Ephrin receptor, EphB4, regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes, and blood vessels.
Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development,however,must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor,EphB4,known to be spatially restricted in expression and critical for organized vessel formation,modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast,blood cell,cardiomyocyte,and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated,but not neuroectoderm-associated,genes. Therefore,EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization.
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产品号#：
06902
06952
00321
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00325
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Meng A et al. (DEC 2003)
Experimental hematology 31 12 1348--56
Ionizing radiation and busulfan inhibit murine bone marrow cell hematopoietic function via apoptosis-dependent and -independent mechanisms.
OBJECTIVE: Ionizing radiation (IR) and busulfan (BU) are commonly used as preconditioning regimens for bone marrow transplantation (BMT). We examined whether induction of apoptosis in murine bone marrow (BM) hematopoietic cells contributes to IR- and BU-induced suppression of their hematopoietic function. METHODS: The hematopoietic functions of hematopoietic stem cells (HSCs) and progenitors were analyzed by the cobblestone area-forming cell (CAFC) assay. Apoptosis was determined by measuring 3,3'-dihexyloxacarbocyanine iodide (DiCO6) uptake,annexin V staining,and/or sub-G(0/1) cells. Four cell types were studied: murine BM mononuclear cells (BM-MNCs),linage-negative hematopoietic cells (Lin-) cells),Lin- Scal+ c-kit+ cells,and Lin- Scal- c-kit+ cells by flow cytometry. RESULTS: Exposure of BM-MNCs to IR (4 Gy) or incubation of the cells with BU (30 microM) resulted in a significant reduction in CAFC frequency (ptextless0.001). The survival fractions of various day-types of CAFC for the irradiated cells were less than 10%,while that for BU-treated cells was 71.3% on day 7 and progressively declined to 5.3% on day 35. Interestingly,IR significantly induced apoptosis in BM-MNCs,Lin- cells,HSCs,and progenitors,whereas BU failed to increase apoptosis in these cells. In addition,preincubation of BM-MNCs with z-Val-Ala-Asp (OCH3)-fluoromethylketone,methyl ester (z-VAD) attenuated IR-induced reduction in CAFC but not that induced by BU. CONCLUSION: IR and BU differentially suppress the hematopoietic function of HSCs and progenitors by fundamentally different mechanisms. IR inhibits the function primarily by the induction of HSC and progenitor apoptosis. In contrast,BU suppresses HSC and progenitor function via an apoptosis-independent mechanism.
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产品类型：
产品号#：
03534
产品名：
MethoCult™ GF M3534
Fang F et al. (APR 2014)
Journal of cell science 127 Pt 7 1428--40
The role of Hath6, a newly identified shear-stress-responsive transcription factor, in endothelial cell differentiation and function.
The key regulators of endothelial differentiation that is induced by shear stress are mostly unclear. Human atonal homolog 6 (Hath6 or ATOH8) is an endothelial-selective and shear-stress-responsive transcription factor. In this study,we sought to elucidate the role of Hath6 in the endothelial specification of embryonic stem cells. In a stepwise human embryonic stem cell to endothelial cell (hESC-EC) induction system,Hath6 mRNA was upregulated synchronously with endothelial determination. Subsequently,gain-of-function and loss-of-function studies of Hath6 were performed using the hESC-EC induction model and endothelial cell lines. The overexpression of Hath6,which mimics shear stress treatment,resulted in an increased CD45(-)CD31(+)KDR(+) population,a higher tubular-structure-formation capacity and increased endothelial-specific gene expression. By contrast,the knockdown of Hath6 mRNA markedly decreased endothelial differentiation. Hath6 also facilitated the maturation of endothelial cells in terms of endothelial gene expression,tubular-structure formation and cell migration. We further demonstrated that the gene encoding eNOS is a direct target of Hath6 through a reporter system assay and western blot analysis,and that the inhibition of eNOS diminishes hESC-EC differentiation. These results suggest that eNOS plays a key role in linking Hath6 to the endothelial phenotype. Further in situ hybridization studies in zebrafish and mouse embryos indicated that homologs of Hath6 are involved in vasculogenesis and angiogenesis. This study provides the first confirmation of the positive impact of Hath6 on human embryonic endothelial differentiation and function. Moreover,we present a potential signaling pathway through which shear stress stimulates endothelial differentiation.
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05850
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mTeSR™1
mTeSR™1
Kimbrel EA et al. (JUL 2014)
Stem Cells and Development 23 14 1611--1624
Mesenchymal Stem Cell Population Derived from Human Pluripotent Stem Cells Displays Potent Immunomodulatory and Therapeutic Properties
Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however,loss of potency and inconsistent quality severely limit their use. To overcome these issues,we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking,scraping,and sorting that other hESC-MSC derivation methods require. Moreover,unlike previous reports on hESC-MSCs,we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected,they dynamically secrete a range of bioactive factors,display enzymatic activity,and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However,they also display unique immunophenotypic properties,as well as a smaller size and textgreater30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition,this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models,including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources.
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05850
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mTeSR™1
mTeSR™1
Miranda C et al. (OCT 2015)
Biotechnology Journal 10 10 1612--1624
Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment
3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work,we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation,after four days of culture,3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm,which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up.
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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.
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05850
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mTeSR™1
mTeSR™1
Lam BS et al. (JAN 2011)
Blood 117 4 1167--75
Pharmacologic modulation of the calcium-sensing receptor enhances hematopoietic stem cell lodgment in the adult bone marrow.
The ability of hematopoietic stem cells (HSCs) to undergo self-renewal is partly regulated by external signals originating from the stem cell niche. Our previous studies with HSCs obtained from fetal liver of mice deficient for the calcium-sensing receptor (CaR) have shown the crucial role of this receptor in HSC lodgment and engraftment in the bone marrow (BM) endosteal niche. Using a CaR agonist,Cinacalcet,we assessed the effects of stimulating the CaR on the function of murine HSCs. Our results show that CaR stimulation increases primitive hematopoietic cell activity in vitro,including growth in stromal cell cocultures,adhesion to extracellular matrix molecules such as collagen I and fibronectin,and migration toward the chemotactic stimulus,stromal cell-derived factor 1α. Receptor stimulation also led to augmented in vivo homing,CXCR4-mediated lodgment at the endosteal niche,and engraftment capabilities. These mechanisms by which stimulating the CaR dictates preferential localization of HSCs in the BM endosteal niche provide additional insights into the fundamental interrelationship between the stem cell and its niche. These studies also have implications in the area of clinical stem cell transplantation,where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM.
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产品类型：
产品号#：
03434
03444
产品名：
MethoCult™ GF M3434
MethoCult™ GF M3434
Ungrin MD et al. (APR 2012)
Biotechnology and bioengineering 109 4 853--66
Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics.
We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation,and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and,with quantitative cell division tracking and fate monitoring,identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion,during directed differentiation,to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.
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产品号#：
27845
27945
27840
27865
27940
27965
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Ermakov A et al. (NOV 2012)
Stem Cell Research 9 3 171--184
A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture
Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-,G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ,intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly,treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs,lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (ptextless0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture.
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产品号#：
05850
05857
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产品名：
mTeSR™1
mTeSR™1
Haraguchi Y et al. (DEC 2015)
Journal of Tissue Engineering and Regenerative Medicine 9 12 1363--1375
Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.
In this study,a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension,only a few aggregated cells were observed. However,after 3 days,culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry,immunocytochemistry and quantitative RT-PCR,and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium,expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore,the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A,BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes,including HCN4,MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes,including pacemakers. Moreover,when cardiac cell sheets were fabricated using differentiated cardiomyocytes,they beat spontaneously and synchronously,indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.
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产品号#：
05850
05857
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07174
60002
60002AD
60002AD.1
60002AZ
60002AZ.1
60002BT
60002BT.1
60002FI
60002FI.1
60002PE
60002PE.1
60002PS
60002PS.1
60002PB
60002PB.1
60062
60062AD
60062AD.1
60062BT
60062FI
60062FI.1
60062PE
60062PE.1
85850
85857
85870
85875
1
产品名：
抗小鼠CD11c抗体，克隆N418
抗小鼠CD11c抗体，clone N418，Alexa Fluor® 488
抗小鼠CD11c抗体，克隆N418，APC
抗小鼠CD11c抗体，克隆N418，APC
抗小鼠CD11c抗体，克隆N418，Biotin
抗小鼠CD11c抗体，克隆N418，FITC
抗小鼠CD11c抗体，克隆N418，PerCP-Cy5.5
抗小鼠CD11c抗体，克隆N418，Pacific Blue™
抗小鼠CD11c抗体，克隆N418，Pacific Blue™
抗人SSEA-4抗体，克隆号MC-813-70，生物素
抗人SSEA-4抗体，克隆号MC-813-70，FITC
抗人SSEA-4抗体, 克隆号MC-813-70，FITC
抗人SSEA-4抗体，克隆号MC-813-70，PE
抗人SSEA-4抗体，克隆号MC-813-70，PE
mTeSR™1
mTeSR™1
Leach LL et al. (MAY 2016)
Journal of Ocular Pharmacology and Therapeutics 32 5 jop.2016.0022
Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods
Abstract Purpose: The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic,with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells,but previous studies have shown variability in iPSC propensity to differentiate into RPE. Methods: This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid,directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE,which were characterized with respect to global gene expression,expression of RPE markers,and cellular function. Results: We found that all 5 iPSC lines (iPSC-1,iPSC-2,iPSC-3,iPSC-4,and iPSC-12) generated RPE using the directed differentiation protocol; however,2 of the 5 iPSC lines (iPSC-4 and iPSC-...
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产品类型：
产品号#：
05850
05857
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85850
85857
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产品名：
mTeSR™1
mTeSR™1
Ma D et al. (JAN 2017)
Stem cell research 18 48--50
Derivation of human induced pluripotent stem cell (iPSC) line with LRRK2 gene R1398H variant in Parkinson's disease.
Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 72-year old female Parkinson's disease (PD) patient with R1398H variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model provides a good platform for studying the mechanism of PD,and also for drug testing and gene therapy studies.
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