letters to nature NATURE | VOL 412 | 16 AUGUST 2001 | www.nature.com 739 mechanically dissociating collected spheres (centrifuged for 10 min at 100g) into a single- cell suspension and replating in basal media containing EGF and FGF-2 (complete medium) as described 1,12 . Spheres were differentiated by transfer to glass coverslips coated with poly-L-ornithine (one sphere per coverslip) in complete medium for 1 d, basal medium for 1 d, then basal medium with 1% FCS for 4±5 d, then assessed by immuno- cytochemistry for neurons and glia. Freshly isolated NSCs from BU5X mice were co-cultured with C2C12 myogenic cells (5 ´ 10 3 cells cm -2 ) for 2 d in DME medium (Gibco) containing 20% heat-inactivated FCS, then in DME supplemented with 1% normal horse serum (CSL) for an additional 2±4 d in vitro. Cultures were ®xed for 5 min with 4% paraformaldehyde then processed for immunocytochemistry. Immunocytochemistry Double-antigen immunocytochemistry on neurospheres was performed as described previously 1,12 , using monoclonal antibody to b-tubulin type III (Sigma), GFAP antisera (Dako) and monoclonal antibody to O4 (immunoglobulin-m, IgM, Boehringer). Neural- derived muscle cells were identi®ed by simultaneous detection of endogenous GFP and muscle cell types identi®ed by mouse monoclonal antibodies to fast MyHC (NCL-MHCf, 1:10, Novocastra) or a-actinin-2 (1:750; ref. 25). These antigens were detected by appropriate TRITC-conjugated IgG secondary antibody (1:200; Southern Biotech). The proportion of neural cells that differentiated into muscle cells was determined by counting the number of GFP + nuclei expressing myogenic markers divided by the number of NSCs plated (counted 6h after plating). To detect the progeny of freshly isolated BU5X NSCs injected into (C57BL/6 ´ DBA/2) F 1 hybrid pups (E12.5), mice were perfused with 4% paraformaldehyde, their brain sectioned (10-mm slices) and immunocytochemistry performed to double-label, donor-derived cells immunoreactive to b-gal (anti-b-gal, Chemicon) with known markers for astrocytes (anti-GFAP, Chemicon) or neurons (anti- NeuN, Chemicon). Images were captured on a Nikon Diaphot microscope using a KX-85 (Apogee) camera. Received 21 March; accepted 29 June 2001. 1. Reynolds, B. A. & Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707±1710 (1992). 2. Richards, L. J., Kilpatrick, T. J. & Bartlett, P. F. De novo generation of neuronalcells from the adult mouse brain. Proc. Natl Acad. Sci. USA 89, 8591±8595 (1992). 3. McKay, R. Stem cells in the central nervous system. Science 276, 66±71 (1997). 4. Gage, F. H. Mammalian neural stem cells. 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We express our gratitude to P. Tam for supplying BU5X mice; S. S. Tan for transplantation assistance; E. Hardeman for supply and assistance with C2C12 myocytes and muscle-speci®c antibodies and J. Coonan for assistance with microinjections. Correspondence and requests for materials should be addressed to P.F.B. (e-mail: bartlett@wehi.edu.au). ................................................................. Antibodies inhibit prion propagation and clear cell cultures of prion infectivity David Peretz*, R. Anthony Williamson², Kiotoshi Kaneko*³, Julie Vergara*, Estelle Leclerc², Gerold Schmitt-Ulms*, Ingrid R. Mehlhorn*, Giuseppe Legname*, Mark R. Wormald§, Pauline M. Rudd§, Raymond A. Dwek§, Dennis R. Burton²k & Stanley B. Prusiner*¶# * Institute for Neurodegenerative Diseases and Departments of ¶ Neurology and # Biochemistry and Biophysics, University of California, San Francisco, California 94143-0518, USA ² Departments of Immunology and k Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA § The Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK .............................................................................................................................................. Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrP Sc ) template and endogenous cellular prion protein (PrP C ) is proposed to drive the formation of nascent infectious prions 1,2 . Reagents speci®cally binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuro- blastoma cells (ScN2a) infected with PrP Sc . Here we show that antibodies binding cell-surface PrP C inhibit PrP Sc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrP Sc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrP C molecules on the cell surface, and with the location of its epitope on PrP C . Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrP C for drug targeting. To study inhibition of prion propagation by antibodies, we used recombinant prion protein-speci®c Fabs D13, D18, R1, R2, E123, E149 and R72 (refs 3±6). The binding epitopes and af®nity constants of the antibodies for recombinant prion protein are shown in Supplementary Information Table 1. Fab R72 does not recognize prion protein in surface plasmon resonance (SPR) or on the cell surface, but does bind to PrP C coated onto the surface of wells for enzyme-linked immunosorbent assay (ELISA) 4 . A range of concentrations of each antibody was added to ScN2a cultures for 7 days. Cells were then collected and the level of PrP Sc in the culture analysed by immunoblotting. The level of PrP Sc in cells ³Present address: National Institute of Neuroscience, Tokyo 187-8502, Japan. © 2001 Macmillan Magazines Ltd