Tsolis KC et al. (JUN 2016)
Journal of Proteome Research 15 6 1995--2007
Proteome changes during transition from human embryonic to vascular progenitor cells
Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However,a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes,we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry,label-free quantification,and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation,which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining textgreater150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis.
View Publication
Self-organization of the human embryo in the absence of maternal tissues.
Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here,we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages,and morphogenetic rearrangements leading to generation of a bilaminar disc,formation of a pro-amniotic cavity within the embryonic lineage,appearance of the prospective yolk sac,and trophoblast differentiation. Using human embryos and human pluripotent stem cells,we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together,our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous,highlighting the remarkable and unanticipated self-organizing properties of human embryos.
View Publication
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Peters DT et al. (MAY 2016)
Development (Cambridge,England) 143 9 1475--81
Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.
Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro,but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal,we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray,and tested their ability to perform mature hepatocyte functions (albumin and urea secretion,cytochrome activity). By these measures,ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation.
View Publication
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Shinkuma S et al. (MAY 2016)
Proceedings of the National Academy of Sciences of the United States of America 113 20 5676--5681
Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.
Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining,leading to reading frame disruption. The approach is applicable to dominant negative disorders,which can be treated simply by knocking out the mutant allele,while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB),which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation,c.80688084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed,respectively,into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting,90% of the iPSCs were edited,and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition,we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.
View Publication
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Mansouri M et al. ( 2016)
Nature Communications 7 May 11529
Highly efficient baculovirus-mediated multigene delivery in primary cells
Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including,synthetic and structural biology,cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity,thus impeding unrestricted multigene expression. We developed MultiPrime,a modular,non-cytotoxic,non-integrating,baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types,including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming,and for genome editing and engineering by CRISPR/Cas9. Moreover,we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool,to deliver multiple genes for a wide range of applications in primary and established mammalian cells.
View Publication
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Maillet A et al. ( 2016)
Scientific reports 6 April 25333
Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes.
Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death,reactive oxygen species production,mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq,as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally,we show that CRISPR-Cas9-mediated disruption of TOP2B,a gene implicated in DIC in mouse studies,significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response.
View Publication
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Friesen TJ et al. (MAY 2016)
The Journal of Experimental Medicine 213 6 913--920
Recent thymic emigrants are tolerized in the absence of inflammation.
T cell development requires a period of postthymic maturation. Why this is the case has remained a mystery,particularly given the rigors of intrathymic developmental checkpoints,successfully traversed by only ∼5% of thymocytes. We now show that the first few weeks of T cell residence in the lymphoid periphery define a period of heightened susceptibility to tolerance induction to tissue-restricted antigens (TRAs),the outcome of which depends on the context in which recent thymic emigrants (RTEs) encounter antigen. After encounter with TRAs in the absence of inflammation,RTEs exhibited defects in proliferation,diminished cytokine production,elevated expression of anergy-associated genes,and diminished diabetogenicity. These properties were mirrored in vitro by enhanced RTE susceptibility to regulatory T cell-mediated suppression. In the presence of inflammation,RTEs and mature T cells were,in contrast,equally capable of inducing diabetes,proliferating,and producing cytokines. Thus,recirculating RTEs encounter TRAs during a transitional developmental stage that facilitates tolerance induction,but inflammation converts antigen-exposed,tolerance-prone RTEs into competent effector cells.
View Publication
产品号#：
19852
19852RF
19853
19853RF
产品名：
EasySep™小鼠CD4+ T细胞分选试剂盒
RoboSep™ 小鼠CD4+ T细胞分选试剂盒
EasySep™小鼠CD8+ T细胞分选试剂盒
RoboSep™ 小鼠CD8+ T细胞分选试剂盒
Tian L et al. (APR 2016)
Stem Cell Reviews and Reports 12 4 500--508
Efficient and Controlled Generation of 2D and 3D Bile Duct Tissue from Human Pluripotent Stem Cell-Derived Spheroids
While in vitro liver tissue engineering has been increasingly studied during the last several years,presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver,but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly,generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions,and has been inefficient so far. Towards generating a fully functional liver containing biliary system,we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver,EpCAM,is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can,not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes),in a 2D differentiation condition,but also form functional ductal structures in a 3D condition. Importantly,this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition,we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues,which may facilitate engineering of complete and functional liver tissue in the future.
View Publication
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Robinson M et al. (AUG 2016)
Stem Cell Reviews and Reports 12 4 476--483
Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells
Pluripotent stem cells can become any cell type found in the body. Accordingly,one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells,which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next,we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture,hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells.
View Publication
产品号#：
05832
05833
05838
05872
05873
05940
07174
07180
07183
07190
27147
07191
36254
07930
07931
07940
07955
07956
07959
07954
27845
27945
27840
27865
27940
27965
05835
05839
08581
08582
100-0483
100-0484
100-1061
07952
100-0763
100-0485
100-1077
产品名：
STEMdiff™ 神经花环选择试剂
STEMdiff™神经前体细胞培养基
STEMdiff™神经祖细胞冻存液
Vitronectin XF™
CellAdhere™ 稀释缓冲液
DMEM/F-12 with 15 mM HEPES
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
CryoStor® CS10
STEMdiff™ 神经诱导培养基
STEMdiff™ 神经诱导培养基
STEMdiff™SMADi神经诱导试剂盒
STEMdiff™SMADi神经诱导试剂盒，2套
Hausser Scientificᵀᴹ 明线血球计数板
ReLeSR™
CryoStor® CS10
CryoStor® CS10
Vitronectin XF™
温和细胞解离试剂
ReLeSR™
Wang W et al. (MAY 2016)
Cell 165 5 1092--105
Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.
Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here,we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells,resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival,respectively. Thus,our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.
View Publication
产品号#：
17953
17953RF
15022
15062
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
RosetteSep™人CD4+ T细胞富集抗体混合物
RosetteSep™人CD4+ T细胞富集抗体混合物
EasySep™人CD8+ T细胞分选试剂盒
Lowe A et al. (MAY 2016)
Stem Cell Reports 6 5 743--756
Intercellular Adhesion-Dependent Cell Survival and ROCK-Regulated Actomyosin-Driven Forces Mediate Self-Formation of a Retinal Organoid
In this study we dissected retinal organoid morphogenesis in human embryonic stem cell (hESC)-derived cultures and established a convenient method for isolating large quantities of retinal organoids for modeling human retinal development and disease. Epithelialized cysts were generated via floating culture of clumps of Matrigel/hESCs. Upon spontaneous attachment and spreading of the cysts,patterned retinal monolayers with tight junctions formed. Dispase-mediated detachment of the monolayers and subsequent floating culture led to self-formation of retinal organoids comprising patterned neuroretina,ciliary margin,and retinal pigment epithelium. Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for the self-organization. Our data supports a hypothesis that newly specified neuroretina progenitors form characteristic structures in equilibrium through minimization of cell surface tension. In long-term culture,the retinal organoids autonomously generated stratified retinal tissues,including photoreceptors with ultrastructure of outer segments. Our system requires minimal manual manipulation,has been validated in two lines of human pluripotent stem cells,and provides insight into optic cup invagination in vivo.
View Publication
A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production.
Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations,convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors,measuring yield and purity outputs of undifferentiated,neuroectoderm,mesendoderm,and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically,small interfering RNA knockdown of RAPTOR,a component of mTOR complex 1,phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold,with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.
View Publication
EasySep™小鼠TIL（CD45）正选试剂盒
沪公网安备31010102008431号