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

The human transferrin receptor (hTfR) is a target for cancer immunotherapy due to its overexpression on the surface of cancer cells. We previously developed an antibody-avidin fusion protein that targets hTfR (anti-hTfR IgG3-Av) and exhibits intrinsic cytotoxicity against certain malignant cells. Gambogic acid (GA),a drug that also binds hTfR,induces cytotoxicity in several malignant cell lines. We now report that anti-hTfR IgG3-Av and GA induce cytotoxicity in a new broader panel of hematopoietic malignant cell lines. Our results show that the effect of anti-hTfR IgG3-Av is iron-dependent whereas that of GA is iron-independent in all cells tested. In addition,we observed that GA exerts a TfR-independent cytotoxicity. We also found that GA increases the generation of reactive oxygen species that may play a role in the cytotoxicity induced by this drug. Additive cytotoxicity was observed by simultaneous combination treatment with these drugs and synergy by using anti-hTfR IgG3-Av as a chemosensitizing agent. In addition,we found a concentration of GA that is toxic to malignant hematopoietic cells but not to human hematopoietic progenitor cells. Our results suggest that these two compounds may be effective,alone or in combination,for the treatment of human hematopoietic malignancies. View Publication

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate all of the hematopoietic lineages in vivo and sustain the production of these cells for the life span of the individual. In the absence of reliable direct markers for HSCs,their identification and enumeration depends on functional long-term,multilineage,in vivo repopulation assays. The extremely low frequency of HSCs in any tissue and the absence of a specific HSC phenotype have made their purification and characterization a highly challenging goal. HSCs and primitive hematopoietic cells can be distinguished from mature blood cells by their lack of lineage-specific markers and presence of certain other cell-surface antigens,such as CD133 (for human cells) and c-kit and Sca-1 (for murine cells). Functional analyses of purified subpopulations of primitive hematopoietic cells have led to the development of several procedures for isolating cell populations that are highly enriched in cells with in vivo stem cell activity. Simplified methods for obtaining these cells at high yield have been important to the practical exploitation of such advances. This article reviews recent progress in identifying human and mouse HSCs and current techniques for their purification. View Publication

MED1/TRAP220,a subunit of the transcriptional Mediator/TRAP complex,is crucial for various biological events through its interaction with distinct activators,such as nuclear receptors and GATA family activators. In hematopoiesis,MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study,we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1(-/-)) mouse embryonic fibroblasts (MEFs) was significantly suppressed compared to the control. Furthermore,the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1(-/-) MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN,as well as Mediator recruitment to the Opn promoter,was specifically attenuated in the Med1(-/-) MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs,both of which were attenuated by the addition of the anti-OPN antibody to Med1(+/+) MEFs and to BM stromal cells. Consequently,MED1 in niche appears to play an important role in supporting HSPCs by upregulating VDR- and Runx2-mediated transcription on the Opn promoter. View Publication

Emerging evidence has shown that resting quiescent hematopoietic stem cells (HSCs) prefer to utilize anaerobic glycolysis rather than mitochondrial respiration for energy production. Compelling evidence has also revealed that altered metabolic energetics in HSCs underlies the onset of certain blood diseases; however,the mechanisms responsible for energetic reprogramming remain elusive. We recently found that Fanconi anemia (FA) HSCs in their resting state are more dependent on mitochondrial respiration for energy metabolism than on glycolysis. In the present study,we investigated the role of deficient glycolysis in FA HSC maintenance. We observed significantly reduced glucose consumption,lactate production,and ATP production in HSCs but not in the less primitive multipotent progenitors or restricted hematopoietic progenitors of Fanca-/- and Fancc-/- mice compared with that of wild-type mice,which was associated with an overactivated p53 and TP53-induced glycolysis regulator,the TIGAR-mediated metabolic axis. We utilized Fanca-/- HSCs deficient for p53 to show that the p53-TIGAR axis suppressed glycolysis in FA HSCs,leading to enhanced pentose phosphate pathway and cellular antioxidant function and,consequently,reduced DNA damage and attenuated HSC exhaustion. Furthermore,by using Fanca-/- HSCs carrying the separation-of-function mutant p53R172P transgene that selectively impairs the p53 function in apoptosis but not cell-cycle control,we demonstrated that the cell-cycle function of p53 was not required for glycolytic suppression in FA HSCs. Finally,ectopic expression of the glycolytic rate-limiting enzyme PFKFB3 specifically antagonized p53-TIGAR-mediated metabolic reprogramming in FA HSCs. Together,our results suggest that p53-TIGAR metabolic axis-mediated glycolytic suppression may play a compensatory role in attenuating DNA damage and proliferative exhaustion in FA HSCs. Stem Cells 2019;37:937-947. View Publication

CD4 T cells play a critical role in promoting the development of autoimmunity in type 1 diabetes. The diabetogenic CD4 T cell clone BDC-2.5,originally isolated from a NOD mouse,has been widely used to study the contribution of autoreactive CD4 T cells and relevant Ags to autoimmune diabetes. Recent work from our laboratory has shown that the Ag for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA) and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP Ag (2.5HIP-coupled PLG NPs) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-coupled PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce proinflammatory cytokines through induction of anergy,leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-gamma+ effector T cells. To our knowledge,this work is the first to use a hybrid insulin peptide,or any neoepitope,to re-educate diabetogenic T cells and may have significant implications for the development of an Ag-specific therapy for type 1 diabetes patients. View Publication

We studied leukemic stem cells (LSCs) in a Smad4(-/-) mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes,there is no cytoplasmic stabilization of HOXA9 in Smad4(-/-) HSPCs,and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore,the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control,we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis,leading to a loss of LSCs. Together,these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia. 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

Mouse embryonic stem (mES) cells have short duration of their cell cycle and are capable of proliferating in the absence of growth factors. To find out which signaling pathways contribute to the regulation of the mES cell cycle,we used pharmacological inhibitors of MAP and PI3 kinase cascades. The MAP kinase inhibitors as well as serum withdrawal did not affect mES cell cycle distribution,whereas the inhibitor of PI3K activity,LY294002,induced accumulation of cells in G(1) phase followed by apoptotic cell death. Serum withdrawal also causes apoptosis,but it does not change the content and activity of cell cycle regulators. In contrast,in mES cells treated with LY294002,the activities of Cdk2 and E2F were significantly decreased. Interestingly,LY294002had a much stronger effect on cell cycle distribution in low serum conditions,implying that serum can promote G(1)--textgreaterS transition of mES cells by a LY294002-resistant mechanism. Thus,proliferation of mES cells is maintained by at least two separate mechanisms: a LY294002-sensitive pathway,which is active even in the absence of serum,and LY294002-resistant,but serum-dependent,pathway. View Publication