing HLA-class I-restricted antigens. View Publication

AbstractIntroduction
Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited,there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast,and express HLA-G,a marker of EVT. The goals of the present study were to further characterize the HLA-G+ cells derived from BMP4-treated hESC,and determine their suitability as a model.
Methods
HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G+ and HLA-G−/TACSTD2+ populations with immunomagnetic beads and expression profiles analyzed by microarray.
Results
There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent,stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7,and the majority were also HLA-G+. Gene expression profiles revealed that HLA-G+,BMP4-treated hESC were similar to,but distinct from,HLA-G+ cells isolated from first trimester placentas. Whereas HLA-G+ and HLA-G− cells from first trimester placentas had highly divergent gene expression profiles,HLA-G+ and HLA-G− cells from BMP4-treated hESC had somewhat similar profiles,and both expressed genes characteristic of early trophoblast development.
Conclusions
We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage. View Publication

Recently,induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer,both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore,the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front,although donor cell types for generating iPSCs are wide-ranging,T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells,which can be applied to novel immunotherapies. In the present study,we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining,had a normal karyotype,and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined,animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application. View Publication

The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover,because of its barrier properties,this endothelial interface restricts uptake of neurotherapeutics. Thus,a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues,including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes,including well-organized tight junctions,appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably,they respond to astrocytes,acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2),and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients. View Publication

OBJECTIVES: Tolerogenic dendritic cells (tolDCs) constitute a promising experimental treatment for targeting autoreactive T cells in autoimmune diseases,including rheumatoid arthritis (RA). The authors' goal is to bring tolDC therapy for RA to the clinic. Here the authors address key translational issues related to the manufacturing of tolDCs from RA patients with current good manufacturing practice (cGMP)-compliant reagents,the stability of tolDCs,and the selection of suitable quality control markers. METHODS: Human monocyte-derived tolDCs were established from RA patients and healthy controls (HCs) using the immunosuppressive drugs dexamethasone and vitamin D₃,and the cGMP-grade immunomodulator,monophosphoryl lipid A,in the cGMP-compliant medium,CellGroDC. The functionality of tolDCs and tolDC-modulated autologous CD4 T cells was determined by flow cytometry,[³H]thymidine incorporation and ELISA. RESULTS: Clinical-grade tolDCs established from patients with RA exhibit a typical tolerogenic phenotype of reduced costimulatory molecules,low production of proinflammatory cytokines and impaired stimulation of autologous antigen-specific T cells,comparable to HC tolDCs. Toll-like receptor 2 (TLR-2) was highly expressed by tolDCs but not mature DCs. Furthermore,tolDCs suppressed mature DC-induced T cell proliferation,interferon γ and interleukin 17 production,and rendered T cells hyporesponsive to further stimulation. Importantly,tolDCs were phenotypically stable in the absence of immunosuppressive drugs and were refractory to further challenge with proinflammatory mediators. CONCLUSIONS: tolDCs established from patients with RA are comparable to those derived from healthy donors. TLR-2 was identified as an ideal marker for quality control of tolDCs. Potently tolerogenic and highly stable,these tolDCs are a promising cellular therapeutic for tailored immunomodulation in the treatment of RA. View Publication

Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes,such as tryptase and chymase,at the cell-cell contact site. This previously unidentified mast cell effector mechanism,which we name the antibody-dependent degranulatory synapse (ADDS),is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably,IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence. View Publication

BACKGROUND This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood,and compare the two methods. METHODS The manual method was optimized for whole blood centrifugation speed,gradient type (Ficoll,Leucosep,CPT),and freezing method (Mr Frosty,Controlled Rate Freezing). Various parameters of the automated protocol using a CPT gradient on a Tecan liquid handler were optimized. Optimal protocols were validated in parallel for reproducibility and robustness. Optimization and validation were assessed in terms of cell yield,viability,recovery,white blood cell (WBC) subpopulation distribution,gene expression,and lymphoblastoid cell line (LCL) transformation. RESULTS An initial centrifugation of whole blood at 2000 g was considered optimal for further processing,allowing isolation of plasma and PBMCs from a single sample. The three gradients gave similar outcomes in terms of cell yield,viability,and WBC subpopulation distribution. Ficoll showed some advantages and was selected for further evaluations. Optimization of the automated protocol script using a CPT gradient gave 61% cell recovery. No significant differences in quality,quantity,and WBC subpopulation distribution were seen between the two freezing methods,and Mr. Frosty was selected. The manual and automated protocols were reproducible in terms of quantity,recovery,viability,WBC subpopulation distribution,gene expression,and LCL transformation. Most (75%-100%) of the 13 robustness parameters were accepted for both methods with an 8 h pre-centrifugation delay versus 38%-85% after 24 h. Differences identified between the automated and manual methods were not considered consequential. CONCLUSIONS We validated the first fully automated method for isolating viable PBMCs,including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection. View Publication

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes,where it is often referred to as an insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types View Publication

Pluripotent cells represent a powerful tool for tissue regeneration,but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study,we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated,bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting,we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate,thereby allowing for functional engraftment of pluripotent cells into regenerating tissue. View Publication