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

BACKGROUND: Conventional regulatory T cells (Tregs) can suppress human immunodeficiency virus type 1 (HIV-1)-specific immune responses but cannot control immune activation in primary HIV infection. Here,we characterized Treg subsets,using recently defined phenotypic delineation,and analyzed the relative contribution of cell subsets to the production of immunosuppressive cytokines in primary HIV infection. METHODS: In a longitudinal prospective study,ex vivo phenotyping of fresh peripheral blood mononuclear cells from patients with primary HIV infection was performed at baseline and month 6 of follow-up to characterize Treg subsets,immune activation,and cytokine production in isolated CD4(+) T cells. RESULTS: The frequency of CD4(+)CD25(+)CD127(low) Tregs and the distribution between the naive,memory,and activated/memory Treg subsets was similar in patients and healthy donors. However,Tregs from patients with primary HIV infection showed peculiar phenotypic profiles,such as elevated FoxP3,ICOS,and CTLA-4 expression,with CTLA-4 expression strikingly increased in all Treg subsets both at baseline and month 6 of follow-up. The great majority of interleukin 10 (IL-10)-producing CD4(+) T cells were FoxP3(neg) (ie,Tr1-like cells). In contrast to conventional Tregs,Tr1-like cells were inversely correlated with immune activation and not associated with lower effector T-cell responses. CONCLUSION: FoxP3(neg) Tr1-like cells-major contributors to IL-10 production-may have a beneficial role by controlling immune activation in early HIV infection. View Publication

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans,with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models,indicating that an appropriate adaptive immune response is required for the control of the disease. However,cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients,the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points,downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice. View Publication

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore,overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria,committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU,TMZ,and MMS,which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy. View Publication

The notion that gliomas could originate from mutated glial precursor cells highlights the possibility of modulating the proliferative and migratory behaviour of glioma cells by acting on the molecular mechanisms operative during the development of the Central Nervous System (CNS),but absent in the normal adult brain. We show that the GL15 glioblastoma derived human cell line displays a high expression of nestin which,combined with the previously demonstrated high expression of vimentin,constitutes a characteristic of astrocyte restricted precursors. We also show that,in analogy with some leukaemia cells,GL15 cells display the constitutively phosphorylated form of Janus kinase 2 (JAK2),a tyrosine kinase expressed during CNS development but undetectable in the normal adult brain. The constitutive activation of JAK2 does not result from chromosomal aberrations involving the JAK2 gene,but most probably from abnormally activated transduction systems operative in glioblastoma cells. We then investigated the effects of tyrphostin AG490,an inhibitor of JAK2 autophosphorylation,on GL15 cell growth. In the absence of exogenous growth factors and cytokines,10 microM tyrphostin AG490 induces an S phase arrest,combined with a partial impairment of the G2 phase of the cell cycle. The abnormally activated JAK2 could then potentially represent a target for a selective pharmacological approach in glioblastoma cells in which a combination of glial precursor characteristics and genetic alterations occurs. View Publication

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs),and their neuronal progeny,will play an important role in disease modeling,drug screening tests,central nervous system development studies,and may even become valuable for regenerative medicine treatments. Nonetheless,it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs,and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here,we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days,and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS),leaving the NP population nearly free of PSCs. View Publication

Nature Communications 6,(2015). doi:10.1038/ncomms6999 View Publication

Traumatic spinal cord injury results in persistent disability due to disconnection of surviving neural elements. Neural stem cell transplantation has been proposed as a therapeutic option,but optimal cell type and mechanistic aspects remain poorly defined. Here,we describe robust engraftment into lesioned immunodeficient mice of human neuroepithelial stem cells derived from the developing spinal cord and maintained in self-renewing adherent conditions for long periods. Extensive elongation of both graft and host axons occurs. Improved functional recovery after transplantation depends on neural relay function through the grafted neurons,requires the matching of neural identity to the anatomical site of injury,and is accompanied by expression of specific marker proteins. Thus,human neuroepithelial stem cells may provide an anatomically specific relay function for spinal cord injury recovery. View Publication

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular,human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency,their self-renewal potential,and their ability to create patient-specific cell lines. Unfortunately,pluripotent stem cell-derived CMs are immature,with characteristics more closely resembling fetal CMs than adult CMs,and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation,as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold,compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes,Junctin,CaV1.2,NCX1,HCN4,SERCA2a,Triadin,and CASQ2. Consistent with this,we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine,likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together,these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease. View Publication

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics,epigenomics,and bioinformatics that inform on genetic pathways directing tissue development and function. When possible,population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American,Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype,verified teratoma formation,pluripotency biomarkers,and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural,pancreatic,and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential. View Publication

Spinal motor neurons (MNs) represent a highly vulnerable cellular population,which is affected in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In this study,we show that the heterozygous loss of SYT13 is sufficient to trigger a neurodegenerative phenotype resembling those observed in ALS and SMA. SYT13+/? hiPSC-derived MNs displayed a progressive manifestation of typical neurodegenerative hallmarks such as loss of synaptic contacts and accumulation of aberrant aggregates. Moreover,analysis of the SYT13+/? transcriptome revealed a significant impairment in biological mechanisms involved in motoneuron specification and spinal cord differentiation. This transcriptional portrait also strikingly correlated with ALS signatures,displaying a significant convergence toward the expression of pro-apoptotic and pro-inflammatory genes,which are controlled by the transcription factor TP53. Our data show for the first time that the heterozygous loss of a single member of the synaptotagmin family,SYT13,is sufficient to trigger a series of abnormal alterations leading to MN sufferance,thus revealing novel insights into the selective vulnerability of this cell population. View Publication

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study,we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg,HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV,thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection,we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients,we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. View Publication