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

Human induced pluripotent stem cell (hiPSC) derived neurons are powerful tools to model disease biology in the drug development space. Here we leveraged a spectrum of neurophysiological tools to characterize iPSC-derived NGN2 neurons. Specifically,we applied these technologies to detect phenotypes associated with presynaptic dysfunction and rescue in NGN2 neurons lacking a synaptic vesicle associated protein MUNC18-1,encoded by syntaxin binding protein 1 gene (STXBP1). STXBP1 homozygous knock out NGN2 neurons lacked miniature post synaptic currents and demonstrated disrupted network bursting as assayed with multielectrode array and calcium imaging. Furthermore,knock out neurons released less glutamate into culture media,consistent with a presynaptic deficit. These synaptic phenotypes were rescued by reconstitution of STXBP1 protein by AAV transduction in a dose-dependent manner. Our results identify a complementary suite of physiological methods suitable to examine the modulation of synaptic transmission in human neurons. View Publication

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals,we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs),which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency,the NANOG-OCT4 Complex,and its associated network are significantly enriched for regulatory variants with large effects,suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks,shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity,and suggests that genetic factors may contribute to cell state transitions in human iPSC lines. Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Here,authors show that pluripotency and self-renewal processes have a high level of regulatory complexity and suggest that genetic factors contribute to cell state transitions in human iPSC lines. View Publication

Oligodendrocytes are the myelinating cells of the central nervous system. Regulation of the early stages of oligodendrocyte development is critical to the function of the cell. Specifically,myelin sheath formation is an energetically demanding event that requires precision,as alterations may lead to dysmyelination. Fatty acid β‐oxidation has been shown to be critical for the function of oligodendrocytes. We previously showed that myeloid cell leukemia‐1 (MCL‐1),a well‐characterized anti‐apoptotic protein,is required for the development of murine oligodendrocytes in vivo. Further,MCL‐1 regulates long‐chain fatty acid β‐oxidation in cancer cells through its interaction with Acyl‐CoA synthetase long‐chain family member 1 (ACSL1),an enzyme responsible for the conversion of free long‐chain fatty acids into fatty acyl‐CoA esters. Here,we introduce an in vitro system to isolate human stem cell‐derived oligodendrocyte progenitor cells (OPCs) and investigate the involvement of MCL‐1 during human oligodendrocyte development. Using this system,we pharmacologically inhibited MCL‐1 in OPCs to investigate its non‐apoptotic function at this developmental stage. We also used a motor neuron‐oligodendrocyte co‐culture system to examine the downstream effects of MCL‐1 at later developmental stages when oligodendrocytes begin to contact axons and generate myelin. We demonstrate that the mitochondrial network changes in human oligodendrocyte development resemble those reported in mouse tissue. Our findings point to MCL‐1 as a critical factor essential for proper oligodendrocyte morphogenesis. A unified model of oligodendrocyte differentiation from human embryonic stem cells revealed that MCL‐1 is critical for regulating the expression of oligodendrocyte‐related genes and the morphogenesis of myelinating oligodendrocytes. View Publication

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo,SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via α6β1 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEC) regulate SVZ cells neurogenic capacities,cocultures of SVZ neurospheres and primary BEC,both obtained from C57BL/6 mice,were performed. The involvement of laminin-integrin interactions in SVZ homeostasis was tested in three ways. Firstly,SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (CHX) prior to coculture,a treatment expected to decrease membrane proteins. Secondly,SVZ cells were cocultured with BEC in the presence of an anti-α6 integrin neutralizing antibody. Thirdly,BEC were cultured with β1-/- SVZ cells. We showed that contact with BEC supports,at least in part,proliferation and stemness of SVZ cells,as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEC. These effects are dependent on BEC-derived laminin binding to α6β1 integrin and are decreased in cocultures incubated with anti-α6 integrin neutralizing antibody and in cocultures with SVZ β1-/- cells. Moreover,BEC-derived laminin sustains stemness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving α6β1 integrin binding to laminin,contribute to SVZ cell proliferation and stemness. View Publication

B-cell functions in antitumor immunity are not well understood. In this study,we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag),D5 mouse melanoma,or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments,spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors),IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells. View Publication

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1,also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution,with,at any given time,a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage,likely under pressure of host restriction factors,which act notably by silencing L1 expression during early embryogenesis. Here,we demonstrate that in human embryonic stem (hES) cells,KAP1 (KRAB [Kruppel-associated box domain]-associated protein 1),the master cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs) previously implicated in the restriction of endogenous retroviruses,represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 million and 7.6 million years ago. In mice,we documented a similar chronologically conditioned pattern,albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP,suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hES cells induced the expression of KAP1-bound L1 elements,but their younger,human-specific counterparts (L1Hs) were unaffected. Instead,they were stimulated by depleting DNA methyltransferases,consistent with recent evidence demonstrating that the PIWI-piRNA (PIWI-interacting RNA) pathway regulates L1Hs in hES cells. Altogether,these data indicate that the early embryonic control of L1 is an evolutionarily dynamic process and support a model in which newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms before KAP1-recruiting protein repressors are selected. View Publication