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

The association between type 2 diabetes (T2D) pathogenesis and immune-mediated tissue damage and insulin resistance suggests that T2D patients might benefit from the suppression of pathogenic inflammation. Foxp3+ Treg cells are crucial suppressors of inflammation,but the differentiation of Foxp3+ Treg cells is not static and is subject to conversion into IL-17-producing Th17-like cells upon receiving external signals. In this study,we examined the production of IL-17 by Treg cells. Compared to non-T2D controls,T2D patients presented significantly higher levels of IL-17-expressing cells in both Foxp3- CD4 T cells and Foxp3+ Treg cells. The frequencies of IL-17-nonexpressing Foxp3+ Treg cells,on the other hand,were not changed. Interestingly,IL-17-expressing Foxp3+ Treg cells were mutually exclusive from IL-10-expressing and TGF-$\beta$-expressing Foxp3+ Treg cells,suggesting that multiple subpopulations exist within the Foxp3+ Treg cells from T2D patients. In T2D patients,the frequencies of IL-17-expressing Foxp3+ Treg cells were positively correlated with the body mass index (BMI) and the HbA1c levels of T2D patients. The frequencies of IL-10-expressing Treg cells,on the other hand,were inversely associated with the BMI of both non-T2D controls and T2D patients. In addition,the suppressive activity of Treg cells was significantly lower in T2D patients than in non-T2D controls. Together,our study uncovered a dysregulation in Foxp3+ Treg cells from T2D patients,characterized by high IL-17 expression and low suppression activity. View Publication

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However,such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here,we present SNIPE,a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells,which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here,the authors present SNIPE,a marker-free method that selects cells based on DNA sequence,enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells. View Publication

Imatinib is a novel tyrosine kinase inhibitor used for the treatment of Philadelphia chromosome-positive leukemias and other malignancies. Side effects are mostly moderate; however,a dose-dependent hematologic toxicity affecting all hematopoietic lineages is observed clinically. The aim of this study was to investigate the effect of imatinib on normal hematopoietic stem and progenitor cells in vitro. A dose-dependent decrease in proliferation potential was found when CD34+ cells were expanded in serum-free medium supplemented with 6 growth factors and imatinib. Functionally,a decrease in colony-forming capacity was observed under increasing doses of imatinib. However,no such effect on more primitive cobblestone area-forming cells was detectable. Both withdrawal of stem cell factor from our expansion cultures or functional inhibition of c-kit led to a similar degree of inhibition of expansion,whereas the effect of imatinib was substantially greater at all dose levels tested. These data suggest a significant inhibitory effect of imatinib on normal CD34+ progenitor (but not stem) cells that is largely independent of c-kit signaling. View Publication

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4,Sox2,Klf4,and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming,but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs,which resembled ground-state mouse ES cells in growth properties,gene expression,and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome. View Publication

BACKGROUND Plasma cell neoplasms (PCNs) encompass a spectrum of disorders including monoclonal gammopathy of undetermined significance,smoldering myeloma,plasma cell myeloma,and plasma cell leukemia. Molecular subtypes have been defined by recurrent cytogenetic abnormalities and somatic mutations that are prognostic and predictive. Karyotype and fluorescence in situ hybridization (FISH) have historically been used to guide management; however,new technologies and markers raise the need to reassess current testing algorithms. METHODS We convened a panel of representatives from international clinical laboratories to capture current state-of-the-art testing from published reports and to put forward recommendations for cytogenomic testing of plasma cell neoplasms. We reviewed 65 papers applying FISH,chromosomal microarray (CMA),next-generation sequencing,and gene expression profiling for plasma cell neoplasm diagnosis and prognosis. We also performed a survey of our peers to capture current laboratory practice employed outside our working group. RESULTS Plasma cell enrichment is widely used prior to FISH testing,most commonly by magnetic bead selection. A variety of strategies for direct,short- and long-term cell culture are employed to ensure clonal representation for karyotyping. Testing of clinically-informative 1p/1q,del(13q) and del(17p) are common using karyotype,FISH and,increasingly,CMA testing. FISH for a variety of clinically-informative balanced IGH rearrangements is prevalent. Literature review found that CMA analysis can detect abnormalities in 85-100{\%} of patients with PCNs; more specifically,in 5-53{\%} (median 14{\%}) of cases otherwise normal by FISH and cytogenetics. CMA results in plasma cell neoplasms are usually complex,with alteration counts ranging from 1 to 74 (median 10-20),primarily affecting loci not covered by FISH testing. Emerging biomarkers include structural alterations of MYC as well as somatic mutations of KRAS,NRAS,BRAF,and TP53. Together,these may be measured in a comprehensive manner by a combination of newer technologies including CMA and next-generation sequencing (NGS). Our survey suggests most laboratories have,or are soon to have,clinical CMA platforms,with a desire to move to NGS assays in the future. CONCLUSION We present an overview of current practices in plasma cell neoplasm testing as well as an algorithm for integrated FISH and CMA testing to guide treatment of this disease. View Publication

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries,but reprogrammed neurons are dissociated and spatially disorganized during transplantation,rendering poor cell survival,functionality and engraftment in vivo. Here,we present the design of three-dimensional (3D) microtopographic scaffolds,using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming,neural network establishment and support neuronal engraftment into the brain. Scaffold-supported,reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices,showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells,and allowed delivery of multiple neuronal subtypes. Thus,3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance. View Publication

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication

During human brain development,multiple signaling pathways generate diverse cell types with varied regional identities. Here,we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor,neuronal,and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together,these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. View Publication

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized,the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV,an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013),productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation,even during early stages of infection,leading to progenitor depletion,disruption of the VZ,impaired neurogenesis,and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly. View Publication

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines,we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene,and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines,even in the absence of patient tissue. View Publication

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here,we show that three members of the Ccr4-Not complex,Cnot1,Cnot2,and Cnot3,play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts,these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs,Cnot1,Cnot2,and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead,they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly,these Cnot genes are also necessary for the maintenance of human ESCs,and silencing them mainly lead to TE and primitive endoderm differentiation. Together,our results indicate that Cnot1,Cnot2,and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs. View Publication