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

Differentiation of embryonic stem (ES) cells typically requires cell-cell aggregation in the form of embryoid bodies (EBs). This process is not very well controlled and final cell numbers can be limited by EB agglomeration and the inability to drive differentiation towards a desired cell type. This study compares three-dimensional (3D) fibrin culture to conventional two-dimensional (2D) suspension culture and to culture in a semisolid methylcellulose medium solution. Two types of fibrin culture were evaluated,including a PEGylated fibrin gel. PEGylation with a difunctional PEG derivative retarded fibrinogen migration during through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a result of crosslinking,similarly,degradation was slowed in the PEGylated gel. ES cell proliferation was higher in both the fibrin and PEGylated fibrin gels versus 2D and methylcellulose controls. FACS analysis and real-time-PCR revealed differences in patterns of differentiation for the various culture systems. Culture in PEGylated fibrin or methylcellulose culture demonstrated features characteristic of less extensive differentiation relative to fibrin and 2D culture as evidenced by the transcription factor Oct-4. Fibrin gels showed gene and protein expression similar to that in 2D culture. Both fibrin and 2D cultures demonstrated statistically greater cell numbers positive for the vascular mesoderm marker,VE-cadherin. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. View Publication

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is an innovative approach for the treatment of a range of diseases that are not curable by current therapies including heart failure. A number of clinical trials have been completed and many others are ongoing; more than 2,000 patients worldwide have been administered with culture-expanded allogeneic or autologous MSCs for the treatment of various diseases,showing feasibility and safety (and some efficacy) of this approach. However,protocols for isolation and expansion of donor MSCs vary widely between these trials,which could affect the efficacy of the therapy. It is therefore important to develop international standards of MSC production,which should be evidence-based,regulatory authority-compliant,of good medical practice grade,cost-effective,and clinically practical,so that this innovative approach becomes an established widely adopted treatment. This review article summarizes protocols to isolate and expand bone marrow-derived MSCs in 47 recent clinical trials of MSC-based therapy,which were published after 2007 onwards and provided sufficient methodological information. Identified issues and possible solutions associated with the MSC production methods,including materials and protocols for isolation and expansion,are discussed with reference to relevant experimental evidence with aim of future clinical success of MSC-based therapy. View Publication

Immunosuppressants are powerful drugs,capable of triggering severe adverse effects. Hence,there is tremendous interest in replacing them with less-toxic agents. Adoptive therapy with CD25(+)CD4(+) T regulatory cells (Tregs) holds promise as an alternative to immunosuppressants. Tregs have been described as the most potent immunosuppressive cells in the human body. In a number of experimental models,they have been found to quench autoimmune diseases,maintain allogeneic transplants,and prevent allergic diseases. A major stumbling block in their clinical application is related to Treg phenotype and the very limited number of these cells in the periphery,not exceeding 1-5% of total CD4(+) T cells. Recent progress in multicolor flow cytometry and cell sorting as well as cellular immunology has found ways of overcoming these obstacles,and has opened the doors to the clinical application of Tregs. In the review,we describe Treg sorting and expansion techniques that have been developed in recent years. In the experience of our laboratory,as well as some published reports,Treg adoptive therapy is a promising tool in immunosuppressive therapy,and should be considered for clinical trials. View Publication

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however,the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore,HSCs in their normal microenvironment activate a LEF-1/TCF reporter,which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain,inhibitors of the Wnt signalling pathway,leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore,activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1,genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo,and provide insight into a potential molecular hierarchy of regulation of HSC development. View Publication

Circulating Tumor Cells (CTCs) represent an easy,repeatable and representative access to information regarding solid tumors. However,their detection remains difficult because of their paucity,their short half-life,and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast,sensitive and affordable technique,ideal for rare cells detection. Adapted to CTCs detection (i.e. extremely rare cells),most FC-based techniques require a time-consuming pre-enrichment step,followed by a 2-hours staining procedure,impeding on the efficiency of CTCs detection. We overcame these caveats and reduced the procedure to less than one hour,with minimal manipulation. First,cells were simultaneously fixed,permeabilized,then stained. Second,using low-speed FC acquisition conditions and two discriminators (cell size and pan-cytokeratin expression),we suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases,this protocol ensures a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and can predict which samples are derived from cancer donors. This proof-of-concept study lays the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses. View Publication

A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene,which was readily identified because of its high degree of homology to the gibbon sequence,also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells,proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3. View Publication

A method for the generation of human induced pluripotent stem (iPS) cells was established. This method employs adenovirus carrying the ecotropic retrovirus receptor mCAT1 and Moloney murine leukemia virus (MMLV)-based retroviral vectors carrying the four transcription factors POU5F1 (OCT3/4),KLF4,SOX2,and MYC (c-Myc) (Masaki H & Ishikawa T Stem Cell Res 1:105-15,2007). The differentiation of human iPS cells into hepatic cells was performed by a stepwise protocol (Song Z et al. Cell Res 19:1233-42,2009). These cells have potential as patient-specific in vitro models for studying disease etiology and could be used in drug discovery programs tailored to deal with genetic variations in drug efficacy and toxicity. View Publication

Human induced pluripotent stem cells (HIPSC) have tremendous value as a source of autologous cells for cellular transplantation in the treatment of degenerative diseases. The protocols described here address methods for large-scale genetic modification of HIPSCs. The first is an optimized method for transfecting HIPSCs cultured in feeder-free conditions. The second method allows nucleofection of trypsinized HIPSCs at an optimal cell density. Both methods enable robust generation of stable HIPSC transfectants within two weeks. Our protocols are highly reproducible and do not require optimization for individual HIPSC and human embryonic stem cell (HESC) lines. View Publication

Human mesenchymal stem cells are thought to be multipotent cells,which are present in adult marrow,that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues,including bone,cartilage,fat,tendon,muscle,and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic,chondrocytic,or osteocytic lineages. Individual stem cells were identified that,when expanded to colonies,retained their multilineage potential. View Publication

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system,hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1,which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype,stable proliferation rate,and high telomerase activity. Similar to cells cultured on feeders,hES cells maintained under feeder-free conditions expressed OCT-4,hTERT,alkaline phosphatase,and surface markers including SSEA-4,Tra 1-60,and Tra 1-81. In addition,hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus,the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production. View Publication

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently,differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately,differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether,our results provide a new platform for the study of kidney diseases and lineage commitment,and open new avenues for the future application of regenerative strategies in the clinic. View Publication