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

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However� View Publication

Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications,such as transplantation of clinically engineered tissues and organs,and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study,the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80 % efficiency,by means of the dual-SMAD inhibition protocol,based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted,and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these contaminating" cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5 % and less than 1.5 % of Tra-1-60 positive cells were present in the purified populations. After re-plating� View Publication

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein,tandem dimer Tomato (tdTomato),are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons,evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. View Publication

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms,yet their clinical translation has been compromised by their biosafety concern. Here,we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/ progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immu-nodeficient mice. Moreover,removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplanta-tion,ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable,depending on the oncogenic load,with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus,transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:694–702 SIGNIFICANCE Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products,especially when reprogrammed with integrating vectors. Two major under-lying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzy-matic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in test-ing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature b cell phenotype would lead to safe islet replacement therapy for diabetes. View Publication

BACKGROUND: We previously identified a novel serine protease inhibitor (serpin),megsin,which is predominantly expressed in the kidney. Megsin expression is up-regulated in human and experimental renal diseases associated with mesangial proliferation and expansion,suggesting that urinary megsin may be a novel diagnostic marker for some renal diseases. METHODS: We established a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for megsin and measured urinary megsin of patients with various renal diseases. RESULTS: Megsin ELISA specifically detected megsin but not other serpins. The detection limit was 0.04 ng/ml,which allowed detection of urinary megsin in 3.6% of healthy individuals. The antigenic epitope in the urine detected by the ELISA was confirmed as megsin protein by time-of-flight mass spectrometry. Among patients with rapidly progressive glomerulonephritis (n = 18),55.6% were urinary megsin-positive,while 24.1% in IgA nephropathy (n = 112) and 15.1% in chronic non-IgA glomerulonephritis (n = 245) were urinary megsin-positive,respectively. Among patients with chronic renal failure due to unknown causes (n = 74),18.9% were positive for urinary megsin. In diabetic patients with or without nephropathy (n = 1073),12.3% were urinary megsin-positive,while positivity of urinary megsin in patients with non-renal diseases (n = 768) was equivalent (3.3%) to that of healthy individuals. Of note,when urinary megsin-positive patients with diabetic nephropathy (n = 71) were classified into four stages by their proteinuria and estimated glomerular filtration rate,urinary megsin excretion increased as the stage progressed up to stage 3A,suggesting correlation of that with mesangial expansion level. Urinary megsin decreased in the advanced stage,probably reflecting development of glomerulosclerosis. CONCLUSION: We established a high-sensitive megsin ELISA,which detects urinary megsin in some patients with renal diseases and in only a few healthy subjects. Megsin ELISA may be a novel diagnostic tool for renal diseases. View Publication

Neural tissue formation is induced by growth factors that activate networks of signal transduction cascades that ultimately lead to the expression of early neural genes,including transcription factors of the SoxB family. Here,we report that fibroblast growth factor (FGF)-induced Erk1/2 (Mapk3 and Mapk1,respectively) mitogen-activated protein kinase (MAPK),but not phosphatidylinositol 3'-OH kinase (PI3K,Pik3r1),signalling is required for neural specification in mouse embryonic stem (ES) cells and in the chick embryo. Further,blocking Erk1/2 inhibits the onset of key SoxB genes in both mouse ES cells (Sox1) and chick embryos (Sox2 and Sox3) and,in both contexts,Erk1/2 signalling is required during only a narrow time window,as neural specification takes place. In the absence of Erk1/2 signalling,differentiation of ES cells stalls following Fgf5 upregulation. Using differentiating ES cells as a model for neural specification,we demonstrate that sustained Erk1/2 activation controls the transition from an Fgf5-positive,primitive ectoderm-like cell state to a neural progenitor cell state without attenuating bone morphogenetic protein (BMP) signalling and we also define the minimum period of Erk1/2 activity required to mediate this key developmental step. Together,these findings identify a conserved,specific and stage-dependent requirement for Erk1/2 signalling downstream of FGF-induced neural specification in higher vertebrates and provide insight into the signalling dynamics governing this process. View Publication

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However,the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1,the only known histone H3 lysine 79 (H3K79) methyltransferase,has been shown to interact with multiple MLL fusion partners including AF9,ENL,AF10,and AF17. In this study,we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9,we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis,suggesting the involvement of Dot1 in survival pathways. In summary,our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target. View Publication

Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless,a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process,including hypoxic stress,fibrosis,and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions,leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology,demonstrating that the phased combination of TGF? inhibitor SB431542,Rho kinase inhibitor Y27632,and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction,providing a crucial platform for discovering therapeutic targets for ischemic heart disease. Engineered human models of hearts are needed to study pathology and repair. Here,the authors develop a model which replicates the phased remodelling process. The model is then used to study signalling pathway modulators for their therapeutic potential in a mini-repair model. View Publication

SummaryThe BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS),a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits,ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes,little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/? induced pluripotent stem cells to model CNCC specification,we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT),a process necessary for CNCC delamination and migration from the neural tube. Furthermore,wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level,these EMT enhancers contain binding motifs for ZIC2,and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers,ZIC2 relocates to neuronal enhancers,triggering aberrant neuronal gene activation. In mice,deletion of Zic2 impairs NCC delamination,while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. Graphical abstract ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition,a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers,resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. View Publication

Chagas disease,caused by the parasite Trypanosoma cruzi,affects millions of people globally. Unfortunately,the available treatment options,especially for the chronic stage of the disease,are suboptimal. Given the chronic nature of the disease and the elusive nature of the parasite,there is a high need for new and safer drugs that deliver sterile cure. Posaconazole was a promising lead in the drug discovery pipeline but ultimately failed in clinical trials due to patient relapses. This failure illustrates the need for a drug screening assay that can predict sterile cure by assessing recrudescence after treatment. Here,we used human induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) as host cells for T. cruzi. The iMACs were highly susceptible to infection by the parasites. By combining red fluorescent protein (RFP)-expressing iMACs with mNeonGreen-expressing T. cruzi,we were able to monitor the dynamics of the infection through live cell imaging. The activity of the compounds benznidazole and posaconazole was consistent with the results of an established infection system using mouse primary macrophages. The post-mitotic nature of iMACs makes them suitable host cells for long-term assays needed to assess recrudescence of parasites. Moreover,their human origin,stable genetic background,and capacity for genetic modification make the iMACs excellent host cells for studying host-pathogen interaction. Author summaryThe parasite Trypanosoma cruzi,the causative agent of Chagas disease,is a global health concern affecting millions each year. Infection with T. cruzi can cause chronic disease,often remaining asymptomatic for decades before resulting in severe cardiac or gastro-intestinal pathologies. To date,only benznidazole and nifurtimox are used for treatment of the infection,but both drugs are suboptimal for curing the chronic stage. Posaconazole showed great promise in preclinical studies but failed to achieve sterile cure in clinical trials,causing patient relapses. These disappointing results underline the need for drug screening assays able to predict sterile cure by evaluating recrudescence post-treatment. We used human induced pluripotent stem cell derived macrophages as host cells for T. cruzi and testing of trypanocidal compounds. This model can be used for long-term in vitro screening assays to find new drug candidates against Chagas disease. The human origin of these cells combined with the possibility of upscaling their production make them great host cells for drug screening campaigns. View Publication

Cellular plasticity contributes to the regenerative capacity of plants,invertebrates,teleost fishes and amphibians. In vertebrates,differentiated cells are known to revert into replicating progenitors,but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells,we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast,direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming,the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states,notably cancer. View Publication