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

Rationale: Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases,inflammatory diseases,etc.,due to their pleiotropic properties. However,largely incongruent data were obtained from different MSC-based clinical trials,which may be partially due to functional heterogeneity among MSC. Here,we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Methods: Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP),which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression,proliferation,migration,multipotency,immunomodulatory activity and global gene expression profile. Moreover,the in vivo therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). Results: We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC),and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly,NMP-MSC display much stronger immunomodulatory activity than BMSC in vitro and in vivo,as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Conclusion: Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies. View Publication

Since the discovery of rapamycin,considerable progress has been made in unraveling the details of the mammalian target of rapamycin (mTOR) signaling network,including the upstream mechanisms that modulate mTOR signaling functions,and the roles of mTOR in the regulation of mRNA translation and other cell growth-related responses. mTOR is found in two different complexes within the cell,mTORC1 and mTORC2,but only mTORC1 is sensitive to inhibition by rapamycin. mTORC1 is a master controller of protein synthesis,integrating signals from growth factors within the context of the energy and nutritional conditions of the cell. Activated mTORC1 regulates protein synthesis by directly phosphorylating 4E-binding protein 1 (4E-BP1) and p70S6K (S6K),translation initiation factors that are important to cap-dependent mRNA translation,which increases the level of many proteins that are needed for cell cycle progression,proliferation,angiogenesis,and survival pathways. In normal physiology,the roles of mTOR in both glucose and lipid catabolism underscore the importance of the mTOR pathway in the production of metabolic energy in quantities sufficient to fuel cell growth and mitotic cell division. Several oncogenes and tumor-suppressor genes that activate mTORC1,often through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway,are frequently dysregulated in cancer. Novel analogs of rapamycin (temsirolimus,everolimus,and deforolimus),which have improved pharmaceutical properties,were designed for oncology indications. Clinical trials of these analogs have already validated the importance of mTOR inhibition as a novel treatment strategy for several malignancies. Inhibition of mTOR now represents an attractive anti-tumor target,either alone or in combination with strategies to target other pathways that may overcome resistance. The far-reaching downstream consequences of mTOR inhibition make defining the critical molecular effector mechanisms that mediate the anti-tumor response and associated biomarkers that predict responsiveness to mTOR inhibitors a challenge and priority for the field. View Publication

Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb,helper-dependent adenoviral vectors with long homology arms are used for gene editing. However,this makes vector construction and recombinant analysis difficult. Conversely,insufficient homology may compromise targeting efficiency. Thus,we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology,the frequencies of targeted recombinants were 50-64.6% after positive selection for vector integration,and 97.4-100% after negative selection against random integrations. With 14.8 kb,the frequencies were 26.9-57.1% after positive selection and 87.5-100% after negative selection. With 9.6 kb,the frequencies were 21.4 and 75% after positive and negative selection,respectively. With only 5.6 kb,the frequencies were 5.6-16.7% after positive selection and 50% after negative selection,but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore,we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However,low frequencies (≤ 1 × 10(-3)) necessitated negative selection for piggyBac-excision product isolation. View Publication

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease,characterized by motor neuron (MN) death,for which there are no truly effective treatments. Here,we describe a new small molecule survival screen carried out using MNs from both wild-type and mutant SOD1 mouse embryonic stem cells. Among the hits we found,kenpaullone had a particularly impressive ability to prolong the healthy survival of both types of MNs that can be attributed to its dual inhibition of GSK-3 and HGK kinases. Furthermore,kenpaullone also strongly improved the survival of human MNs derived from ALS-patient-induced pluripotent stem cells and was more active than either of two compounds,olesoxime and dexpramipexole,that recently failed in ALS clinical trials. Our studies demonstrate the value of a stem cell approach to drug discovery and point to a new paradigm for identification and preclinical testing of future ALS therapeutics. View Publication

The use of antiretroviral therapy (ART) has remarkably decreased the morbidity associated with HIV-1 infection,however,the prevalence of HIV-1-associated neurocognitive disorders (HAND) is still increasing. The blood-brain barrier (BBB) is the major impediment for penetration of antiretroviral drugs,causing therapeutics to reach only suboptimal level to the brain. Conventional antiretroviral drug regimens are not sufficient to improve the treatment outcomes of HAND. In our recent report,we have developed a poloxamer-PLGA nanoformulation loaded with elvitegravir (EVG),a commonly used antiretroviral drug. The nanoformulated EVG is capable of elevating intracellular drug uptake and simultaneously enhance viral suppression in HIV-1-infected macrophages. In this work,we identified the clinical parameters including stability,biocompatibility,protein corona,cellular internalization pathway of EVG nanoformulation for its potential clinical translation. We further assessed the ability of this EVG nanoformulation to cross the in vitro BBB model and suppress the HIV-1 in macrophage cells. Compared with EVG native drug,our EVG nanoformulation demonstrated an improved BBB model penetration cross the in vitro BBB model and an enhanced HIV-1 suppression in HIV-1-infected human monocyte-derived macrophages after crossing the BBB model without altering the BBB model integrity. Overall,this is an innovative and optimized treatment strategy that has a potential for therapeutic interventions in reducing HAND. View Publication

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MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal,pluripotency,and differentiation. However,with respect to induced pluripotent stem cells (iPSC),in which embryonic-like cells are reprogrammed from adult cells using defined factors,the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge,we use miRNA microarrays to compare the microRNA-omes" of human iPSCs View Publication

Herpes simplex type 1 (HSV-1) amplicon vectors possess a number of features that make them excellent vectors for the delivery of transgenes into stem cells. HSV-1 amplicon vectors are capable of efficiently transducing both dividing and nondividing cells and since the virus is quite large,152 kb,it is of sufficient size to allow for incorporation of entire genomic DNA loci with native promoters. HSV-1 amplicon vectors can also be used to incorporate and deliver to cells a variety of sequences that allow extrachromosomal retention. These elements offer advantages over integrating vectors as they avoid transgene silencing and insertional mutagenesis. The construction of amplicon vectors carrying extrachromosomal retention elements,their packaging into HSV-1 viral particles,and the use of HSV-1 amplicons for stem cell transduction will be described. View Publication

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes,and forced expression of OCT4,KLF4,and KLF2 allows maintenance of human cells in a naïve state [Hanna J,et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid,followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics,antibody labeling profile,gene expression,X-inactivation profile,mitochondrial morphology,microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive,but attainable,process,leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible. View Publication

Despite major progress in the management of human liver disease,the only cure for a critically failing organ is liver transplantation. While a highly successful approach,the use of cadaveric organs as a routine treatment option is severely limited by organ availability. Therefore,the use of cell-based therapies has been explored to provide support for the failing liver. In addition to developing new treatments,there is also an imperative to develop better human models 'in a dish'. Such approaches will undoubtedly lead to a better understanding of the disease process,offering new treatment or preventative strategies. With both approaches in mind,we have developed robust hepatocyte differentiation methodologies for use with pluripotent stem cells. Importantly,our procedure is highly efficient (∼ 90%) and delivers active,drug-inducible,and predictive human hepatocyte populations. View Publication