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Rensselaer, NY, United States

Gay L.,University of Oregon | Gay L.,Howard Hughes Medical Institute | Miller M.R.,University of Oregon | Miller M.R.,Howard Hughes Medical Institute | And 9 more authors.
Genes and Development

Transcriptional profiling is a powerful approach for understanding development and disease. Current celltype-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging" a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT+ cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre+ brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT+ bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice. © 2013 by Cold Spring Harbor Laboratory Press. Source

De Genst E.,University of Cambridge | Messer A.,Neural Stem Cell Institute | Messer A.,University at Albany | Dobson C.M.,University of Cambridge
Biochimica et Biophysica Acta - Proteins and Proteomics

Protein misfolding disorders, including the neurodegenerative conditions Alzheimer's disease (AD) and Parkinson's disease (PD) represent one of the major medical challenges or our time. The underlying molecular mechanisms that govern protein misfolding and its links with disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species within the crowded environment of the cell and tissue. Nevertheless, much progress has been made in understanding these events in recent years through innovative experiments and therapeutic strategies, and in this review we present an overview of the key roles of antibodies and antibody fragments in these endeavors. We discuss in particular how these species are being used in combination with a variety of powerful biochemical and biophysical methodologies, including a range of spectroscopic and microscopic techniques applied not just in vitro but also in situ and in vivo, both to gain a better understanding of the mechanistic nature of protein misfolding and aggregation and also to design novel therapeutic strategies to combat the family of diseases with which they are associated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. Source

Stern J.H.,Neural Stem Cell Institute | Temple S.,Neural Stem Cell Institute

Retinal degenerative disease has limited therapeutic options and the possibility of stem cell-mediated regenerative treatments is being actively explored for these blinding retinal conditions. The relative accessibility of this central nervous system tissue and the ability to visually monitor changes after transplantation make the retina and adjacent retinal pigment epithelium prime targets for pioneering stem cell therapeutics. Prior work conducted for several decades indicated the promise of cell transplantation for retinal disease, and new strategies that combine these established surgical approaches with stem cell-derived donor cells is ongoing. A variety of tissue-specific and pluripotent-derived donor cells are being advanced to replace lost or damaged retinal cells and/or to slow the disease processes by providing neuroprotective factors, with the ultimate aim of long-term improvement in visual function. Clinical trials are in the early stages, and data on safety and efficacy are widely anticipated. Positive outcomes from these stem cell-based clinical studies would radically change the way that blinding disorders are approached in the clinic. © 2011 The American Society for Experimental NeuroTherapeutics, Inc. Source

Stern J.,Neural Stem Cell Institute | Temple S.,Neural Stem Cell Institute
Developments in Ophthalmology

Remarkable progress over the past decade has led to the first clinical studies of stem cell therapy for retinal disease. The unique access retina offers for implantation, monitoring, and ablation is well suited for stem cell trials, and retinal applications have now moved to the forefront of the field of regenerative medicine. Retinal progeny derived from either pluripotent stem cells or tissue-specific retinal and retinal pigment epithelium (RPE) stem cells have the capacity both to replace damaged retina and to provide trophic support that slows disease progression. In contrast, bone marrow and neural stem cells produce nonretinal progeny that provide trophic support but with limited integration and capacity to differentiate into retinal progeny that can replace damaged retinal tissue. Embryonic and induced pluripotent stem cells differentiated into neural retinal and RPE progeny provide an unlimited supply of human cells for transplantation and disease modeling but raise the risks of aberrant differentiation and over proliferation. Tissue-specific stem cells isolated from neural retina or RPE that are naturally committed to retinal fates have a restricted lineage potential that improves the margin of safety. This improved safety of retina and RPE stem cells is balanced, however, by a restricted proliferative potential, which limits the quantity of progeny produced. In this chapter, we review the types of stem cells under development for retinal therapy. © 2014 S. Karger AG, Basel. Source

Bjornsson C.S.,Neural Stem Cell Institute | Apostolopoulou M.,Neural Stem Cell Institute | Tian Y.,University at Albany | Temple S.,Neural Stem Cell Institute
Developmental Cell

Although many features of neurogenesis during development and in the adult are intrinsic to the neurogenic cells themselves, the role of the microenvironment is irrefutable. The neurogenic niche is a melting pot of cells and factors that influence CNS development. How do the diverse elements assemble and when? How does the niche change structurally and functionally during embryogenesis and in adulthood? In this review, we focus on the impact of non-neural cells that participate in the neurogenic niche, highlighting how cells of different embryonic origins influence this critical germinal space. © 2015 Elsevier Inc. Source

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