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

We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 10(5) progenitors,cardiomyocytes or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Myocardial function was assessed at 2-week and 4-week post-infarction by using echocardiography and pressure-volume catheterization. Early myocardial remodelling was observed at 2-week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 $\$,P textless 0.05) and cardiomyocyte (19.52 ± 3.97 $\$,P textless 0.05) groups,but not in progenitor group (25.65 ± 3.61 $\$),significantly deteriorated as compared to sham control group (28.41 ± 4.41 $\$). Consistently,pressure-volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 $\$,P textless 0.05; ESV: 17.08 ± 5.82 $\$,P textless 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 $\$,P textless 0.05; ESV: 18.03 ± 6.58 $\$,P textless 0.05) groups by 4-week post-infarction as compared to control (EDV: 15.26 ± 2.96 $\$; ESV: 8.41 ± 2.94 $\$). In contrast,cardiac progenitors (EDV: 20.09 ± 7.76 $\$; ESV: 13.98 ± 6.74 $\$) persistently protected chamber geometry against negative cardiac remodelling. Similarly,as compared to sham control (54.64 ± 11.37%),LV ejection fraction was preserved in progenitor group from 2-(38.68 ± 7.34%) to 4-week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%,P textless 0.05) and saline (35.34 ± 11.86%,P textless 0.05) groups deteriorated early at 2-week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm(2) to 25.48 ± 2.08/mm(2) myocardial tissue,P textless 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone. View Publication

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here,memory B cells are activated and amplified using Epstein-Barr virus infection,co-cultured with CHO-muCD40L cells,and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells,and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly,our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family,influenza A neutralizing antibodies,contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. View Publication

Regulation of gene expression in immune cells is known to be under genetic control,and likely contributes to susceptibility to autoimmune diseases such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice,more accurately reflecting genetically diverse human populations,we provide an extensive characterization of the genetic regulation of gene expression in five different naive immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control,exhibiting diverse patterns: global,cell-specific and sex-specific. Bioinformatic analysis of the genetically controlled transcript networks reveals reduced cell type specificity and inflammatory activity in wild-derived PWD/PhJ mice,compared with the conventional laboratory strain C57BL/6J. Additionally,candidate MS-GWAS (genome-wide association study candidate genes for MS susceptibility) genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared with PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis,the principal model of MS,and skewing of the encephalitogenic T-cell responses. Taken together,our results provide functional insights into the genetic regulation of the immune transcriptome,and shed light on how this in turn contributes to susceptibility to autoimmune disease.Genes and Immunity advance online publication,22 September 2016; doi:10.1038/gene.2016.37. View Publication

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable,synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells,including impaired neural rosette formation,increased susceptibility to growth factor withdrawal,and deficits in mitochondrial respiration,are rescued in isogenic controls. Importantly,using genome-wide expression analysis,we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines,suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities,and the importance of isogenic controls for disease modeling using hiPSCs. View Publication

Garcinol is a polyisoprenylated benzophenone derivative found in Garcinia indica fruit rind and other species. The potential antioxidative and neuroprotective effects of garcinol in rat cortical astrocyte were demonstrated in our laboratory recently. Here,the effects of garcinol on the neuritogenesis process in cultured cortical progenitor cells were investigated to understand the roles of garcinol in neuronal survival and differentiation. These cells,derived from embryonic day 17 rats,differentiated into EGF-responsive neural precursor cells,would further form neurospheres. Our data exhibited garcinol induced neurite outgrowth in early developing EGF-treated neurospheres and significantly enhanced the expression of neuronal proteins,microtubule-associated protein 2 (MAP-2),and glial fibrillary acidic protein (GFAP). Furthermore,the neuronal marker,high-molecular-weight subunit of neurofilaments (NFH),was highly expressed after 5 μM garcinol treatment in neural precursor cells for 20 days. To identify the extracellular mechanism,rat cortical progenitor cells were treated garcinol and accordingly mediated the sustained activation of extracellular signal-regulated kinase (ERK) for different periods up to 20 h. In this regard,NMDA receptor-mediated calcium influx led to excitotoxic death and activated tyrosine phosphatase which limited the duration of ERK in cultured neurons. MK801,the NMDA receptor antagonist,treatment also induced the sustained phosphorylation of ERK and therefore enhanced neuronal survival. In our observation,garcinol treatment reduced growth factor deprivation-mediated cell death and nuclear import of C/EBPβ levels. Noteworthy,garcinol could promote neurite outgrowth in EGF-responsive neural precursor cells and modulate the ERK pathway in the enhancement of neuronal survival. View Publication

Pluripotency,the ability of a cell to differentiate and give rise to all embryonic lineages,defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes,accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells,as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency,we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation,except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression,suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers,and observed much greater dynamics in chromatin modifications,especially H3K4me1 and H3K27ac,which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency. View Publication

Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower,less toxic,doses. Experimentally,high doses induce rapid DNA damage and cytotoxicity,which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses,without causing immediate cytotoxicity,produce an antitumor memory" response� View Publication

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine,especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells,but these predominantly rely on biochemical cues. Recently,differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However,the means by which these topographical cues improve neuronal yield remains unknown. In this study,we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 ??m gratings increase the rate of neural differentiation,and that an additional culture period of 2 ??m gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added,thus generating hPSC-derived neural cells in a more cost effective and efficient manner. ?? 2012 Elsevier Ltd. View Publication

Articular cartilage,which is mainly composed of collagen II,enables smooth skeletal movement. Degeneration of collagen II can be caused by various events,such as injury,but degeneration especially increases over the course of normal aging. Unfortunately,the body does not fully repair itself from this type of degeneration,resulting in impaired movement. Microfracture,an articular cartilage repair surgical technique,has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However,the therapeutic outcomes of all these techniques vary in different patients depending on their age,health,lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage,both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone,or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs),which are able to self-renew and differentiate into multiple cell types,provides a potentially valuable cell resource for drug screening in a more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis." View Publication

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined,growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling,drug screening and cell-based therapeutic applications. View Publication

In this study,we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor $$-positive (ER$$(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ER$$,HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly,expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover,aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5,indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT,stemness and ultimately tumor progression. Importantly,pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ER$$ expression,downregulation of HER-2/Neu,inhibition of EMT and impaired self-renewal ability,resulting in the suppression of distant metastases. Taken together,our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ER$$(+) breast cancer cells. Moreover,this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ER$$(+) breast cancer patients resistant to conventional endocrine therapy. View Publication

Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here,we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system,when optimized in human U87 cells,provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs,targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs,the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette,demonstrating the feasibility of cassette exchange. Moreover,as assessed by measuring γ-H2AX expression levels,genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency,flexibility in transgene exchange and low genome toxicity,our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells. View Publication