The role of ARX in human pancreatic endocrine specification
The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG),affecting many cell types including those of the pancreas. Indeed,XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin,insulin,and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development,we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected),glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult $$-cells. Differentiated ARX knockout cells upregulated PAX4,NKX2.2,ISL1,HHEX,PCSK1,PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide,somatostatin,glucagon and insulin positive cells from hESCs.
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05850
05857
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07923
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Dispase (1 U/mL)
mTeSR™1
mTeSR™1
Kitamura T et al. (AUG 1989)
Journal of cellular physiology 140 2 323--34
Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin.
We have established a novel cell line,designated as TF-1,from a patient with erythroleukemia,which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1,but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion,and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I,the morphological and cytochemical features,and the constitutive expression of globin genes,indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate,TF-1 entered two different pathways. Specifically,hemin and delta-aminolevulinic acid induced hemoglobin synthesis,whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary,TF-1 is a cell line of immature erythroid origin that requires GM-CSF,IL-3,or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3,GM-CSF,and EPO or the signal transduction of these hemopoietic growth factors.
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产品号#：
02503
02603
02532
02832
02625
02630
02830
02731
02931
产品名：
Grandy RA et al. (FEB 2015)
Molecular and Cellular Biology 36 December MCB.00877--15
Genome-wide Studies Reveal that H3K4me3 Modification in Bivalent Genes is Dynamically Regulated During the Pluripotent Cell Cycle and Stabilized Upon Differentiation
textlessptextgreaterStem cell phenotypes are reflected by post-translational histone modifications,and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs),bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remains to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2,mitotic,and G1 phases of the cell cycle,we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle–dependent fashion. Interestingly,bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore,the histone-modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle–independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in maintenance of pluripotency.textless/ptextgreater
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05850
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产品名：
mTeSR™1
mTeSR™1
Wculek SK and Malanchi I (DEC 2015)
Nature advance on 7582 413--417
Neutrophils support lung colonization of metastasis-initiating breast cancer cells
Despite progress in the development of drugs that efficiently target cancer cells,treatments for metastatic tumours are often ineffective. The now well-established dependency of cancer cells on their microenvironment suggests that targeting the non-cancer-cell component of the tumour might form a basis for the development of novel therapeutic approaches. However,the as-yet poorly characterized contribution of host responses during tumour growth and metastatic progression represents a limitation to exploiting this approach. Here we identify neutrophils as the main component and driver of metastatic establishment within the (pre-)metastatic lung microenvironment in mouse breast cancer models. Neutrophils have a fundamental role in inflammatory responses and their contribution to tumorigenesis is still controversial. Using various strategies to block neutrophil recruitment to the pre-metastatic site,we demonstrate that neutrophils specifically support metastatic initiation. Importantly,we find that neutrophil-derived leukotrienes aid the colonization of distant tissues by selectively expanding the sub-pool of cancer cells that retain high tumorigenic potential. Genetic or pharmacological inhibition of the leukotriene-generating enzyme arachidonate 5-lipoxygenase (Alox5) abrogates neutrophil pro-metastatic activity and consequently reduces metastasis. Our results reveal the efficacy of using targeted therapy against a specific tumour microenvironment component and indicate that neutrophil Alox5 inhibition may limit metastatic progression.
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产品号#：
01700
01705
01702
产品名：
ALDEFLUOR™ 试剂盒
ALDEFLUOR™ DEAB试剂, 1.5 mM, 1 mL
ALDEFLUOR™检测缓冲液
Gren ST et al. ( 2015)
PloS one 10 12 e0144351
A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets.
Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-,intermediate CD14++CD16+,and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16,and unique gene expression profile. So far,the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study,the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers,and proteins involved in immune regulation. Within the three human monocyte subsets,our descriptive findings show multimodal expression of key immune response genes,such as CD40,NFⱪB1,RELA,TLR4,TLR8 and TLR9. Furthermore,we discovered one subgroup of cells within the classical monocytes,which showed alterations of 22 genes e.g. IRF8,CD40,CSF1R,NFⱪB1,RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes,respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation,independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance,and can be useful in unravelling inter-cellular variation in leukocyte populations,identifying subpopulations involved in disease pathogenesis and help tailor new therapies.
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Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.
Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However,upgrading them to pluripotency confers refractoriness toward senescence,higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling,such as Down syndrome or $\$-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing,feeder-dependent culture. Here,we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium,a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4,Nanog,Sox2,SSEA-1,SSEA-4,TRA-1-60,TRA-1-81 in a pattern typical for human primed PSC. Additionally,the cells formed teratomas,and were deemed pluripotent by PluriTest,a global expression microarray-based in-silico pluripotency assay. However,we found that the PluriTest scores were borderline,indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology,non-integrating reprogramming and chemically defined culture are more acceptable.
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产品号#：
05850
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07180
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27147
07191
07930
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85850
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100-1061
07952
100-0763
产品名：
Vitronectin XF™
CellAdhere™ 稀释缓冲液
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
mTeSR™1
mTeSR™1
CryoStor® CS10
CryoStor® CS10
Vitronectin XF™
Higuchi A et al. (DEC 2015)
Scientific Reports 5 18136
Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity
The tentative clinical application of human pluripotent stem cells (hPSCs),such as human embryonic stem cells and human induced pluripotent stem cells,is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore,we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture,whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture.
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产品号#：
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产品名：
mTeSR™1
mTeSR™1
Kang H et al. (DEC 2015)
Molecular therapy. Nucleic acids 4 October e268
CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.
The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here,we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA,12.5% of cell colonies demonstrated CCR5 editing,of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells,we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells,including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication,macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro,and generation of HIV-resistant cells for potential therapeutic applications.
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产品号#：
05850
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84445
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mTeSR™1
mTeSR™1
McCabe KL et al. (DEC 2015)
PloS one 10 12 e0145266
Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.
AIM To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. MATERIALS AND METHODS Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology,expression of corneal endothelial markers,and microarray analysis of gene expression. RESULTS hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells,expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPase$\$1 (ATPA1) on the apical surface in monolayer culture,and produced the key proteins of Descemet's membrane,Collagen VIII$\$1 and VIII$\$2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. CONCLUSION hESC-CECs are morphologically similar,express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.
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Self-reactive IgE exacerbates interferon responses associated with autoimmunity.
Canonically,immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE),IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs),a type of cell of the immune system linked to viral defense,which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE,followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses.
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产品号#：
17953
17953RF
100-0710
产品名：
EasySep™人CD8+ T细胞分选试剂盒
RoboSep™ 人CD8+ T细胞分选试剂盒
EasySep™人CD8+ T细胞分选试剂盒
Chandran A et al. (DEC 2015)
Frontiers in cellular and infection microbiology 5 90
Mycobacterium tuberculosis Infection Induces HDAC1-Mediated Suppression of IL-12B Gene Expression in Macrophages.
Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live,but not killed,virulent MTB. Additionally,we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live,virulent MTB,and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases,and by knockdown of crucial transcriptional regulators,we demonstrate that protein kinase-A (PKA),CREB,and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis,we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live,virulent MTB to subvert the immune system to survive and replicate in the host.
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产品号#：
70025
70025.1
70025.2
70025.3
70047
70047.1
70047.2
70048
70048.1
70048.2
产品名：
冻存的人外周血单个核细胞
冻存的人外周血单个核细胞
冻存的人外周血单个核细胞
冻存的人外周血单个核细胞
Ignatius Irudayam J et al. (DEC 2015)
Data in Brief 5 871--878
Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells
Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs) to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5),hepatoblast (day 15) and hepatocyte-like cells (day 21) were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type,the hepatic cells (days 15 and 21) had significantly higher expression of acute phase protein genes including complement factors,coagulation factors,serum amyloid A and serpins. Furthermore,hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1,IL1R1,IL1RAP,IL2RG,IL6R,IL6ST and IL10RB. These cells also produced CCL14,CCL15,and CXCL- 1,2,3,16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors,CXCR4 and CXCR7,than that of hepatic cells. Sirtuin family of genes involved in aging,inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF) family as well as downstream signaling factors TRAF2,TRAF4,FADD,NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.
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EasySep™小鼠TIL（CD45）正选试剂盒
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