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

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However,acquired resistance and treatment failure are commonplace. To address this,we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line,MDS-L,and identify TOPORS as a loss-of-function target that synergizes with HMAs,reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable,we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall,our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML. Subject terms: Myelodysplastic syndrome,Acute myeloid leukaemia,Sumoylation View Publication

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system,neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay,that all neurospheres are derived from a NSC,and provided evidence that it overestimates NSC frequency,rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA),which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs,thereby facilitating further advances in the promising application of NSCs for therapeutic use. View Publication

Mesenchymal stem cells (MSC) can differentiate into osteoblasts,adipocytes,chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro,MSC differentiate to osteoblasts when exposed to bone-inducing medium. However,adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering,but may also be relevant in the search for new treatments of osteoporosis. View Publication

Little is known on the embryonic origin and related heterogeneity of adult neural stem cells (aNSCs). We use conditional genetic tracing,activated in a global or mosaic fashion by cell type-specific promoters or focal laser uncaging,coupled with gene expression analyses and Notch invalidations,to address this issue in the zebrafish adult telencephalon. We report that the germinal zone of the adult pallium originates from two distinct subtypes of embryonic progenitors and integrates two modes of aNSC formation. Dorsomedial aNSCs derive from the amplification of actively neurogenic radial glia of the embryonic telencephalon. On the contrary,the lateral aNSC population is formed by stepwise addition at the pallial edge from a discrete neuroepithelial progenitor pool of the posterior telencephalic roof,activated at postembryonic stages and persisting lifelong. This dual origin of the pallial germinal zone allows the temporally organized building of pallial territories as a patchwork of juxtaposed compartments. View Publication

PU.1 ( SPI1 ) is pivotal in hematopoiesis,yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1 ’s regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis,we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells,respectively - as downstream targets of SPI1 . Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically,KLF1 overexpression restores myeloid lineage potential,while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1 - LYL1 axis in the regulatory network in in vivo EHT. Taken together,our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT,potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs). Subject areas: Biological sciences,Molecular biology,Molecular interaction,Cell biology; View Publication

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties,yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME,we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway,possibly by modulating availability of hyaluronic acid (HA),the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships,and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions,respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction,whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo,targeting CD44 decreased PI3K-4EBP1-SOX2 signaling,tumor growth,and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally,a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes,respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44,TAM,and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery. View Publication

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent postnatal stem cells that have been used for the treatment of bone defects and graft-versus-host diseases in clinics. In this study,we found that subcutaneously transplanted human BMMSCs are capable of organizing hematopoietic progenitors of recipient origin. These hematopoietic cells expressed multiple lineages of hematopoietic cell associated markers and were able to rescue lethally irradiated mice,with successful engraftment in the recipient,suggesting a potential bone marrow (BM) resource for stem cell therapies. Furthermore,we found that platelet-derived growth factor (PDGF) promotes the formation of BMMSC-generated BM niches through upregulation of beta-catenin,implying that the PDGF pathway contributes to the formation of ectopic BM. These results indicate that the BMMSC-organized BM niche system represents a unique hematopoietic progenitor resource possessing potential clinical value. View Publication

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony-stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants. However,G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors,precluding autologous transplantation in those donors or substantially delaying transplant recovery time. Consequently,new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice,we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF-mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF,implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment,genetic reduction of EGFR activity in HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42),and genetic Cdc42 deficiency in vivo also enhanced G-CSF-induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes. View Publication

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis,fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro,preferentially promoting B-lymphocyte development,enhancing self-renewal of B-cell precursors,and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However,it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis,TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. View Publication

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid,beta-glycerophosphate,and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after,but not before,14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair. View Publication

Retinitis pigmentosa,other inherited retinal diseases,and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here,we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx+ photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells,facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling. STEM CELLS2013;31:408–414 View Publication

Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus,salivary glands,lung,thymus,parathyroid and thyroid. Despite its importance,reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here,we describe a novel protocol to derive a subdomain of AFE,identified by expression of Pax9,from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells,which when transplanted in vivo,can form several distinct complex AFE-derived epithelia,including mucosal glands and stratified squamous epithelium. Finally,we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus,this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders. View Publication