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

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however,an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4 + MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity,but MP cells retain less compromised mitochondria compared to effector CD4 + T cells,and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells. Subject areas: immunity,cell biology View Publication

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this,the molecular mechanisms that allow H . influenzae to establish persistent infections of human epithelia are not well understood. Here,we have investigated how H . influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H . influenzae infection with an initial,‘unproductive’ inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently,an apparent tolerance to large extracellular and intraepithelial burdens of H . influenzae developed,with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations,and appears to involve interruption of NFκB signalling. This is the first time that large-scale,persistence-promoting immunomodulatory effects of H . influenzae during infection have been observed,and we were able to demonstrate that only infections with live,but not heat-killed H . influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β,IL-36γ and TNFα. Interestingly,NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection,resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H . influenzae in the host,our data further indicate the presence of infection stage-specific gene expression modules,highlighting fundamental similarities between immune responses in NHNE and canonical immune cells,which merit further investigation. View Publication

Mounting evidence indicates that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exosomes) combine the advantages of hucMSC pluripotency with their nanoscale dimensions,enhancing their clinical potential through prolonged circulation half-life. Despite these promising characteristics,research on their immunological toxicity remains insufficient. This study focuses on the impact of hucMSC-exosomes on the general toxicity and immunopathological indicators. When mice received tail vein injections of 6 × 10 10 hucMSC-exosomes particles,we observed no significant changes in body weight,feed intake,blood composition,organ indices,or histopathological findings throughout the 14 days observation period. Similarly,blood levels of immunoglobulins,cytokines,and lymphocyte subpopulations remained stable. The hucMSC-exosomes produced no detectable negative effects on immune organs including the thymus,spleen,and bone marrow. These findings indicate that intravenous administration of 6 × 10 10 particles of hucMSC-exosomes appears relatively safe at the murine level. This assessment of safety and immunological impact following intravenous hucMSC-exosomes infusion offers experimental support for potential clinical applications and future analyses in this field. View Publication

BACKGROUND Recent studies have suggested that both poly(l-lactide-co-1,5-dioxepan-2-one) (or poly(LLA-co-DXO)) and poly(l-lactide-co-$\epsilon$-caprolactone) (or poly(LLA-co-CL)) porous scaffolds are good candidates for use as biodegradable scaffold materials in the field of tissue engineering; meanwhile,their surface properties,such as hydrophilicity,need to be further improved. METHODS We applied several different concentrations of the surfactant Tween 80 to tune the hydrophilicity of both materials. Moreover,the modification was applied not only in the form of solid scaffold as a film but also a porous scaffold. To investigate the potential application for tissue engineering,human bone marrow mesenchymal stem cells (hMSCs) were chosen to test the effect of hydrophilicity on cell attachment,proliferation,and differentiation. First,the cellular cytotoxicity of the extracted medium from modified scaffolds was investigated on HaCaT cells. Then,hMSCs were seeded on the scaffolds or films to evaluate cell attachment,proliferation,and osteogenic differentiation. The results indicated a significant increasing of wettability with the addition of Tween 80,and the hMSCs showed delayed attachment and spreading. PCR results indicated that the differentiation of hMSCs was stimulated,and several osteogenesis related genes were up-regulated in the 3{\%} Tween 80 group. Poly(LLA-co-CL) with 3{\%} Tween 80 showed an increased messenger Ribonucleic acid (mRNA) level of late-stage markers such as osteocalcin (OC) and key transcription factor as runt related gene 2 (Runx2). CONCLUSION A high hydrophilic scaffold may speed up the osteogenic differentiation for bone tissue engineering. View Publication

We used synthetic lethal high-throughput screening to interrogate 23,550 compounds for their ability to kill engineered tumorigenic cells but not their isogenic normal cell counterparts. We identified known and novel compounds with genotype-selective activity,including doxorubicin,daunorubicin,mitoxantrone,camptothecin,sangivamycin,echinomycin,bouvardin,NSC146109,and a novel compound that we named erastin. These compounds have increased activity in the presence of hTERT,the SV40 large and small T oncoproteins,the human papillomavirus type 16 (HPV) E6 and E7 oncoproteins,and oncogenic HRAS. We found that overexpressing hTERT and either E7 or LT increased expression of topoisomerase 2alpha and that overexpressing RAS(V12) and ST both increased expression of topoisomerase 1 and sensitized cells to a nonapoptotic cell death process initiated by erastin. View Publication

The challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited their clinical application. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by transducing OCT4 (hHFMSCs OCT4 ) and pre-inducing with FLT3LG/SCF,and differentiated into erythrocytes. These intermediate cells exhibit gene expression patterns similar to pre-HSCs,making them promising for artificial haematopoiesis. However,further investigation is required to elucidate the in vitro proliferation ability and mechanism underlying the self-renewal of pre-HSCs derived from hHFMSCs. hHFMSCs OCT4 were pre-treated with FLT3LG and SCF cytokines,followed by characterization and isolation of the floating cell subsets for erythroid differentiation through stimulation with hematopoietic cytokines and nutritional factors. Cell adhesion was assessed through disassociation and adhesion assays. OCT4 expression levels were measured using immunofluorescence staining,RT-qPCR,and Western blotting. RNA sequencing and Gene Ontology (GO) enrichment analysis were then conducted to identify proliferation-related biological processes. Proliferative capacity was evaluated through CCK-8,colony formation assays,Ki67 index,and cell cycle analysis. Cytoskeleton was observed through Wright‒Giemsa,Coomassie brilliant blue,and phalloidin staining. Expression of adherens junction (AJ) core members was confirmed through RT‒qPCR,Western blotting,and immunofluorescence staining before and after ZO-1 knockdown. A regulatory network was constructed to determine relationships among cytoskeleton,proliferation,and the AJ pathway. Student’s t tests (GraphPad Prism 8.0.2) were used for group comparisons. The results were considered significant at P < 0.05. Pre-treatment of hHFMSCs OCT4 with FLT3LG and SCF leads to the emergence of floating cell subsets exhibiting small,globoid morphology,suspended above adherent cells,forming colonies,and displaying minimal expression of CD45. Excessive OCT4 expression weakens adhesion in floating hHFMSCs OCT4 . Floating cells moderately enhanced proliferation and undergo cytoskeleton remodelling,with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 could impact the expressions of F-actin,E-cadherin,and β-catenin genes,as well as the nuclear positioning of β-catenin,leading to variations in the cytoskeleton and cell cycle. Finally,a regulatory network revealed that the AJ pathway cored with ZO-1 critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner. ZO-1 improved the self-renewal of pre-HSCs from OCT4-overexpressing hHFMSCs by remodeling the cytoskeleton via the ZO-1-regulated AJ pathway,suggesting floating hHFMSCs OCT4 as the promising seed cells for artificial hematopoiesis. The online version contains supplementary material available at 10.1186/s13287-024-04080-w. View Publication

Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4,CD25,and the transcription factor,FoxP3. However,these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4(+) T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3(+),including those that express low levels or no CD25. A combination of CD4,CD25,and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact,cells separated based solely on CD4 and CD127 expression were anergic and,although representing at least three times the number of cells (including both CD25(+)CD4(+) and CD25(-)CD4(+) T cell subsets),were as suppressive as the classic" CD4(+)CD25(hi) T reg cell subset. Finally� View Publication

In the present study,we investigated the in vitro effect of resveratrol (RSVL),a polyphenolic phytoestrogen,on cell proliferation and osteoblastic maturation in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures. RSVL (10(-8)-10(-5) M) increased cell growth dose-dependently,as measured by [(3)H]-thymidine incorporation,and stimulated osteoblastic maturation as assessed by alkaline phosphatase (ALP) activity,calcium deposition into the extracellular matrix,and the expression of osteoblastic markers such as RUNX2/CBFA1,Osterix and Osteocalcin in HBMSCs cell cultures. Further studies found that RSVL (10(-6)M) resulted in a rapid activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling in HBMSCs cultures. The effects of RSVL were mimicked by 17beta-estrodial (10(-8) M) and were abolished by estrogen receptor (ER) antagonist ICI182780. An ERK1/2 pathway inhibitor,PD98059,significantly attenuated RSVL-induced ERK1/2 phosphorylation,consistent with the reduction of cell proliferation and osteoblastic differentiation as well as expression of osteoblastic markers. In contrast,SB203580,a p38 MAPK pathway blocker,blocked RSVL-induced p38 phosphorylation,but resulted in an increase of cell proliferation and a more osteoblastic maturation. These data suggest that RSVL stimulates HBMSCs proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation. View Publication

Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection,we used human neural progenitor cells (NPC),derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation,ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2 + cells was demonstrated by activation of caspases 3/7,8 and 9,and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2 + cells was prevented by incubation with the pan-caspase inhibitor,Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities,including multipolar spindle,chromosome laggards,micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy,such as monosomy,trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain,including microcephaly. View Publication

Endothelial cells (ECs) line the luminal surface of blood vessels and have an active role in the recruitment of leukocytes,including immune cell activation. Regulatory T cells (Tregs) are immune suppressor cells that maintain peripheral tolerance and must interact with the endothelium as they traffic into tissue. We hypothesized that human ECs could modulate Tregs and their suppressor function. Cocultures of CD4+ T cells with human umbilical vein ECs (HUVECs) or dermal microvascular ECs (HDMECs) were conducted and analyzed for activation and proliferation after 72 and 120 h using flow cytometry. In monocyte-depleted cultures,human ECs were found to support CD4+ T cell proliferation in the presence of external mitogens phytohemagglutinin or anti-CD3/28 antibodies (aCD3/28). Activation was shown by CD25 expression in these cells that also transiently expressed the Treg transcription factor FOXP3. HUVECs supported the specific concurrent proliferation of both effector T cells and Tregs when cocultured with aCD3/28. Purified Tregs were also functionally activated by prior coculture with EC to suppress effector T (Teff) cell proliferation. Both direct coculture and indirect coculture of EC and Treg showed activation of the Treg suppressive phenotype. However,whereas HUVEC showed enhancement of suppression by both mechanisms,HDMEC only supported Treg suppressive activity via the contact-independent mechanism. In the contact-independent cultures,the soluble mediators IL-6,GM-CSF,or G-CSF released from ECs following interferon-gamma$ activation were not responsible for the enhanced Treg suppressor function. Following direct coculture,Treg expression of inhibitory receptors PD-1 and OX40 was elevated while activated EC expressed the counter ligands programmed death ligand (PD-L)1 and PD-L2. Therefore,human ECs have a role in supporting T cell proliferation and increasing Treg suppressor function. This ability of EC to enhance Treg function could offer novel targets to boost Treg activity during inflammatory disorders. View Publication

Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore,we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics,BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm),yet,24 hr later,suppressed endoderm and induced mesoderm. At lineage bifurcations,cross-repressive signals separated mutually exclusive fates; TGF-?? and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations,thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of pre-enhancer" states before activation� View Publication

Incurable neurological disorders such as Parkinson's disease (PD),Huntington's disease (HD),and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases,we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor,valproic acid (VPA),and inhibitor of p160-Rho associated coiled-coil kinase (ROCK),Y-27632,after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology,cell surface antigens,pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542,inhibitors of the SMAD signaling pathway,HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction,neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture,which had the ability to differentiate further into NPCs and neurons,as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays. View Publication