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

Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors,nerve growth factor and brain-derived neurotrophic factor,share substantial amino acid sequence identity. We have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene,neurotrophin-3,that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain,suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development. View Publication

Background: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4,KChIP2 and Kv4.3 were quantified from BCs. Results: BCs exhibited spontaneous beating (60.5??2.6bpm) and maximum-diastolic-potential (MDP) of 67.8??0.8mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7??1.9 pA/pF at +40mV; n=14). Recovery of Ito from inactivation (at -80mV) showed slow kinetics (??1=200??110ms (12%) and ??2=2380??240ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion: The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype. ?? 2013 Elsevier Ltd. View Publication

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system,but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells,we assessed fucosylated (2′FL and 3FL),sialylated (3′SL and 6′SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity,to stimulate the secretion of chemokines from intestinal epithelial cells,and to modulate cytokine release from LPS-activated dendritic cells (DCs),M1 macrophages (MØs),and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10,CCL20 and CXCL8 from intestinal epithelial cells,promoted the secretion of several cytokines (including IL-10,IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs,and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus,3′SL and 6′SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively,our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life. View Publication

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless,dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS),taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated,the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition,transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together,our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Subject terms: Neuroimmunology,Innate immunity,Neural stem cells View Publication

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however,unipotent neutrophil progenitors are not well defined. Here,we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly,we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models,including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases,including cancer. View Publication

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes,ARGFX,CPHX1,CPHX2,DPRX,DUXA,DUXB,NOBOX,TPRX1 and TPRX2,were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1,CPHX2,ARGFX) or repressors (DPRX,DUXA,TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development. View Publication

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence,comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end,here,we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages,including bone,muscle,and heart. We defined the extrinsic signals controlling each binary lineage decision,enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%???99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in??vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation,a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively,this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. Video Abstract View Publication

Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes,in vitro testing of novel therapies,and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward,scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study,we describe the use of a gammaretroviral vector encoding a fluorescent marker,mRFP1,to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming. View Publication

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date,most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-β (TGFβ) signaling pathways,and none provide for replacement of FGF/TGFβ ligands with chemical compounds. The Wnt/β-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however,the role of Wnt/β-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/β-catenin signaling in mouse embryonic stem cells,we identified a compound,ID-8,that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8,we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFβ activation. These culture conditions do not include xenobiotic supplements,serum,serum replacement,or albumin. Using this culture system,we have shown that several human pluripotent cell lines maintained pluripotency (textgreater20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds. View Publication

SummaryMolecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation,presence of a putative ciliary margin zone,emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data,we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development,and to show that chromatin accessibility is highly correlated to the developing human retina,but with some differences in the temporal emergence and abundance of some of the retinal neurons. Graphical abstract Highlights•Single cell analyses reveal putative ciliary margin (pCM) presence in retinal organoids•PCM harbors early RPCs which differentiate to late RPCs and retinal neurons•Single cell ATAC-seq data reveal novel TF binding motifs in RPCs and retinal neurons•RO development largely recapitulates retinogenesis Genetics; Molecular biology; Neuroscience; Cell biology; Omics View Publication

Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies,but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present,it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can recapitulate the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC,with decreased cell viability,impaired mitochondrial and metabolic function,impaired calcium handling,decreased antioxidant pathway activity,and increased reactive oxygen species production. Taken together,our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC. View Publication

The thyroid hormone,3,5,3'-Triiodo-L-thyronine (T3),is essential for growth,differentiation,and regulation of metabolic functions in multicellular organisms,although the specific mechanisms of this control are still unknown. In this study,treatment of a human pancreatic duct cell line (hPANC-1) with T3 blocks cell growth by an increase of cells in G(0)/G(1) cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a beta-cell-like phenotype,we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion,our results suggest,for the first time,that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC-1) into pancreatic-beta-cell-like cells. These findings have important implications in cell-therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes. View Publication