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

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However,direct injection of hESCs,and cells differentiated from hESCs,into living organisms has thus far been hampered by significant cell death,teratoma formation,and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization,proliferation,and viability. Molecular imaging has given investigators a high-throughput,inexpensive,and sensitive means for tracking in vivo cell proliferation over days,weeks,and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment,proliferation,and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes,under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery,are introduced into cells using a variety of vector and non-vector methods. Once in the cell,reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions,depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive,noninvasive instrumentation (e.g.,CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging,bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase,derived from the firefly Photinus pyralis,encodes an enzyme that catalyzes D-luciferin to the optically active metabolite,oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells,allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore,because expression of the reporter gene product is required for signal generation,only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video,the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described. View Publication

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-gamma secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family,such as NKp46,by ligands expressed on tumour cells. However,it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here,we investigated the early events occurring during T cell-tumour cell interactions,and their impact on NK cell functions. We observed that on co-culture with some melanomas,activated CD4(+) T cells promoted degranulation,and NKG2D- and NKp46-dependent IFN-gamma secretion by NK cells,probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4(+),CD8(+) and resting T cells,which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new,so far,unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. View Publication

BACKGROUND AIMS Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products,which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities,there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment,and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS This study compared cryopreservation of PBSC in a novel intracellular-like,fully defined,serum- and protein-free preservation solution,CryoStor (BioLife Solutions Inc.),with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly,human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard,Normosol-R,5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed,and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC),viability,CD34 and granulocytes by flow cytometry,along with colony-forming activity in methylcellulose. RESULTS The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+),colony-forming units (CFU) and granulocytes. Specifically,relative to the FHCRC standard formulation,cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005),a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition,it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins,and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however,these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections,along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO. View Publication

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes,indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model,transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (ptextless0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition,transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease. View Publication

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However,it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias,we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition,overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover,PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia. View Publication

The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration,trafficking,and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice,the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro,we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration,abnormal cell adhesion,and increased homotypic aggregation. In vivo,immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eμ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eμ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL. View Publication

Hematopoietic progenitor cell trafficking is an important phenomenon throughout life. It is thought to occur in sequential steps,similar to what has been described for mature leukocytes. Molecular actors have been identified for each step of leukocyte migration; recently,CD99 was shown to play a part during transendothelial migration. We explored the expression and role of CD99 on human hematopoietic progenitors. We demonstrate that (1) CD34+ cells express CD99,albeit with various intensities; (2) subsets of CD34+ cells with high or low levels of CD99 expression produce different numbers of erythroid,natural killer (NK),or dendritic cells in the in vitro differentiation assays; (3) the level of CD99 expression is related to the ability to differentiate toward B cells; (4) CD34+ cells that migrate through an endothelial monolayer in response to SDF-1alpha and SCF display the highest level of CD99 expression; (5) binding of a neutralizing antibody to CD99 partially inhibits transendothelial migration of CD34+ progenitors in an in vitro assay; and (6) binding of a neutralizing antibody to CD99 reduces homing of CD34+ progenitors xenotransplanted in NOD-SCID mice. We conclude that expression of CD99 on human CD34+ progenitors has functional significance and that CD99 may be involved in transendothelial migration of progenitors. View Publication

Autoimmune diseases (AIDs),a heterogeneous group of immune-mediated disorders,are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs,the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However,stem cell transplantation therapy has limitations,including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology,which allows the generation of patient-specific pluripotent stem cells,could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS),Sjogren's syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally,all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS,SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs,including AS,SS and SLE. View Publication

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here,we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes,where they trap T cells by releasing the CCR7 ligand,CCL19,and to inflamed central nervous system,where they locally limit immunopathogenesis through interleukin-10 production,thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment,where proB cells develop,is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition,these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. View Publication

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However,the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum,blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus,RTM protects the normal arterial intima,and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions. View Publication

MicroRNAs (miRNA) are central regulators of diverse biological processes and are important in the regulation of stem cell self-renewal. One of the widely studied miRNA-protein regulators is the Lin28-Let-7 pair. In this study,we demonstrate that contrary to the well-established models of mouse ES cells (mESC) and transformed human cancer cells,the pluripotent state of human ES cells (hESC) involves expression of mature Let-7 family miRNAs with concurrent expression of all LIN28 proteins. We show that mature Let-7 miRNAs are regulated during hESC differentiation and have opposite expression profile with LIN28B. Moreover,mature Let-7 miRNAs fine tune the expression levels of LIN28B protein in pluripotent hESCs,whereas silencing of LIN28 proteins have no effect on mature Let-7 levels. These results bring novel information to the highly complex network of human pluripotency and suggest that maintenance of hESC pluripotency differs greatly from the mESCs in regard to LIN28-Let-7 regulation. View Publication

No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is,however,limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel),0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker,MATH5. Furthermore,0.5% RGD-alginate hydrogel encapsulation did not adversely affect the expression of other neural retina markers (PROX1,CRX,RCVRN,AP2α or VSX2) as determined by qRT-PCR,or the percentage of VSX2 positive cells as determined by flow cytometry. 1% RGD-alginate increased the percentage of EBs with OVs and/or RPE,but did not significantly influence any other measures of retinal differentiation. HA-based hydrogels had no significant effect on retinal tissue development. The results indicated that derivation of retinal tissue from hESCs/hiPSCs can be enhanced by culture in 0.5% RGD-alginate hydrogel. This RGD-alginate scaffold may be useful for derivation,transport and transplantation of neural retina and RPE,and may also enhance formation of other pigmented,neural or epithelial tissue. STATEMENT OF SIGNIFICANCE The burden of retinal disease is ever growing with the increasing age of the world-wide population. Transplantation of retinal tissue derived from human pluripotent stem cells (PSCs) is considered a promising treatment. However,derivation of retinal tissue from PSCs using defined media is a lengthy process and often variable between different cell lines. This study indicated that alginate hydrogels enhanced retinal tissue development from PSCs,whereas hyaluronic acid-based hydrogels did not. This is the first study to show that 3D culture with a biomaterial scaffold can improve retinal tissue derivation from PSCs. These findings indicate potential for the clinical application of alginate hydrogels for the derivation and subsequent transplantation retinal tissue. This work may also have implications for the derivation of other pigmented,neural or epithelial tissue. View Publication