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

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 this report,we sought to optimize gene transfer into primitive human umbilical cord blood (UCB) cells. Initially,we found that fresh UCB isolated with the CD34brCD38 CD33 phenotype were highly enriched for hematopoietic progenitors detected in extended long-term cultures (8-week LTCs). In addition,following ex vivo gene transfer,this population possessed virtually all the 8-week LTC activity of the cultured cells. A multiparameter FACS assay was developed to efficiently screen the effects of alternative retroviral vector gene transfer procedures on the transduction efficiency and maintenance of CD34brCD38 CD33 cells. Proliferation of the CD34brCD38 CD33 cells was found to be a prerequisite for efficient transduction. However,in all conditions tested,proliferation of the CD34brCD38 CD33 cells was associated with a progressive loss of primitive cell properties including a reduction in CD34 expression,an increase in CD38/CD33 expression,and a decline in the ability to sustain 8-week LTCs. These observations indicate that it will be necessary to define conditions that more effectively support the self-renewal capacity of CD34brCD38 CD33 cells to optimize retroviral vector gene transfer in these cells. Evaluating these conditions and reagents will be facilitated by the multiparameter FACS assay described in this report. View Publication

Despite its wide use as a marker for hematopoietic stem cells (HSCs),the function of stem cell antigen-1 (Sca-1) (also known as lymphocyte activation protein-6A [Ly-6A]) in hematopoiesis remains poorly defined. We have previously established that Sca-1(-/-) T cells develop normally,although they are hyperresponsive to antigen. Here,we report detailed analysis of hematopoiesis in Sca-1-deficient animals. The differentiation potential of Sca-1-null bone marrow was determined from examination of the most mature precursors (culture colony-forming units [CFU-Cs]) to less committed progenitors (spleen CFUs [CFU-Ss]) to long-term repopulating HSCs. Sca-1-null mice are mildly thrombocytopenic with a concomitant decrease in megakaryocytes and their precursors. Bone marrow cells derived from Sca-1(-/-) mice also have decreased multipotential granulocyte,erythroid,macrophage,and megakaryocyte CFU (GEMM-CFU) and CFU-S progenitor activity. Competitive repopulation assays demonstrated that Sca-1(-/-) HSCs are at a competitive disadvantage compared with wild-type HSCs. To further analyze the potential of Sca-1(-/-) HSCs,serial transplantations were performed. While secondary repopulations using wild-type bone marrow completely repopulated Sca-1(-/-) mice,Sca-1(-/-) bone marrow failed to rescue one third of lethally irradiated wild-type mice receiving secondary bone marrow transplants from irradiation-induced anemia and contributed poorly to the surviving transplant recipients. These data strongly suggest that Sca-1 is required for regulating HSC self-renewal and the development of committed progenitor cells,megakaryocytes,and platelets. Thus,our studies conclusively demonstrate that Sca-1,in addition to being a marker of HSCs,regulates the developmental program of HSCs and specific progenitor populations. 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

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors,and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions,yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore,we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1,and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors,leading to target cell death. View Publication