Aoyama A et al. ( 2014)
Molecular cancer therapeutics 13 12 2978--2990
Tivantinib (ARQ 197) exhibits antitumor activity by directly interacting with tubulin and overcomes ABC transporter-mediated drug resistance.
Tivantinib (ARQ197) was first reported as a highly selective inhibitor of c-MET and is currently being investigated in a phase III clinical trial. However,as recently reported by us and another group,tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules,we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Consistent with our previous report,tivantinib reduced tubulin polymerization in cells and in mouse xenograft tumors in vivo. To determine if tivantinib directly binds to tubulin,we performed an in vitro competition assay. Tivantinib competitively inhibited colchicine but not vincristine or vinblastine binding to purified tubulin. These results imply that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin,we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fitted into the colchicine binding pocket of tubulin without steric hindrance. Furthermore,tivantinib showed similar IC50 values against parental and multidrug-resistant cells. In contrast,other microtubule-targeting drugs,such as vincristine,paclitaxel,and colchicine,could not suppress the growth of cells overexpressing ABC transporters. Moreover,the expression level of ABC transporters did not correlate with the apoptosis-inducing ability of tivantinib different from other microtubule inhibitor. These results suggest that tivantinib can overcome ABC transporter-mediated multidrug-resistant tumor cells and is potentially useful against various tumors.
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产品号#：
73482
73484
产品名：
Watson CL et al. (NOV 2014)
Nature Medicine 20 11 1310--4
An in vivo model of human small intestine using pluripotent stem cells.
Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes,for pharmacologic studies and as a potential resource for therapeutic transplant. To date,limited in vivo models exist for human intestine,all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here,we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme,both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme,as demonstrated by differentiated intestinal cell lineages (enterocytes,goblet cells,Paneth cells,tuft cells and enteroendocrine cells),presence of functional brush-border enzymes (lactase,sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore,transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection,suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology,disease and translational studies.
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产品号#：
05854
05855
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产品名：
mFreSR™
mFreSR™
mTeSR™1
mTeSR™1
Varela I et al. (DEC 2014)
Cellular reprogramming 16 6 447--455
Generation of human $\$-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.
Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with $$-thalassemia ($$-thal) with the aim to generate trangene-free $$-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4,Klf4,Sox2,cMyc,and Lin28 resulted in formation of five iPSC colonies,from which three were picked up and expanded in $$-thal-iPSC lines. After 10 serial passages in vitro,$$-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs,whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%,but with a decreased hematopoietic colony-forming capability. In conclusion,we report herein the generation of transgene-free $$-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover,it was demonstrated that the mRNA-based reprogramming method,used mainly in fibroblasts,is also suitable for reprogramming of human BM-MSCs.
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产品号#：
04230
05850
05857
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07923
60062
60062AD
60062AD.1
60062BT
60062FI
60062FI.1
60062PE
60062PE.1
85850
85857
85870
85875
05270
05275
产品名：
MethoCult™ H4230
Dispase (1 U/mL)
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
STEMdiff™ APEL™2 培养基
STEMdiff™ APEL™2 培养基
Badja C et al. (DEC 2014)
Stem cells translational medicine 3 12 1467--72
Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.
For years,our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular,much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells,hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction,expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days,neuronal precursors can be expanded,frozen,and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction,cells express typical voltage-gated and ionotrophic receptors for GABA,glycine,and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications,especially for modeling human pathologies.
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产品号#：
05850
05857
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产品名：
mTeSR™1
mTeSR™1
McCracken KW et al. (DEC 2014)
Nature 516 7531 400--4
Modelling human development and disease in pluripotent stem-cell-derived gastric organoids.
Gastric diseases,including peptic ulcer disease and gastric cancer,affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis,and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF,WNT,BMP,retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains,proliferative zones containing LGR5-expressing cells,surface and antral mucous cells,and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease,we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor,activation of signalling and induction of epithelial proliferation. Together,these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.
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产品号#：
05850
05857
05870
05875
07920
85850
85857
85870
85875
07922
产品名：
ACCUTASE™
mTeSR™1
mTeSR™1
ACCUTASE™
Pettinato G et al. (NOV 2014)
PLoS ONE 9 11 e100742
ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells
We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin,+ROCKi/-spin,-ROCKi/+spin,and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions,including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation,elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment,and low-cost scalability,which will directly support automated,large-scale production of hEBs and hESC-derived cells needed for clinical,research,or therapeutic applications.
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产品号#：
05850
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产品名：
mTeSR™1
mTeSR™1
Sarker D et al. ( 2015)
Clinical cancer research : an official journal of the American Association for Cancer Research 21 1 77--86
First-in-human phase I study of pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors.
PURPOSE: This first-in-human dose-escalation trial evaluated the safety,tolerability,maximal-tolerated dose (MTD),dose-limiting toxicities (DLT),pharmacokinetics,pharmacodynamics,and preliminary clinical activity of pictilisib (GDC-0941),an oral,potent,and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). PATIENTS AND METHODS: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily,initially on days 1 to 21 every 28 days and later,using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET,and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. RESULTS: Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea,rash,and fatigue,whereas the DLT was grade 3 maculopapular rash (450 mg,2 of 3 patients; 330 mg,1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed textgreater90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC textgreater20 htextperiodcenteredμmol/L. Significant increase in plasma insulin and glucose levels,and textgreater25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria,respectively. CONCLUSION: Pictilisib was safely administered with a dose-proportional pharmacokinetic profile,on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily.
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产品号#：
73152
产品名：
GDC- 0941
Courtot A-M et al. (OCT 2014)
BioResearch open access 3 5 206--216
Morphological analysis of human induced pluripotent stem cells during induced differentiation and reverse programming.
The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming.
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05850
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产品名：
mTeSR™1
mTeSR™1
Zhou Y et al. (NOV 2014)
Scientific reports 4 6978
Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay.
Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically,various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods,we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods,which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming,thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.
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Miyazaki S et al. (DEC 2015)
Annals of surgical oncology 22 Suppl 3 S3 S1394----401
A Cancer Reprogramming Method Using MicroRNAs as a Novel Therapeutic Approach against Colon Cancer: Research for Reprogramming of Cancer Cells by MicroRNAs.
BACKGROUND We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4,Sox2,c-Myc,and Klf4). In this study,we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential. METHODS Mature microRNAs (mir-302a-d,369-3p and 5p,and mir-200c,as needed) were introduced into colon cancer cells (DLD-1,RKO,and HCT116) using lipofection. RESULTS The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog,Oct3/4,SOX2,and Klf4,as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers,indicating that reprogramming of the cancer cells was partially successful. Moreover,we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo,accompanied by increased expression of the tumor suppressor genes p16 (ink4a) and p21 (waf1) . miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil,possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1,RKO,and HCT116 cells exhibited reduced c-Myc expression,in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors. CONCLUSIONS Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application,because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression.
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产品名：
mTeSR™1
mTeSR™1
Griesi-Oliveira K et al. (NOV 2014)
Molecular psychiatry 20 March 1--16
Modeling non-syndromic autism and the impact of TRPC6 disruption in human neurons.
An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs),and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here,we report a de novo balanced translocation disruption of TRPC6,a cation channel,in a non-syndromic autistic individual. Using multiple models,such as dental pulp cells,induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models,we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development,morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin,a TRPC6-specific agonist,suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome,revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population,and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together,these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.Molecular Psychiatry advance online publication,11 November 2014; doi:10.1038/mp.2014.141.
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mTeSR™1
mTeSR™1
Jung J-H et al. (APR 2015)
Stem cells and development 24 8 948--61
CXCR2 and its related ligands play a novel role in supporting the pluripotency and proliferation of human pluripotent stem cells.
Basic fibroblast growth factor (bFGF) is a crucial factor sustaining human pluripotent stem cells (hPSCs). We designed this study to search the substitutive factors other than bFGF for the maintenance of hPSCs by using human placenta-derived conditioned medium without exogenous bFGF (hPCCM-),containing chemokine (C-X-C motif) receptor 2 (CXCR2) ligands,including interleukin (IL)-8 and growth-related oncogene $\$(GRO$\$),which were developed on the basis of our previous studies. First,we confirmed that IL-8 and/or GRO$\$ independent roles to preserve the phenotype of hPSCs. Then,we tried CXCR2 blockage of hPSCs in hPCCM- and verified the significant decrease of pluripotency-associated genes expression and the proliferation of hPSCs. Interestingly,CXCR2 suppression of hPSCs in mTeSR™1 containing exogenous bFGF decreased the proliferation of hPSCs while maintaining pluripotency characteristics. Lastly,we found that hPSCs proliferated robustly for more than 35 passages in hPCCM- on a gelatin substratum. Higher CXCR2 expression of hPSCs cultured in hPCCM- than those in mTeSR™1 was observable. Our findings suggest that CXCR2 and its related ligands might be novel factors comparable to bFGF supporting the characteristics of hPSCs and hPCCM- might be useful for the maintenance of hPSCs as well as for the accurate evaluation of CXCR2 role in hPSCs without the confounding influence of exogenous bFGF.
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