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

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter,technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple,effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple,but important step results in remarkable protein deposition efficiencies often exceeding 50%,whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays,and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions,as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies. View Publication

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In stem cell biology,a long-held structure–function relationship is the domed colony morphology and naïve pluripotency for mouse or human pluripotent stem cells. This link has provided a convenient way to recognize bona fide naïve pluripotent cells during derivation,passaging and characterization. However,the molecular basis of this link remains poorly understood. Results: We show that a loss of domed morphology may not impact the overall genetic architecture of naïve pluripotency in mouse embryonic stem cells (mESCs). We first generated stable mESC lines by knocking out Myh9 that encodes non-muscle myosin heavy chain IIA,resulting in colonies deprived of the typical domed morphology,but competent to differentiate into the three germ layers and chimeric mice. Modulating cell morphologies with inhibitors against kinases known to regulate myosin pathway also phenocopy the knockout in wild type mESCs. Conclusions: These results provide evidence that the domed morphology and potency can be uncoupled and suggest that domed structure is not a pre-requisite for acquiring and maintaining naïve pluripotency. View Publication

Autophagy-based targeted degradation offers a powerful complement to proteasomal degradation leveraging the capacity and versatility of lysosomes to degrade complex cargo. However,it remains unclear which components of the autophagy-lysosomal pathway are most effective for targeted degradation. Here,we describe two orthogonal induced-proximity strategies to identify autophagy effectors capable of degrading organelles and soluble targets. Recruitment of autophagy cargo receptors,ATG8-like proteins,or the kinases ULK1 and TBK1 is sufficient to trigger mitophagy,while only autophagy cargo receptors capable of self-oligomerization degrade soluble cytosolic proteins. We further report a single-domain antibody against p62 and its use as a heterobifunctional degrader to clear mitochondria. Fusing the p62 single-domain antibody to PINK1 enables selective targeting of damaged mitochondria. Our study highlights the importance of avidity for targeted autophagy and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for organelles or protein aggregates. Using proximity-based screening,protein engineering,and structural analysis,this study describes the development of a p62-based biodegrader for the clearance of organelles and aggregated proteins by autophagy-targeted degradation. View Publication

Acute myeloid leukemia (AML) is associated with unfavorable patient outcomes primarily related to disease relapse. Since specific types of leukemic hematopoietic stem and progenitor cells (HSPCs) are suggested to contribute to AML propagation,this study aimed to identify and explore relapse-initiating HSPC subpopulations present at diagnosis,using single-cell analysis (SCA). We developed unique high-resolution techniques capable of tracking single-HSPC-derived subclones during AML evolution. Each subclone was evaluated for chemo-resistance,in vivo leukemogenic potential,mutational profile,and the cell of origin. In BM samples of 15 AML patients,GMP-like and MLP-like HSPC subpopulations were identified as prevalent at relapse,exhibiting chemo-resistance to commonly used chemotherapy agents cytosine arabinoside (Ara-C) and daunorubicin. Reconstruction of phylogenetic lineage trees combined with genetic analysis of single HSPCs and single-HSPC-derived subclones demonstrated two distinct clusters,originating from MLP-like or GMP-like subpopulations,observed both at diagnosis and relapse. These subpopulations induced leukemia development ex vivo and in vivo. Genetic SCA showed that these relapse-related subpopulations harbored mutated EZH2 and TP53,detected already at diagnosis. This study,using combined molecular,functional,and genomic analyses at the level of single cells,identified patient-specific chemo-resistant HSPC subpopulations at the time of diagnosis,promoting AML relapse. View Publication

OVERVIEW: All current antipsychotic medications work by binding to Gi-coupled dopamine (DA) D2 receptors. Such medications are thought to affect cellular function primarily by decreasing DA-mediated regulation of intracellular cyclic adenosine monophosphate (cAMP).However,several studies indicate that cAMP signal transduction abnormalities in schizophrenia may not be limited to D2-containing cells. The current study examines the potential of using non-receptor-based agents that modify intracellular signal transduction as potential antipsychotic medications. METHODS: The indirect DA agonist amphetamine has been used to model the auditory sensory processing deficits in schizophrenia. Such pharmacologically induced abnormalities are reversed by current antipsychotic treatments. This study examines the ability of the phosphodiesterase-4 inhibitor,rolipram,to reverse amphetamine-induced abnormalities in auditory-evoked potentials that are characteristic of schizophrenia. RESULTS: Rolipram reverses amphetamine-induced reductions in auditory-evoked potentials. CONCLUSION: This finding could lead to novel approaches to receptor-independent treatments for schizophrenia. View Publication

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells,Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b,which in turn activates pro-caspase-1 to mediate cell death3,but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples,but not in cells from many other lineages,and that these inhibitors inhibit human AML progression in mouse models. Overall,this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML. View Publication

RNA-binding motif protein 15 (RBM15) is involved in the RBM15-megakaryoblastic leukemia 1 fusion in acute megakaryoblastic leukemia. Although Rbm15 has been reported to be required for B-cell differentiation and to inhibit myeloid and megakaryocytic expansion,it is not clear what the normal functions of Rbm15 are in the regulation of hematopoietic stem cell (HSC) and megakaryocyte development. In this study,we report that Rbm15 may function in part through regulation of expression of the proto-oncogene c-Myc. Similar to c-Myc knockout (c-Myc-KO) mice,long-term (LT) HSCs are significantly increased in Rbm15-KO mice due to an apparent LT-HSC to short-term HSC differentiation defect associated with abnormal HSC-niche interactions caused by increased N-cadherin and beta(1) integrin expression on mutant HSCs. Both serial transplantation and competitive reconstitution capabilities of Rbm15-KO LT-HSCs are greatly compromised. Rbm15-KO and c-Myc-KO mice also share related abnormalities in megakaryocyte development,with mutant progenitors producing increased,abnormally small low-ploidy megakaryocytes. Consistent with a possible functional interplay between Rbm15 and c-Myc,the megakaryocyte increase in Rbm15-KO mice could be partially reversed by ectopic c-Myc. Thus,Rbm15 appears to be required for normal HSC-niche interactions,for the ability of HSCs to contribute normally to adult hematopoiesis,and for normal megakaryocyte development; these effects of Rbm15 on hematopoiesis may be mediated at least in part by c-Myc. View Publication