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

The recently developed ability to differentiate primary adult stem cells and induced pluripotent stem cells (iPSCs) into cardiomyocytes is providing unprecedented opportunities to produce an unlimited supply of cardiomyocytes for use in patients with heart disease. Here,we examine the evidence for the preclinical use of such cells for successful heart regeneration. We also describe advances in the identification of new cardiac molecular and cellular targets to induce proliferation of cardiomyocytes for heart regeneration. Such new advances are paving the way for a new innovative drug development process for the treatment of heart disease. View Publication

Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P,the immediate precursor of PtdIns-4,5-P2. Here we report the cloning of a novel,ubiquitously expressed PtdIns 4-kinase (PI4Kbeta). The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene. The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli,and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The biochemical properties of PI4Kbeta are characteristic of a type III enzyme. Interestingly,both recombinant PI4Kbeta and the endogenous protein are inhibited by 150 nM wortmannin,suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi,S.,Catt,J. K.,and Balla,T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,5317-5321). View Publication

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC),which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover,immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids,which will advance the use of kidney organoids for transplantation research. View Publication

Propionic acidemia (PA) is a metabolic disorder caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC) due to mutations in the PCCA or PCCB genes,which encode the two PCC subunits. PA may lead to several types of cardiomyopathy and has been linked to cardiac electrical abnormalities such as QT interval prolongation,life-threatening arrhythmias,and sudden cardiac death. To gain insights into the mechanisms underlying PA-induced proarrhythmia,we recorded action potentials (APs) and ion currents using whole-cell patch-clamp in ventricular-like induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) from a PA patient carrying two pathogenic mutations in the PCCA gene (p.Cys616_Val633del and p.Gly477Glufs*9) (PCCA cells) and from a healthy subject (healthy cells). In cells driven at 1 Hz,PCC deficiency increased the latency and prolonged the AP duration (APD) measured at 20% of repolarization,without modifying resting membrane potential or AP amplitude. Moreover,delayed afterdepolarizations appeared at the end of the repolarization phase in unstimulated and paced PCCA cells. PCC deficiency significantly reduced peak sodium current (INa) but increased the late INa (INaL) component. In addition,L-type Ca2+ current (ICaL) density was reduced,while the inward and outward density of the Na+/Ca2+ exchanger current (INCX) was increased in PCCA cells compared to healthy ones. In conclusion,our results demonstrate that at the cellular level,PCC deficiency can modify the ion currents controlling cardiac excitability,APD,and intracellular Ca2+ handling,increasing the risk of arrhythmias independently of the progressive late-onset cardiomyopathy induced by PA disease. View Publication

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO,Ac-DEVD-CHO,Ac-YVAD-CHO,t-butoxycarbonyl-IETD-CHO,and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes,with dissociation constants ranging from 75 pM to {\textgreater}10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor,with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki {\textless} 20 nM) and selective inhibitor of Group I caspases (caspase-1,-4,and -5) and most Group III caspases (caspase-8,-9,and -10),suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response. View Publication

A large number of human embryonic stem cell (hESC) lines have been derived worldwide since the first hESC line establishment in 1998. Despite many common characteristics,most important of which is the pluripotency,hESC lines vary significantly in their transcriptional profiles,genetic,and epigenetic state. These differences may arise both from individual genetics of the cell lines and from variations in their handling such as isolation and cultivation. In order to minimize the latter differences,the standardized protocols of cultivation and inter-laboratory comprehensive studies should be performed. In this report,we summarized our experience of derivation and characterization of hESC lines as well as of adaptation of hESCs to novel cultivation protocols. We have successfully derived five hESC lines and characterized them by previously established criteria,including expression of specific markers and the capacity to differentiate both in vitro and in vivo. Four of these lines,namely hESM01-04,were initially derived using mouse fibroblasts as a feeder and currently are maintained under feeder-free,serum-free conditions using mTeSR1 and Matrigel. The fifth line,hESMK05 was derived in feeder-free,serum-free conditions using mTeSR1 and Matrigel. Cell lines retain their pluripotent status and normal karyotype for more than 70 passages and are available to the scientific community. View Publication

Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development,but these events are essentially impossible to study at the molecular level in vivo in humans. The first EMT event that has been described morphologically in human development occurs just prior to generation of the primitive streak. Because human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are thought to most closely resemble cells found in epiblast-stage embryos prior to formation of the primitive streak,we sought to determine whether this first human EMT could be modeled in vitro with pluripotent stem cells. The data presented here suggest that generating embryoid bodies from hESCs or hiPSCs drives a procession of EMT events that can be observed within 24-48 hours after EB generation. These structures possess the typical hallmarks of developmental EMTs,and portions also display evidence of primitive streak and mesendoderm. We identify PTK7 as a novel marker of this EMT population,which can also be used to purify these cells for subsequent analyses and identification of novel markers of human development. Gene expression analysis indicated an upregulation of EMT markers and ECM proteins in the PTK7+ population. We also find that cells that undergo this developmental EMT retain developmental plasticity as sorting,dissociation and re-plating reestablishes an epithelial phenotype. View Publication

BACKGROUND: The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them,fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However,downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study,we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2,an inhibitor of protein kinase C (PKC),GF109203X (GFX),increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β),suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator,phorbol 12-myristate 13-acetate whereas Gö6976,a selective inhibitor of PKCα,β,and γ isoforms could not counteract the effect of PMA. Intriguingly,functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ,PKCε,and ζ,the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ,and the phosphorylation of AKT was reduced by PKCε in hPS cells. CONCLUSIONS/SIGNIFICANCE: Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT,mitogen-activated protein kinase/ERK-1/2 kinase (MEK),PKC/ERK-1/2 kinase,and PKC/GSK-3β. Addition of GFX with a MEK inhibitor,U0126,in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells. View Publication

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4,MEF2C,and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However,GMT alone appears insufficient in human fibroblasts,at least in vitro. Here,we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells,fetal heart,and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming,including sarcomere formation,calcium transients,and action potentials,although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore,we found that transforming growth factor β signaling was important for,and improved the efficiency of,human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage,and lay the foundation for future refinements in vitro and in vivo. textcopyright 2013 The Authors. View Publication

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors,particularly radial glia (RG),are rare and are defined by a combination of intracellular markers,position and morphology. To circumvent these problems,we developed Fixed and Recovered Intact Single-cell RNA (FRISCR),a method for profiling the transcriptomes of individual fixed,stained and sorted cells. Using FRISCR,we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB,and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles,an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers. View Publication