Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease.
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产品号#：
04230
产品名：
MethoCult™ H4230
Walker A et al. (JAN 2010)
Nature communications 1 6 71
Non-muscle myosin II regulates survival threshold of pluripotent stem cells.
Human pluripotent stem (hPS) cells such as human embryonic stem (hES) and induced pluripotent stem (hiPS) cells are vulnerable under single cell conditions,which hampers practical applications; yet,the mechanisms underlying this cell death remain elusive. In this paper,we demonstrate that treatment with a specific inhibitor of non-muscle myosin II (NMII),blebbistatin,enhances the survival of hPS cells under clonal density and suspension conditions,and,in combination with a synthetic matrix,supports a fully defined environment for self-renewal. Consistent with this,genetically engineered mouse embryonic stem cells lacking an isoform of NMII heavy chain (NMHCII),or hES cells expressing a short hairpin RNA to knock down NMHCII,show greater viability than controls. Moreover,NMII inhibition increases the expression of self-renewal regulators Oct3/4 and Nanog,suggesting a mechanistic connection between NMII and self-renewal. These results underscore the importance of the molecular motor,NMII,as a novel target for chemically engineering the survival and self-renewal of hPS cells.
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05850
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72402
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(-)-Blebbistatin
(-)-Blebbistatin
mTeSR™1
mTeSR™1
Ciampi O et al. (JUN 2016)
Stem Cell Research 17 1 130--139
Generation of functional podocytes from human induced pluripotent stem cells
Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here,we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol,which induced their differentiation into intermediate mesoderm,then into nephron progenitors and,finally,into mature podocytes. After differentiation,cells expressed the main podocyte markers,such as synaptopodin,WT1,α-Actinin-4,P-cadherin and nephrin at the protein and mRNA level,and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall,these findings demonstrate the establishment of a robust protocol that,mimicking developmental stages,makes it possible to derive functional podocytes in vitro.
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05850
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mTeSR™1
mTeSR™1
STEMdiff™ APEL™2 培养基
STEMdiff™ APEL™2 培养基
Baker D et al. (NOV 2016)
Stem cell reports 7 5 998--1012
Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells.
Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1,12,17,and 20. Thus,hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes,there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding,qPCR,fluorescence in situ hybridization,and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods,a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine.
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05850
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mTeSR™1
mTeSR™1
Ruiz S et al. (JAN 2010)
PLoS ONE 5 12 e15526
High-efficient generation of induced pluripotent stem cells from human astrocytes.
The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells,including fibroblasts,keratinocytes and peripheral blood cells,with variable reprogramming efficiencies and kinetics. Here,we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells,with similar efficiencies to keratinocytes,which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure.
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mTeSR™1
mTeSR™1
Zweigerdt R et al. (MAY 2011)
Nature protocols 6 5 689--700
Scalable expansion of human pluripotent stem cells in suspension culture.
Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones,three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation,but it does not require culture preadaptation,use of microcarriers or any other matrices. Over a time course of 4-7 d,hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly,hPSCs maintain pluripotency and karyotype stability for more than ten passages.
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mTeSR™1
mTeSR™1
Schinzel RT et al. (JAN 2011)
PloS one 6 12 e27495
Efficient culturing and genetic manipulation of human pluripotent stem cells.
Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However,the lack of simple,robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells,2) significantly increases viability and replating efficiency,3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation,we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%,with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions,and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.
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05850
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mTeSR™1
mTeSR™1
Ahfeldt T et al. (FEB 2012)
Nature cell biology 14 1476-4679 (Electronic) 209--219
Programming human pluripotent stem cells into white and brown adipocytes.
The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression,could be maintained in culture for several weeks,expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice,the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease
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05850
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mTeSR™1
mTeSR™1
Sandt C et al. (JAN 2012)
PLoS ONE 7 4 e30743
Identification of spectral modifications occurring during reprogramming of somatic cells.
Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However,research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly,this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.
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产品类型：
产品号#：
05850
05857
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产品名：
mTeSR™1
mTeSR™1
Howden SE and Thomson JA ( 2014)
1114 37--55
Gene targeting of human pluripotent stem cells by homologous recombination.
The ability of human embryonic stem cells and induced pluripotent stem cells to differentiate into all adult cell types greatly facilitates the study of human development,disease pathogenesis,and the generation of screening systems to identify novel therapeutic agents. Autologous cell therapies based on patient-derived induced pluripotent stem cells also hold great promise for the treatment and correction of many inherited and acquired diseases. The full potential of human pluripotent stem cells can be unleashed by genetically modifying a chosen locus with minimal impact on the remaining genome,which can be achieved by targeting genes by homologous recombination. This chapter will describe a protocol for gene modification of pluripotent stem cells by homologous recombination and several methods for the screening and identification of successfully modified clones.
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