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

Recent compelling evidence indicates that Th17 confer host immunity against a variety of microbes,including extracellular and intracellular pathogens. Therefore,understanding mechanisms for the induction and activation of Ag-specific Th17 is important for the rational design of vaccines against pathogens. To study this,we employed an in vitro system in which influenza hemagglutinin (HA) 1 was delivered to dendritic cells (DCs) via Dectin-1 using anti-human Dectin-1 (hDectin-1)-HA1 recombinant fusion proteins. We found that healthy individuals maintained broad ranges of HA1-specific memory Th17 that were efficiently activated by DCs targeted with anti-hDectin-1-HA1. Nonetheless,these DCs were not able to induce a significant level of HA1-specific Th17 responses even in the presence of the Th17-promoting cytokines IL-1? and IL-6. We further found that the induction of surface IL-1R1 expression by signals via TCRs and common ?-chain receptors was essential for naive CD4(+) T cell differentiation into HA1-specific Th17. This process was dependent on MyD88,but not IL-1R-associated kinase 1/4. Thus,interruptions in STAT3 or MyD88 signaling led to substantially diminished HA1-specific Th17 induction. Taken together,the de novo generation of pathogen-specific human Th17 requires complex,but complementary,actions of multiple signals. Data from this study will help us design a new and effective vaccine strategy that can promote Th17-mediated immunity against microbial pathogens. View Publication

AbstractBackgroundParkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra,which promotes a sustained inflammatory environment in the central nervous system. Regulatory T cells (Tregs) play an important role in the control of inflammation and might play a neuroprotective role. Indeed,a decrease in Treg number and function has been reported in PD. In this context,pramipexole,a dopaminergic receptor agonist used to treat PD symptoms,has been shown to increase peripheral levels of Treg cells and improve their suppressive function. The aim of this work was to determine the effect of pramipexole on immunoregulatory Treg cells and its possible neuroprotective effect on human dopaminergic neurons differentiated from human embryonic stem cells.MethodsTreg cells were sorted from white blood cells of healthy human donors. Assays were performed with CD3/CD28?activated and non?activated Treg cells treated with pramipexole at concentrations of 2 or 200 ng/mL. These regulatory cells were co?cultured with in vitro?differentiated human dopaminergic neurons in a cytotoxicity assay with 6?hydroxydopamine (6?OHDA). The role of interleukin?10 (IL?10) was investigated by co?culturing activated IL?10?producing Treg cells with neurons. To further investigate the effect of treatment on Tregs,gene expression in pramipexole?treated,CD3/CD28?activated Treg cells was determined by Fluidigm analysis.ResultsPramipexole?treated CD3/CD28?activated Treg cells showed significant protective effects on dopaminergic neurons when challenged with 6?OHDA. Pramipexole?treated activated Treg cells showed neuroprotective capacity through mechanisms involving IL?10 release and the activation of genes associated with regulation and neuroprotection.ConclusionAnti?CD3/CD28?activated Treg cells protect dopaminergic neurons against 6?OHDA?induced damage. In addition,activated,IL?10?producing,pramipexole?treated Tregs also induced a neuroprotective effect,and the supernatants of these co?cultures promoted axonal growth. Pramipexole?treated,activated Tregs altered their gene expression in a concentration?dependent manner,and enhanced TGF??related dopamine receptor regulation and immune?related pathways. These findings open new perspectives for the development of immunomodulatory therapies for the treatment of PD. Pramipexole?treated,activated regulatory T cells protect dopaminergic neurons against 6 OHDA damage and promote primary neurite length. This could be due to the production of the regulatory cytokine IL?10 and an increased expression of genes related to regulation and neuroprotection. View Publication

BackgroundOrganoids,as near-physiological 3D culture systems,offer new opportunities to study the pathogenesis of various organs in mimicking the cellular complexity and functionality of human organs.MethodHere we used a quite simple and very practicable method to successfully generate induced pluripotent stem cell (iPSC)-derived human lung organoids (LuOrg) in a matrix-free manner as an alternative to the widely used preclinical mouse models in order to investigate normal lung damage in detail and as close as possible to the patient. We performed detailed morphological and molecular analyses,including bulk and single cell RNA sequencing,of generated lung organoids and evaluated the quality and robustness of our model as a potential in vitro platform for lung diseases,namely radiation-induced lung injury.ResultsA matrix-free method for differentiation of iPSCs can be used to obtain lung organoids that morphologically reflect the target tissue of the human lung very well,especially with regard to the cellular composition. The different cellular fates were investigated following the genotoxic stress induced by radiation and revealed further insights in the radiation-sensitivity of the different lung cells. Finally,we provide cellular gene sets found to be induced in the different lung organoid cellular subsets after irradiation,which could be used as additional RT response and particularly senescence gene sets in future studies.ConclusionBy establishing these free-floating LuOrgs for the investigation of cancer therapeutic approaches as a new and patient-oriented in vitro platform particularly in experimental radiooncology,not only a reduction in the number of experimental animals,but also an adequately and meaningfully replacement of corresponding animal experiments can be achieved. View Publication

Thymosin $\beta$4 (T$\beta$4) is a G-actin sequestering protein that contributes to diverse cellular activities,such as migration and angiogenesis. In this study,the beneficial effects of combined cell therapy with T$\beta$4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with T$\beta$4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly,T$\beta$4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with T$\beta$4 significantly increased cell migration and sprouting from microbeads. Moreover,additional treatment with T$\beta$4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the T$\beta$4-hASCs combination on vessel recruitment,dorsal window chambers were transplanted,and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with T$\beta$4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together,the therapeutic application of hASCs combined with T$\beta$4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia. View Publication

The lack of understanding of the molecular and cellular characteristics of human retinal progenitor cells (RPCs) has hindered their application in cell therapy for retinal degenerative diseases. This study aims to employ retinal organoids (ROs) derived from a VSX2-enhanced green fluorescent protein (eGFP) reporter human induced pluripotent stem cell (hiPSC) line for positive selection of human RPCs,investigate their features,and facilitate their applications. Methods: hiPSCs were differentiated into three-dimensional ROs following established protocols. The fidelity of the VSX2-eGFP reporter was confirmed through immunostaining. Fluorescence-activated cell sorting was employed to select VSX2-eGFP-positive (+) cells at distinct developmental stages,followed by bulk RNA sequencing (RNA-seq) analysis to assess their transcriptome profile. Immunostaining and flow cytometry were utilized to validate the identity of VSX2-eGFP+ cells and potential cluster of differentiation (CD) biomarkers for identifying human RPCs. Results: hiPSCs were successfully differentiated into ROs containing abundant RPCs. The spatiotemporal activity of the VSX2-eGFP reporter recapitulated the dynamic expression of endogenous VSX2 protein. Compared to VSX2-eGFP-negative (-) cells,VSX2-eGFP+ cells mainly exhibited characteristics of RPCs at early stages of retinal development and of bipolar cells at late stages. RNA-seq analysis revealed transcriptional heterogeneity within VSX2-eGFP+ cells across four distinct developmental stages. Moreover,the dynamic expression of 394 known CD biomarkers in VSX2-eGFP+ cells at distinct developmental stages was analyzed herein for the first time. One CD biomarker,TNFRSF1B,which has never been reported to be expressed in RPCs,was found to be highly expressed in RPCs at the early stages and might serve as a candidate CD biomarker for sorting RPCs. Conclusions: This study provides valuable insights into the molecular and cellular characteristics of human RPCs,especially their expression profiles of CD biomarkers,laying a foundation for research on retinal development and the clinical translation of hiPSC-derived RPCs. View Publication

The sodium-iodine symporter (NIS) is expressed on the cell membrane of many thyroid cancer cells,and is responsible for the radioactive iodine accumulation. However,treatment of anaplastic thyroid cancer is ineffective due to the low expression of NIS on cell membranes of these tumor cells. Human embryonic stem cells (ESCs) provide a potential vehicle to study the mechanisms of NIS expression regulation during differentiation. Human ESCs were maintained on feeder-independent culture conditions. RT-qPCR and immunocytochemistry were used to study differentiation marker expression,(125)I uptake to study NIS function. We designed a two-step protocol for human ESC differentiation into thyroid-like cells,as was previously done for mouse embryonic stem cells. First,we obtained definitive endoderm from human ESCs. Second,we directed differentiation of definitive endoderm cells into thyroid-like cells using various factors,with thyroid stimulating hormone (TSH) as the main differentiating factor. Expression of pluripotency,endoderm and thyroid markers and (125)I uptake were monitored throughout the differentiation steps. These approaches did not result in efficient induction of thyroid-like cells. We conclude that differentiation of human ESCs into thyroid cells cannot be induced by TSH media supplementation alone and most likely involves complicated developmental patterns that are yet to be understood. View Publication

To directly study the biological properties of purified hematopoietic colony-forming cell precursors,cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting,and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3),IL-6,erythropoietin,and mast cell growth factor (a c-kit ligand),cell numbers increased approximately 10(6)-fold over a period of 4 wk,and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly,the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured,extensive cell production was repeated,again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool. View Publication

Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX),the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene ( LRPPRC ). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions,tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study,we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC,homozygous for the LRPPRC *354V allele,and from a control,homozygous for the LRPPRC *A354 allele. Specifically,for both of these fibroblast lines we generated hiPSC,hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines,as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells,with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC,reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts,this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC. View Publication

Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system,along with their G-coupled cannabinoid receptors (CB�? and CB₂) and the enzymes involved in their biosynthesis and degradation. However,their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here,we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol),whereas CB₂ receptors are expressed in human and murine HSPCs. On ligand stimulation with CB₂ agonists,CB₂ receptors induced chemotaxis,migration,and enhanced colony formation of bone marrow cells,which were mediated via ERK,PI3-kinase,and Gαi-Rac1 pathways. In vivo,the CB₂ agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition,granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB₂ antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together,these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB₂/CB₂ agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus,CB₂ agonists may be therapeutically applied in clinical conditions,such as bone marrow transplantation. View Publication

Irradiation with X-rays has been widely utilized in the clinical treatment of solid tumors and certain hematopoietic malignancies. However,this method fails to completely distinguish between malignant and normal cells. Prolonged or repeated exposure to radiation,whether due to occupational hazards or therapeutical interventions,can cause damage to normal tissues,particularly impacting the hematopoietic system. Therefore,it is important to investigate the effects of total body irradiation on the hematopoietic system of mice and to compare the inhibitory effects of various doses of irradiation on this system. In this study,we primarily employed flow cytometry to analyze mature lineage cells in the peripheral blood,as well as immature hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and spleen. Additionally,we evaluated the multilineage differentiation capacity of HSPCs through colony-forming cell assays. Our results indicated that peripheral B and T cells demonstrated increased sensitivity to irradiation,with significant cell death observed 1-day post-irradiation. Common lymphoid progenitor cells exhibited greater radiotolerance compared to other progenitor cell types,enabling them to maintain a certain population even at elevated doses. Moreover,notable differences were observed between intramedullary and extramedullary hematopoietic stem cells and common lymphoid progenitor cells regarding the extent of damage and recovery rate following irradiation. The multilineage differentiation capacity of HSPCs was also compromised during radiation exposure. In conclusion,different types of mature blood cells,along with immature HSPCs,exhibited varying degrees of sensitivity and tolerance to irradiation,resulting in distinct alterations in cell percentages and numbers. View Publication

Expansion of Human Hematopoietic Stem Cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche,but how to apply this to HSC maintenance and expansion is yet to be explored. We demonstrate a role for the GFL receptor,RET,at the cell surface of HSCs,in mediating sustained cellular growth,resistance to stress and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/co-receptor complex,GDNF/GFRa1,show improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally,we demonstrate that RET drives a multi-faceted intracellular signalling pathway,including key signalling intermediates AKT,ERK1/2,NFkB and p53,responsible for a wide range of cellular and genetic responses which improve cell growth and survival under culture conditions. View Publication

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However,responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here,we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction,cell-cycle progression,and transcriptional responses in HSPC subpopulations,with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation,constraining proliferation,yield,and engraftment of edited HSPCs. However,functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs. View Publication