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

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells,functions as an autocrine growth factor for Th17 cells,and plays critical roles in autoimmune diseases. In this study,we investigated the differentiation and characteristics of IL-21-producing CD4(+) T cells by intracellular staining. Unexpectedly,we found that under Th17-polarizing conditions,the majority of IL-21-producing CD4(+) T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21-producing CD4(+) T cells without the induction of IL-4,IFN-gamma,IL-17A,or IL-17F production. On the other hand,TGF-beta inhibited IL-6- and IL-21-induced development of IL-21-producing CD4(+) T cells. IL-2 enhanced the development of IL-21-producing CD4(+) T cells under Th17-polarizing conditions. Finally,IL-21-producing CD4(+) T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6,but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21-producing CD4(+) T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6-rich environment devoid of TGF-beta,and that IL-21 functions as an autocrine growth factor for IL-21-producing CD4(+) T cells. View Publication

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state,instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date,only mouse iPSC lines are known to be truly pluripotent. However,initial mouse iPSC lines failed to form chimeric offspring,but did generate teratomas and differentiated embryoid bodies,and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore,there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1,SOX2,NANOG,KLF4,LIN28,and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high,85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies,genetic engineering,and other aspects of stem cell and developmental biology. View Publication

CONTEXT: The osteoanabolic properties of PTH may be due to increases in the number and maturity of circulating osteogenic cells. Hypoparathyroidism is a useful clinical model because this hypothesis can be tested by administering PTH. OBJECTIVE: The objective of the study was to characterize circulating osteogenic cells in hypoparathyroid subjects during 12 months of PTH (1-84) administration. DESIGN: Osteogenic cells were characterized using flow cytometry and antibodies against osteocalcin,an osteoblast-specific protein product,and stem cell markers CD34 and CD146. Changes in bone formation from biochemical markers and quadruple-labeled transiliac crest bone biopsies (0 and 3 month time points) were correlated with measurements of circulating osteogenic cells. SETTING: The study was conducted at a clinical research center. PATIENTS: Nineteen control and 19 hypoparathyroid patients were included in the study. INTERVENTION: Intervention included the administration of PTH (1-84). RESULTS: Osteocalcin-positive cells were lower in hypoparathyroid subjects than controls (0.7 ± 0.1 vs. 2.0 ± 0.1%; P textless 0.0001),with greater coexpression of the early cell markers CD34 and CD146 among the osteocalcin-positive cells in the hypoparathyroid subjects (11.0 ± 1.0 vs. 5.6 ± 0.7%; P textless 0.001). With PTH (1-84) administration,the number of osteogenic cells increased 3-fold (P textless 0.0001),whereas the coexpression of the early cell markers CD34 and CD146 decreased. Increases in osteogenic cells correlated with circulating and histomorphometric indices of osteoblast function: N-terminal propeptide of type I procollagen (R(2) = 0.4,P ≤ 0.001),bone-specific alkaline phosphatase (R(2) = 0.3,P textless 0.001),osteocalcin (R(2) = 0.4,P textless 0.001),mineralized perimeter (R(2) = 0.5,P textless 0.001),mineral apposition rate (R(2) = 0.4,P = 0.003),and bone formation rate (R(2) = 0.5,P textless 0.001). CONCLUSIONS: It is likely that PTH stimulates bone formation by stimulating osteoblast development and maturation. Correlations between circulating osteogenic cells and histomorphometric indices of bone formation establish that osteoblast activity is being identified by this methodology. View Publication

BACKGROUND Emerging evidence of antibody-independent functions,as well as the clinical efficacy of anti-CD20 depleting therapies,helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. OBJECTIVE To investigate whether CD19+ B cells may share expression of the serine-protease granzyme-B (GzmB),resembling classical cytotoxic CD8+ T lymphocytes,in the peripheral blood from relapsing-remitting MS (RRMS) patients. METHODS In this study,104 RRMS patients during different treatments and 58 healthy donors were included. CD8,CD19,Runx3,and GzmB expression was assessed by flow cytometry analyses. RESULTS RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8+GzmB+ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19+GzmB+ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA),untreated RRMS patients,and healthy donors but not when compared to interferon-$\beta$ (IFN). Moreover,regarding Runx3,the transcriptional factor classically associated with cytotoxicity in CD8+ T lymphocytes,the expression of GzmB was significantly higher in CD19+Runx3+-expressing B cells when compared to CD19+Runx3- counterparts in RRMS patients. CONCLUSIONS CD19+ B cells may exhibit cytotoxic behavior resembling CD8+ T lymphocytes in MS patients during different treatments. In the future,monitoring cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions." View Publication

Preclinical in vitro models provide an essential tool to study cancer cell biology as well as aid in translational research,including drug target identification and drug discovery efforts. For any model to be clinically relevant,it needs to recapitulate the biology and cell heterogeneity of the primary tumor. We recently developed and described a conditional reprogramming (CR) cell technology that addresses many of these needs and avoids the deficiencies of most current cancer cell lines,which are usually clonal in origin. Here,we used the CR cell method to generate a collection of patient-derived cell cultures from non-small cell lung cancers (NSCLC). Whole exome sequencing and copy number variations are used for the first time to address the capability of CR cells to keep their tumor-derived heterogeneity. Our results indicated that these primary cultures largely maintained the molecular characteristics of the original tumors. Using a mutant-allele tumor heterogeneity (MATH) score,we showed that CR cells are able to keep and maintain most of the intra-tumoral heterogeneity,suggesting oligoclonality of these cultures. CR cultures therefore represent a pre-clinical lung cancer model for future basic and translational studies. View Publication

The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment,particularly during early life. To investigate this concept,we developed a model of neonatal exposure to a common pathogen-associated molecular pattern,LPS,and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE,induced at 12 wk,compared with vehicle-exposed animals. Spinal cord transcript levels of CD3epsilon and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c(+) cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4(+)FoxP3(+) T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE,and this neuroprotective effect was associated with an increased number of CD3(+)FoxP3(+) immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes,albeit reduced in MS compared with non-MS patients' brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS. View Publication

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs,monocytes,and endothelial cells,which express CD105,was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes,and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs,monocytes,and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added,which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME,but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells. View Publication

Small intestinal CD103(+) dendritic cells (DCs) have the selective ability to promote de novo generation of regulatory T cells via the production of retinoic acid (RA). Considering that aldehyde dehydrogenase (ALDH) activity controls the production of RA,we used a flow cytometry-based assay to measure ALDH activity at the single-cell level and to perform a comprehensive analysis of the RA-producing DC populations present in lymphoid and nonlymphoid mouse tissues. RA-producing DCs were primarily of the tissue-derived,migratory DC subtype and can be readily found in the skin and in the lungs as well as in their corresponding draining lymph nodes. The RA-producing skin-derived DCs were capable of triggering the generation of regulatory T cells,a finding demonstrating that the presence of RA-producing,tolerogenic DCs is not restricted to the intestinal tract as previously thought. Unexpectedly,the production of RA by skin DCs was restricted to CD103(-) DCs,indicating that CD103 expression does not constitute a universal" marker for RA-producing mouse DCs. Finally� View Publication

BACKGROUND Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor. Although numerous postoperative therapeutic strategies have already been developed,including radiotherapy,tumors inevitably recur after several years of treatment. The coinhibitory molecule B7-H4 negatively regulates T cell immune responses and promotes immune escape. Exosomes mediate intercellular communication and initiate immune evasion in the tumor microenvironment (TME). OBJECTIVE This study aimed to determine whether B7-H4 is upregulated by radiation and loaded into exosomes,thus contributing to immunosuppression and enhancing tumor growth. METHODS Iodixanol density-gradient centrifugation and flow cytometry were used to verify exosomal B7-H4. Na{\{i}}ve T cells were differentiated into Th1 cells with or without exosomes. T cell-secreted cytokines and markers of T cell subsets were measured. Mechanistically the roles of B7-H4 and ALIX in GBM were analyzed using databases and tissue samples. Co-immunoprecipitation and pull-down assays were used to tested the direct interactions between ATM and ALIX or STAT3. In vitro ATM kinase assays western blotting and site-directed mutation were used to assess ATM-mediated STAT3 phosphorylation. Finally the contribution of exosomal B7-H4 to immunosuppression and tumor growth was investigated in vivo. RESULTS Exosomes from irradiated GBM cells decreased the anti-tumor immune response of T cell in vitro and in vivo via delivered B7-H4. Mechanistically irradiation promoted exosome biogenesis by increasing the ATM-ALIX interaction. Furthermore the ATM-phosphorylated STAT3 was found to directly binds to the B7-H4 promoter to increase its expression. Finally the radiation-induced increase in exosomal B7-H4 induced FoxP3 expression during Th1 cell differentiation via the activated STAT1 pathway. In vivo exosomal B7-H4 decreased the radiation sensitivity of GBM cells and reduced the survival of GBM mice model. CONCLUSION This study showed that radiation-enhanced exosomal B7-H4 promoted immunosuppression and tumor growth hence defining a direct link between irradiation and anti-tumor immune responses. Our results suggest that co-administration of radiotherapy with anti-B7-H4 therapy could improve local tumor control and identify exosomal B7-H4 as a potential tumor biomarker." View Publication

One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However,the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs,we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here,we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably,RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth,both in vitro and in vivo. Thus,our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment,suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment. View Publication

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently,conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However,the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here,we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay,monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly,pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast,brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion,patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions,limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus,CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells,as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells. View Publication

The identification and targeted therapy of cells involved in hepatocellular carcinoma (HCC) recurrence remain challenging. Here,we generated a monoclonal antibody against recurrent HCC,1B50-1,that bound the isoform 5 of the α2δ1 subunit of voltage-gated calcium channels and identified a subset of tumor-initiating cells (TICs) with stem cell-like properties. A surgical margin with cells detected by 1B50-1 predicted rapid recurrence. Furthermore,1B50-1 had a therapeutic effect on HCC engraftments by eliminating TICs. Finally,α2δ1 knockdown reduced self-renewal and tumor formation capacities and induced apoptosis of TICs,whereas its overexpression led to enhanced sphere formation,which is regulated by calcium influx. Thus,α2δ1 is a functional liver TIC marker,and its inhibitors may serve as potential anti-HCC drugs. View Publication