技术资料

The 26S proteasome,a multicatalytic enzyme complex,is the main intracellular proteolytic system involved in the degradation of ubiquitinated proteins. The ability of proteasome inhibitors to induce apoptosis has been exploited in the recent development of chemotherapeutic agents. Here,we show that inhibition of proteasome by MG132 blocks DNA damage-induced apoptosis. Blockage of apoptosis by MG132 correlates with p53 stabilization and upregulation of p21/WAF1,a p53 transcriptional target. Surprisingly,in the absence of MG132,robust apoptosis induced by a high dose of UV irradiation correlate with rapid p53 degradation. This is in sharp contrast to p53 stabilization when cells were exposed to lower levels of UV irradiation. Our findings highlight a scenario in which severe UV damage can induce rapid p53 degradation by the proteasome. Importantly,these data suggest that the 26S proteasome plays a key role in promoting apoptosis induced by high doses of UV irradiation. View Publication

We tested the interaction of 72 kinase inhibitors with 442 kinases covering textgreater80% of the human catalytic protein kinome. Our data show that,as a class,type II inhibitors are more selective than type I inhibitors,but that there are important exceptions to this trend. The data further illustrate that selective inhibitors have been developed against the majority of kinases targeted by the compounds tested. Analysis of the interaction patterns reveals a class of 'group-selective' inhibitors broadly active against a single subfamily of kinases,but selective outside that subfamily. The data set suggests compounds to use as tools to study kinases for which no dedicated inhibitors exist. It also provides a foundation for further exploring kinase inhibitor biology and toxicity,as well as for studying the structural basis of the observed interaction patterns. Our findings will help to realize the direct enabling potential of genomics for drug development and basic research about cellular signaling. View Publication

The recent development of porcine induced pluripotent stem cells (piPSCs) capable of generating chimeric animals,a feat not previously accomplished with embryonic stem cells or iPSCs in a species outside of rodents,has opened the doors for in-depth study of iPSC tumorigenicity,autologous transplantation,and other key aspects to safely move iPSC therapies to the clinic. The study of iPSC tumorigenicity is critical as previous research in the mouse showed that iPSC-derived chimeras possessed large numbers of tumors,rising significant concerns about the safety of iPSC therapies. Additionally,piPSCs capable of generating germline chimeras could revolutionize the transgenic animal field by enabling complex genetic manipulations (e.g.,knockout or knockin of genes) to produce biomedically important large animal models or improve livestock production. In this study,we demonstrate for the first time in a nonrodent species germline transmission of iPSCs with the live birth of a transgenic piglet that possessed genome integration of the human POU5F1 and NANOG genes. In addition,gross and histological examination of necropsied porcine chimeras at 2,7,and 9 months showed that these animals lacked tumor formation and demonstrated normal development. Tissue samples positive for human POU5F1 DNA showed no C-MYC gene expression,further implicating C-MYC as a cause of tumorigenicity. The development of germline-competent porcine iPSCs that do not produce tumors in young chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals. View Publication

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy,including generation of pancreatic β-cells for diabetes research and treatment. In this study,we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation,and then combined treatment with retinoic acid,a bone morphogenic protein inhibitor,and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover,several compounds,including forskolin,dexamethasone,and a TGF-β inhibitor,were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds,more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition,all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol. View Publication

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. View Publication

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. View Publication

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines,from 38 laboratories worldwide,for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal,but there was a progressive tendency to acquire changes on prolonged culture,commonly affecting chromosomes 1,12,17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants,determined from the SNP arrays,also appeared sporadically. No common variants related to culture were observed on chromosomes 1,12 and 17,but a minimal amplicon in chromosome 20q11.21,including three genes expressed in human ES cells,ID1,BCL2L1 and HM13,occurred in textgreater20% of the lines. Of these genes,BCL2L1 is a strong candidate for driving culture adaptation of ES cells. View Publication

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Recent studies have reported the anti-tumor effects of the AhR in breast cancer. In this study,we investigated the anti-tumor effect of AhR activation based on the cancer stem cell hypothesis. We show that AhR activation suppressed mammosphere formation of MCF-7 cells and decreased the proportion of cells with high ALDH-1 (aldehyde dehydrogenase 1) activity. In addition,we also demonstrate that AhR activation regulates self-renewal signaling by down-regulating Wnt/$$-catenin and Notch. View Publication

New drugs for preserving and restoring pancreatic β-cell function are critically needed for the worldwide epidemic of type 2 diabetes and the cure for type 1 diabetes. We previously identified a family of neurogenic 3,5-disubstituted isoxazoles (Isx) that increased expression of neurogenic differentiation 1 (NeuroD1,also known as BETA2); this transcription factor functions in neuronal and pancreatic β-cell differentiation and is essential for insulin gene transcription. Here,we probed effects of Isx on human cadaveric islets and MIN6 pancreatic β cells. Isx increased the expression and secretion of insulin in islets that made little insulin after prolonged ex vivo culture and increased expression of neurogenic differentiation 1 and other regulators of islet differentiation and insulin gene transcription. Within the first few hours of exposure,Isx caused biphasic activation of ERK1/2 and increased bulk histone acetylation. Although there was little effect on histone deacetylase activity,Isx increased histone acetyl transferase activity in nuclear extracts. Reconstitution assays indicated that Isx increased the activity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism. In summary,we have identified a small molecule with antidiabetic activity,providing a tool for exploring islet function and a possible lead for therapeutic intervention in diabetes. View Publication

Because of recent declining malaria transmission in Latin America,some authorities have recommended against chemoprophylaxis for most travelers to this region. However,the predominant parasite species in Latin America,Plasmodium vivax,can form hypnozoites sequestered in the liver,causing malaria relapses. Additionally,new evidence shows the potential severity of vivax infections,warranting continued consideration of prophylaxis for travel to Latin America. Individualized travel risk assessments are recommended and should consider travel locations,type,length,and season,as well as probability of itinerary changes. Travel recommendations might include no precautions,mosquito avoidance only,or mosquito avoidance and chemoprophylaxis. There are a range of good options for chemoprophylaxis in Latin America,including atovaquone-proguanil,doxycycline,mefloquine,and--in selected areas--chloroquine. Primaquine should be strongly considered for nonpregnant,G6PD-nondeficient patients traveling to vivax-endemic areas of Latin America,and it has the added benefit of being the only drug to protect against malaria relapses. View Publication

Mutations in human induced pluripotent stem cells (iPSCs) pose a risk for their clinical use due to preferential reprogramming of mutated founder cell and selection of mutations during maintenance of iPSCs in cell culture. It is unknown,however,if mutations in iPSCs are due to stress associated with oncogene expression during reprogramming. We performed whole exome sequencing of human foreskin fibroblasts and their derived iPSCs at two different passages. We found that in vitro passaging contributed 7% to the iPSC coding point mutation load,and ultradeep amplicon sequencing revealed that 19% of the mutations preexist as rare mutations in the parental fibroblasts suggesting that the remaining 74% of the mutations were acquired during cellular reprogramming. Simulation suggests that the mutation intensity during reprogramming is ninefold higher than the background mutation rate in culture. Thus the factor induced reprogramming stress contributes to a significant proportion of the mutation load of iPSCs. View Publication

Realizing the potential of human pluripotent stem cell (hPSC)-based therapy requires the development of defined scalable culture systems with efficient expansion,differentiation and isolation protocols. We report an engineered 3D microfiber system that efficiently supports long-term hPSCs self-renewal under chemically defined conditions. The unique feature of this system lies in the application of a 3D ECM-like environment in which cells are embedded,that affords: (i) uniform high cell loading density in individual cell-laden constructs (∼10 7 cells/ml); (ii) quick recovery of encapsulated cells (textless10min at 37°C) with excellent preservation of cell viability and 3D multicellular structure; (iii) direct cryopreservation of the encapsulated cells in situ in the microfibers with textgreater17-fold higher cell viability compared to those cultured on Matrigel surface; (iv) long-term hPSC propagation under chemically defined conditions. Four hPSC lines propagated in the microfibrous scaffold for 10 consecutive passages were capable of maintaining an undifferentiated phenotype as demonstrated by the expression of stem cell markers and stable karyotype invitro and the ability to form derivatives of the three germ layers both invitro and invivo. Our 3D microfibrous system has the potential for large-scale cultivation of transplantable hESCs and derivatives for clinical applications. textcopyright 2011 Elsevier Ltd. View Publication