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

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here,we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model,HMBA did not reactivate HIV-1,yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However,HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells,hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside,HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall,our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells,providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug. View Publication

Recently,direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors. This approach may provide alternative cell resources for drug discovery and regenerative medicine,but applications could be limited by the genetic manipulation involved. Here,we show that mouse fibroblasts can be directly converted into neuronal cells using only a cocktail of small molecules,with a yield of up to textgreater90% being TUJ1-positive after 16 days of induction. After a further maturation stage,these chemically induced neurons (CiNs) possessed neuron-specific expression patterns,generated action potentials,and formed functional synapses. Mechanistically,we found that a BET family bromodomain inhibitor,I-BET151,disrupted the fibroblast-specific program,while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes. Overall,our findings provide a proof of principle" for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation� View Publication

Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However,CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here,we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation,proliferation,and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen,we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99 days,whereas median survival with polyclonal Treg infusion was 35 days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors,A2-CAR Tregs slightly increased median graft survival from 11 to 14 days,which was further extended to >100 days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs,alone or in combination with immunosuppressive agents,toward protecting vascularized grafts in fully immunocompetent recipients. View Publication

Multiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here,we use antibodies tagged with a hashtag oligonucleotide (Ab-HTO) to label each sample,and 10?— Genomics technology to analyze single-cell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein,we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development. For complete details on the use and execution of this protocol,please refer to Nozais et al. (2021). View Publication

In recent years,immunotherapy has shown considerable promise in the management of several malignancies. However,the majority of preclinical studies have been conducted in rodents,the results of which often translate poorly to patients given the substantial differences between murine and human immunology. As the porcine immune system is far more analogous to that of humans,pigs may serve as a supplementary preclinical model for future testing of such therapies. We have generated the genetically modified Oncopig with inducible tumor formation resulting from concomitant KRAS(G12D) and TP53(R167H) mutations under control of an adenoviral vector Cre-recombinase (AdCre). The objective of this study was to characterize the tumor microenvironment in this novel animal model with respect to T-cell responses in particular and to elucidate the potential use of Oncopigs for future preclinical testing of cancer immunotherapies. In this study,we observed pronounced intratumoral T-cell infiltration with a strong CD8$\beta$(+) predominance alongside a representation of highly differentiated $\gamma$$\delta$ T cells. The infiltrating CD8$\beta$(+) T cells displayed increased expression of the cytotoxic marker perforin when compared with the peripheral T-cell pool. Similarly,there was robust granzyme B staining localizing to the tumors; affirming the presence of cytotoxic immune cells within the tumor. In parallel with this antitumor immune response,the tumors displayed enrichment in FOXP3-expressing T cells and increased gene expression of indoleamine 2,3-dioxygenase 1 (IDO1),cytotoxic T-lymphocyte-associated protein 4 (CTLA4),and programmed death-ligand 1 (PDL1). Finally,we investigated the Oncopig immune system in mediating antitumor immunity. We observed pronounced killing of autologous tumor cells,which demonstrates the propensity of the Oncopig immune system to recognize and mount a cytotoxic response against tumor cells. Together,these findings suggest innate and adaptive recognition of the induced tumors with a concomitant in vivo suppression of T-cell effector functions. Combined,the data support that the Oncopig may serve as a valuable model for future preclinical testing of immunotherapies aimed at reactivating tumor-directed cytotoxicity in vivo. View Publication

Adhesion properties of hematopoietic stem cells (HSCs) in the bone marrow (BM) niches control their migration and affect their cell-cycle dynamics. The serum response factor (Srf) regulates growth factor-inducible genes and genes controlling cytoskeleton structures involved in cell spreading,adhesion,and migration. We identified a role for Srf in HSC adhesion and steady-state hematopoiesis. Conditional deletion of Srf in BM cells resulted in a 3-fold expansion of the long- and short-term HSCs and multipotent progenitors (MPPs),which occurs without long-term modification of cell-cycle dynamics. Early differentiation steps to myeloid and lymphoid lineages were normal,but Srf loss results in alterations in mature-cell production and severe thrombocytopenia. Srf-null BM cells also displayed compromised engraftment properties in transplantation assays. Gene expression analysis identified Srf target genes expressed in HSCs,including a network of genes associated with cell migration and adhesion. Srf-null stem cells and MPPs displayed impair expression of the integrin network and decreased adherence in vitro. In addition,Srf-null mice showed increase numbers of circulating stem and progenitor cells,which likely reflect their reduced retention in the BM. Altogether,our results demonstrate that Srf is an essential regulator of stem cells and MPP adhesion,and suggest that Srf acts mainly through cell-matrix interactions and integrin signaling. View Publication

Lipid droplets,also called lipid bodies (LB) in inflammatory cells,are important cytoplasmic organelles. However,little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here,we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system,the maturing MCs,derived from 18 different donors,invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore,the MCs transcribe the genes for perilipins (PLIN)1-4,but not PLIN5,and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation,the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion,and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary,we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions,with particular emphasis on AA metabolism,eicosanoid biosynthesis,and subsequent release of proinflammatory lipid mediators from these cells. View Publication

Metformin has been widely used as an oral drug for diabetes mellitus for approximately 60 years. Interestingly,recent reports showed that metformin exhibited an anti-tumor action in a wide range of malignancies including hepatocellular carcinoma (HCC). In the present study,we investigated its impact on tumor-initiating HCC cells. Metformin suppressed cell growth and induced apoptosis in a dose-dependent manner. Flow cytometric analysis showed that metformin treatment markedly reduced the number of tumor-initiating epithelial cell adhesion molecule (EpCAM)(+) HCC cells. Non-adherent sphere formation assays of EpCAM(+) cells showed that metformin impaired not only their sphere-forming ability,but also their self-renewal capability. Consistent with this,immunostaining of spheres revealed that metformin significantly decreased the number of component cells positive for hepatic stem cell markers such as EpCAM and α-fetoprotein. In a xenograft transplantation model using non-obese diabetic/severe combined immunodeficient mice,metformin and/or sorafenib treatment suppressed the growth of tumors derived from transplanted HCC cells. Notably,the administration of metformin but not sorafenib decreased the number of EpCAM(+) cells and impaired their self-renewal capability. As reported,metformin activated AMP-activated protein kinase (AMPK) through phosphorylation; however its inhibitory effect on the mammalian target of rapamycin (mTOR) pathway did not necessarily correlate with its anti-tumor activity toward EpCAM(+) tumor-initiating HCC cells. These results indicate that metformin is a promising therapeutic agent for the elimination of tumor-initiating HCC cells and suggest as-yet-unknown functions other than its inhibitory effect on the AMPK/mTOR pathway. 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

In the present study,the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal,(iii) mesodermal and (iv) endodermal differentiation media. (i) Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media,keratinocyte-like cells (filaggrin(+)/involucrin(+)) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®. (ii) When hSKP were subsequently exposed to NeuroCult® media,cells underwent a weak neuro-ectodermal differentiation expressing nestin,myelin binding protein (MBP),vimentin and alpha-foetoprotein (AFP). Sequential exposure to NPMM® and NPDM® generated cells with an inferior neuro-ectodermal phenotype (nestin(+)/vimentin(+)/MBP(-)/AFP(-)). (iii) Upon exposure of hSKP to insulin-transferrin-selenite (ITS) and dexamethasone,small lipid droplets were observed,suggesting their differentiation potential towards adipocyte-like cells. (iv) Finally,after sequential exposure to hepatogenic growth factors and cytokines,an immature hepatic cell population was generated. The presence of pre-albumin suggests that a sequential exposure strategy is here superior to a cocktail approach. In summary,a considerable impact of different (non)commercial media on the lineage-specific differentiation efficiency of hSKP is shown. In addition,we demonstrate here for the first time that,in a suitable keratinocyte stimulating micro-environment,hSKP can generate keratinocyte-like progeny in vitro. View Publication

Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges,including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer,functional,and tunable alternative for gene silencing without the need for DNA editing. As a proof of concept for multiplex gene silencing,we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors,enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models. Silenced (S) M5CAR T cells retained antitumor functionality comparable to,and in some cases exceeding,that of KO cells. In vivo,S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs). Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells,with potential advantages in potency,persistence,metastasis prevention,and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks. View Publication

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation,and by day 6,more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1,GATA-3,and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis,indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors,including interleukin-3 (IL-3),IL-1,IL-6,IL-11,erythropoietin,and Kit ligand. At days 10 and 14 of differentiation,EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first,approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next,and precursors of the mast cell lineage develop last. The kinetics of precursor development,as well as the growth factor responsiveness of these early cells,is similar to that found in the yolk sac and early fetal liver,indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo. View Publication