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Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population,and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular,disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors,aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits,we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as globulomers"� View Publication

Pluripotent stem cells have the potential to become most of the cell types that make up an organism. However,the signals that trigger these cells to turn into neurons rather than lung cells or muscle cells,for example,are not fully understood. Proteins called growth factors are known to have a role in this process,as are transcription factors,but it is not clear if other factors are also involved. In an attempt to identify additional mechanisms that could contribute to the formation of neurons,Sun et al. screened more than 2,000 small molecules for their ability to transform mouse pluripotent stem cells into neurons in cell culture. Surprisingly,they found that a compound called selamectin,which is used to treat parasitic flatworm infections,also triggered stem cells to turn into neurons. Selamectin works by blocking a particular type of ion channel in flatworms,but this ion channel is not found in vertebrates,which means that selamectin must be promoting the formation of neurons in mice via a different mechanism. Given that a drug related to selamectin is known to act on a subtype of receptors for the neurotransmitter GABA,Sun et al. wondered whether these receptors—known as GABAA receptors—might also underlie the effects of selamectin. Consistent with this idea,drugs that increased GABAA activity stimulated the formation of neurons,whereas drugs that reduced GABAA function blocked the effects of selamectin. In addition,Sun et al. showed that selamectin triggers human embryonic stem cells to become neurons,and that it also promotes the formation of new neurons in developing zebrafish in vivo. As well as revealing an additional mechanism for the formation of neurons from stem cells,the screening technique introduced by Sun et al. could help to identify further pro-neuronal molecules,which could aid the treatment of neurodevelopmental and neurodegenerative disorders. DOI: [http://dx.doi.org/10.7554/eLife.00508.002][1] [1]: /lookup/doi/10.7554/eLife.00508.002 View Publication

The physiologic function of BUBR1,a key component of the spindle checkpoint,was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro,BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes,which was correlated with an increase in megakaryocytic,but a decrease in erythroid,progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However,enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood,which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together,these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis. View Publication

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. View Publication

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation,and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%,but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single,larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture,or aggregates of ES cells in hanging drop culture,they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus,initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however,hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension,whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient,scalable bioprocesses for ES cell differentiation,and inform novel methods for the production of hematopoietic tissues. View Publication

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication

Embryonic stem cells can be induced in vitro,by coculture with the stromal line RP.0.10 and a mixture of interleukins 3,6,and 7,to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+,PgP-1+,c-kit+,Joro75-,B-220-,F4/80-,Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny. View Publication