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

The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However,clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis,a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC,maintained in an optimised cytokine cocktail,to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4,Sox2,Klf4,L-Myc,and Lin28) in a feeder-free system,without any requirement for small molecules,we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells,we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function. View Publication

BACKGROUND: Many retinal degenerative diseases are caused by the loss of retinal ganglion cells (RGCs). Autosomal dominant optic atrophy is the most common hereditary optic atrophy disease and is characterized by central vision loss and degeneration of RGCs. Currently,there is no effective treatment for this group of diseases. However,stem cell therapy holds great potential for replacing lost RGCs of patients. Compared with embryonic stem cells,induced pluripotent stem cells (iPSCs) can be derived from adult somatic cells,and they are associated with fewer ethical concerns and are less prone to immune rejection. In addition,patient-derived iPSCs may provide us with a cellular model for studying the pathogenesis and potential therapeutic agents for optic atrophy.backslashnbackslashnMETHODS: In this study,iPSCs were obtained from patients carrying an OPA1 mutation (OPA1 (+/-) -iPSC) that were diagnosed with optic atrophy. These iPSCs were differentiated into putative RGCs,which were subsequently characterized by using RGC-specific expression markers BRN3a and ISLET-1.backslashnbackslashnRESULTS: Mutant OPA1 (+/-) -iPSCs exhibited significantly more apoptosis and were unable to efficiently differentiate into RGCs. However,with the addition of neural induction medium,Noggin,or estrogen,OPA1 (+/-) -iPSC differentiation into RGCs was promoted.backslashnbackslashnCONCLUSIONS: Our results suggest that apoptosis mediated by OPA1 mutations plays an important role in the pathogenesis of optic atrophy,and both noggin and β-estrogen may represent potential therapeutic agents for OPA1-related optic atrophy. View Publication

Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress,which results in enhanced production of inflammatory cytokines,including interleukin-6 (IL-6),and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis,resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs),Morrbid,in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo,pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells,including clonal hematopoiesis. View Publication

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules,including PD-1,are preferentially expressed at the surface of persistently infected cells. However,whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals,we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely,PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption. View Publication

We report a considerable number of cells in the ventricular and the subventricular zones (SVZ) of newborn mice to stain positive for the PDGF beta-receptor (PDGFRB). Many of them also stained for nestin and/or GFAP but less frequently for the neuroblast marker doublecortin and for the mitotic marker Ki-67. The SVZ of mice with nestin-Cre conditional deletion of PDGFRB expressed the receptor only on blood vessels and was devoid of any morphological abnormality. PDGFRB(-/-) neurospheres showed a higher rate of apoptosis without any significant decrease in proliferation. They demonstrated reduced capacities of migration and neuronal differentiation in response to not only PDGF-BB but also bFGF. Furthermore,the PDGFR kinase inhibitor STI571 blocked the effects of bFGF in control neurosphere cultures. bFGF increased the activity of the PDGFRB promoter as well as the expression and phosphorylation of PDGFRB. These results suggest the presence of the signaling convergence between PDGF and FGF. PDGFRB is needed for survival,and the effects of bFGF in migration and neural differentiation of the cells may be potentiated by induction of PDGFRB. View Publication

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling,drug discovery,and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state,it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons,the cell type destroyed in ALS. View Publication

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor-mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice,reduced numbers of PMF CD34+ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice,whereas similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4,but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA),but not treatment with small molecule inhibitors of JAK2,resulted in the generation of increased numbers of CD34+CXCR4+ cells,which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment,JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents. View Publication

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of ER,PR,and HER2 expression. Its aggressive behavior,high degree of tumor heterogeneity,and immunosuppressive tumor microenvironment (TME) are associated with poor clinical outcomes,rapid disease progression,and limited therapeutic options. Although chimeric antigen receptor (CAR)-engineered T cell therapy has shown certain promise,its applicability in TNBC is hindered by antigen escape,TME-mediated suppression,and the logistical constraints of autologous cell production. In this study,we employed hematopoietic stem and progenitor cell (HSPC) gene engineering and a feeder-free HSPC differentiation culture to generate allogeneic IL-15-enhanced,mesothelin-specific CAR-engineered invariant natural killer T ( Allo15 MCAR-NKT) cells. These cells demonstrated robust and multifaceted antitumor activity against TNBC,mediated by CAR- and NK receptor-dependent cytotoxicity,as well as selective targeting of CD1d + TME immunosuppressive cells through their TCR. In both orthotopic and metastatic TNBC xenograft models,Allo15 MCAR-NKT cells demonstrated potent antitumor activity,associated with robust effector and cytotoxic phenotypes,low exhaustion,and a favorable safety profile without inducing graft-versus-host disease. Together,these results support Allo15 MCAR-NKT cells as a next-generation,off-the-shelf immunotherapy with strong therapeutic potential for TNBC,particularly in the context of metastasis,immune evasion,and treatment resistance. The online version contains supplementary material available at 10.1186/s13045-025-01736-9. View Publication

BACKGROUND Longitudinal studies have verified the pivotal role of mesenchymal stem/stromal cells (MSCs) in the bone marrow microenvironment for hematopoiesis and coordinate contribution to leukemia pathogenesis. However,the precise characteristics and alternation of MSCs during acquired aplastic anemia (AA) remain obscure. METHODS In this study,we originally collected samples from both healthy donors (HD) and AA patients to dissect the hematological changes. To systematically evaluate the biological defects of AA-derived MSCs (AA-MSCs),we analyzed alterations in cellular morphology,immunophenotype,multi-lineage differentiation,cell migration,cellular apoptosis,and chromosome karyocyte,together with the immunosuppressive effect on the activation and differentiation of lymphocytes. With the aid of whole genome sequencing and bioinformatic analysis,we try to compare the differences between AA-MSCs and HD-derived MSCs (HD-MSCs) upon the molecular genetics,especially the immune-associated gene expression pattern. In addition,the efficacy of umbilical cord-derived MSC (UC-MSC) transplantation on AA mice was evaluated by utilizing survivorship curve,histologic sections,and blood cell analyses. RESULTS In coincidence with the current reports,AA patients showed abnormal subsets of lymphocytes and higher contents of proinflammatory cytokines. Although with similar immunophenotype and chromosome karyotype to HD-MSCs,AA-MSCs showed distinguishable morphology and multiple distinct characteristics including genetic properties. In addition,the immunosuppressive effect on lymphocytes was significantly impaired in AA-MSCs. What is more,the cardinal symptoms of AA mice were largely rescued by systemic transplantation of UC-MSCs. CONCLUSIONS Herein,we systematically investigated the signatures and efficacy of MSCs to dissect the alterations occurred in AA both at the cellular and molecular levels. Different from HD-MSCs,AA-MSCs exhibited multifaceted defects in biological characteristics and alterative molecular genetics in the whole genome. Our findings have provided systematic and overwhelming new evidence for the defects of AA-MSCs,together with effectiveness assessments of UC-MSCs on AA as well. View Publication

Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs),the major antigen-presenting cells of the immune system,sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here,we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac53FaxNeu5Ac. Ac53FaxNeu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8+ T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation,cell-adhesion,and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8+ T cells. This increased avidity was detectable in the absence of antigens,but was especially pronounced in antigen-dependent interactions. Together,our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8+ T cell responses. View Publication

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however,a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study,we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically,GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (TH17 cell) and TH22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells,GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity,resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis. View Publication

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential,it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin,expressed on the surfaces of multiple cell types,possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore,we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-,CD45-,and CD11b/c-negative and CD90-,CD29-,CD44-,CD54-,CD73-,and CD105-positive. Osteoblast,adipocyte,and chondrocyte differentiation was observed in these cells. In total,yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly,MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106,whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover,senescence-associated $\beta$-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs. View Publication