Graichen R et al. (APR 2008)
Differentiation 76 4 357--70
Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.
Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here,we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules,we have identified SB203580,a specific p38 MAP kinase inhibitor,as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations textless10 microM,induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers,so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly,transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly,cardiomyogenesis was strongly inhibited at SB203580 concentrations textgreater or =15 microM. Thus,modulation of the p38MAP kinase pathway,in combination with factors released by END2 cells,plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.
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产品类型:
产品号#:
72222
72632
72634
产品名:
SB203580 (Hydrochloride)
SB202190
SB202190
文献
Ishii Y et al. (MAR 2008)
Molecular and cellular neurosciences 37 3 507--18
Characterization of neuroprogenitor cells expressing the PDGF beta-receptor within the subventricular zone of postnatal mice.
We report a considerable number of cells in the ventricular and the subventricular zones (SVZ) of newborn mice to stain positive for the PDGF beta-receptor (PDGFRB). Many of them also stained for nestin and/or GFAP but less frequently for the neuroblast marker doublecortin and for the mitotic marker Ki-67. The SVZ of mice with nestin-Cre conditional deletion of PDGFRB expressed the receptor only on blood vessels and was devoid of any morphological abnormality. PDGFRB(-/-) neurospheres showed a higher rate of apoptosis without any significant decrease in proliferation. They demonstrated reduced capacities of migration and neuronal differentiation in response to not only PDGF-BB but also bFGF. Furthermore,the PDGFR kinase inhibitor STI571 blocked the effects of bFGF in control neurosphere cultures. bFGF increased the activity of the PDGFRB promoter as well as the expression and phosphorylation of PDGFRB. These results suggest the presence of the signaling convergence between PDGF and FGF. PDGFRB is needed for survival,and the effects of bFGF in migration and neural differentiation of the cells may be potentiated by induction of PDGFRB.
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产品类型:
产品号#:
05700
05701
05702
产品名:
NeuroCult™ 基础培养基(小鼠和大鼠)
NeuroCult™ 扩增添加物(小鼠和大鼠)
NeuroCult™扩增试剂盒(小鼠和大鼠)
文献
Kallas A et al. (APR 2011)
PLoS ONE 6 4 e19114
Nocodazole treatment decreases expression of pluripotency markers nanog and Oct4 in human embryonic stem cells
Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells,which also expressed Oct4,SSEA-3 and SSEA-4. We also found another population expressing SSEA-4,but without Nanog,Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog,Oct4,SSEA-3,SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block,the cell cycle of hESC normalised,but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition,the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle,which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.
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产品类型:
产品号#:
85850
85857
产品名:
mTeSR™1
mTeSR™1
文献
Panopoulos AD et al. (JAN 2012)
Cell Research 22 1 168--177
The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming
Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence.
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产品类型:
产品号#:
85850
85857
产品名:
mTeSR™1
mTeSR™1
文献
Tan Y et al. (JAN 2012)
Journal of biomechanics 45 1 123--8
Probing the mechanobiological properties of human embryonic stem cells in cardiac differentiation by optical tweezers.
Human embryonic stem cells (hESC) and hESC-derived cardiomyocytes (hESC-CM) hold great promise for the treatment of cardiovascular diseases. However the mechanobiological properties of hESC and hESC-CM remains elusive. In this paper,we examined the dynamic and static micromechanical properties of hESC and hESC-CM,by manipulating via optical tweezers at the single-cell level. Theoretical approaches were developed to model the dynamic and static mechanical responses of cells during optical stretching. Our experiments showed that the mechanical stiffness of differentiated hESC-CM increased after cardiac differentiation. Such stiffening could associate with increasingly organized myofibrillar assembly that underlines the functional characteristics of hESC-CM. In summary,our findings lay the ground work for using hESC-CMs as models to study mechanical and contractile defects in heart diseases.
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产品类型:
产品号#:
85850
85857
产品名:
mTeSR™1
mTeSR™1
文献
Moschidou D et al. (OCT 2012)
Molecular therapy : the journal of the American Society of Gene Therapy 20 10 1953--67
Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach.
Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However,due to risks of random integration of the reprogramming transgenes into the host genome,the low efficiency of the process,and the potential risk of virally induced tumorigenicity,alternative methods have been developed to generate pluripotent cells using nonintegrating systems,albeit with limited success. Here,we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors,by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion,they maintain genetic stability,protein level expression of key pluripotency factors,high cell-division kinetics,telomerase activity,repression of X-inactivation,and capacity to differentiate into lineages of the three germ layers,such as definitive endoderm,hepatocytes,bone,fat,cartilage,neurons,and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies,pharmaceutical screening,and disease modeling.
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产品类型:
产品号#:
85850
85857
产品名:
mTeSR™1
mTeSR™1
文献
Heng BC et al. (JUL 2013)
Metabolic Engineering 18 9--24
MRNA transfection-based, feeder-free, induced pluripotent stem cells derived from adipose tissue of a 50-year-old patient
Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique,but with defined culture medium without feeder cells,during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells,starting from 150,000 cells. However,only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining,flow cytometry,qRT-PCR,in vitro differentiation assay,and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses,thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13,chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence,this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic. ?? 2013 Elsevier Inc.
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产品类型:
产品号#:
85850
85857
产品名:
mTeSR™1
mTeSR™1
文献
Deng Y et al. (NOV 2013)
Acta Biomaterialia 9 11 8840--8850
Long-term self-renewal of human pluripotent stem cells on peptide-decorated poly(OEGMA-co-HEMA) brushes under fully defined conditions
Realization of the full potential of human induced pluripotent stem cells (hiPSC) in clinical applications requires the development of well-defined culture conditions for their long-term growth and directed differentiation. This paper describes a novel fully defined synthetic peptide-decorated substrate that supports self-renewal of hiPSC in commercially available xeno-free,chemically defined medium. The Au surface was deposited by a poly(OEGMA-co-HEMA) film,using the surface-initiated polymerization method (SIP) with the further step of carboxylation. The hiPSC generated from umbilical cord mesenchymal cells were successfully cultured for 10 passages on the peptide-tethered poly(OEGMA-co-HEMA) brushes for the first time. Cells maintained their characteristic morphology,proliferation and expressed high levels of markers of pluripotency,similar to the cells cultured on Matrigel???. Moreover,the cell adhesion could be tuned by the pattern and peptide concentration on the substrate. This well-defined,xeno-free and safe substrate,which supports long-term proliferation and self-renewal of hiPSC,will not only help to accelerate the translational perspectives of hiPSC,but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation via SIP technology. ?? 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Lai Z et al. (MAR 2002)
Proceedings of the National Academy of Sciences of the United States of America 99 6 3651--6
Design of an HIV-1 lentiviral-based gene-trap vector to detect developmentally regulated genes in mammalian cells.
The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery,mutagenesis,and other applications in mammalian systems. Here we describe two HIV-1-based lentiviral vector derivatives,pZR-1 and pZR-2,that can be used in gene-trap experiments in mammalian cells in vitro and in vivo. Each lentiviral gene-trap vector contains a reporter gene,either beta-lactamase or enhanced green fluorescent protein (EGFP),that is inserted into the U3 region of the 3' long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique. Appropriate insertion of the vector into genes causes EGFP or beta-lactamase expression. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream,cell-specific promoter during cell culture and cell differentiation,which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment,as well as for the discovery of new genes.
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