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We analyzed the proliferative response of the growth factor-dependent murine cell lines FDCp1,DA1-a,32DC1,Ea3.15,7TD1,BCL1 and of femural bone marrow cells for their sensitivity to various cytokines,viz. rhIL-1 beta,rhTNF,rhIL-2,mIL-3,rmIL-4,rmIL-5,rhIL-6,rhG-CSF and rmGM-CSF. We also tested for IL-1 and TNF-mediated cytokine secretion by several T cell lines and thymocytes. In all T cell systems,IL-1 alpha and IL-1 beta were equally active in the induction of cytokine production,except for the rat/mouse T cell hybridoma PC60. This cell line exhibited a 10-fold difference in specific activity for the induction of cytokine secretion between rhIL-1 alpha and the other human or murine IL-1 species. Furthermore,IL-1 and IL-2 synergistically induced PC60 cells to produce a factor,which was preferentially active on FDCp1-cells,provisionally called FDCp1-growth factor. SDS-PAGE analysis of partially purified FDCp1-GF showed 19 kDa and 24 kDa-associated biological activities. Amino-terminal and internal amino acid sequences of both bands were determined and on this basis,we identified FDCp1-GF as rat GM-CSF. 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

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however,methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible,floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors,we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system,we insert the myogenic regulator,Myf5,into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate. View Publication

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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

PURPOSE: To investigate the distribution of CD44(+)/CD24(-) cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy. METHODS: CD44(+)/CD24(-) tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays,respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays,and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST). RESULTS: In 27/78 cases,complete remission (CR) was identified using RECIST. However,18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy,24 cases involved cancer cells that were confined to the tumor outline,and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition,there were 6 patients who were insensitive to chemotherapy,and in these cases,both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy. CONCLUSION: CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion. View Publication

Low oxygen (O(2)) levels characterize the microenvironment of both stem cells and rapidly growing tumors. Moreover,hypoxia is associated with the maintenance of stem cell-like phenotypes and increased invasion,angiogenesis and metastasis in cancer patients. Metastatic cancers,such as breast cancer and melanoma,aberrantly express the embryonic morphogen Nodal,and the presence of this protein is correlated with metastatic disease. In this paper,we demonstrate that hypoxia induces Nodal expression in melanoma and breast cancer cells concomitant with increased cellular invasion and angiogenic phenotypes. Of note,Nodal expression remains up-regulated up to 48 h following reoxygenation. The oxygen-mediated regulation of Nodal expression occurs via a combinatorial mechanism. Within the first 24 h of exposure to low O(2),there is an increase in protein stability. This increase in stability is accompanied by an induction of transcription,mediated by the HIF-1α-dependent activation of Notch-responsive elements in the node-specific enhancer of the Nodal gene locus. Finally,Nodal expression is maintained upon reoxygenation by a canonical SMAD-dependent feed-forward mechanism. This work provides insight into the O(2)-mediated regulation of Nodal,a key stem cell-associated factor,and reveals that Nodal may be a target for the treatment and prevention of hypoxia-induced tumor progression. View Publication

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

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus,hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks,however,conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology,retained a normal karyotype,and expressed characteristic hESC markers (OCT4,SSEA3,SSEA4 and TRA-1-60). Moreover,the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus,the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells. View Publication