In Vitro Myelination by Oligodendrocyte Precursor Cells Transfected With the Neurotrophin-3 Gene NAZARIO RUBIO,* RODRIGO RODRIGUEZ, AND MARIA ANGELES AREVALO Instituto Cajal, Consejo Superior de Investigaciones Cientificas (CSIC), Madrid, Spain KEY WORDS neurotrophin-3; oligodendrocyte precursors; myelination; myelin basic protein; regeneration ABSTRACT Oligodendrocyte precursor cells require exogenous neurotrophin-3 (NT-3) for differentiation into oligodendrocytes. We transfected precursor cells with the gene for NT-3 and looked for changes in their development into myelin-forming cells. The expression of NT-3 in transfected cells was demonstrated by reverse transcription followed by PCR as well as by Northern blots. Direct synthesis of the neurotrophin product and its release to the culture supernatants were also shown by specific ELISA. Transfection converts precursor cells into actively dividing cells that can incorporate 3 H-thymidine into DNA. In the absence of growth factors, a parallel increase in the survival of the transfected cultures was also demonstrated by the MTT test. The final demonstration of biological changes in transfected versus untreated cells was a 10-fold increase in myelin basic protein production observed in Western blots and the direct observation by phase-contrast and electron microscopy of myelin membranes in cocul- tures with hippocampal neurons. We discuss the future use of this transfected cells in regeneration and functional recovery in experimental models of multiple sclerosis. © 2004 Wiley-Liss, Inc. INTRODUCTION Oligodendrocytes are nervous system cells responsi- ble for the synthesis of myelin. They arise from progen- itors or precursors that proliferate, differentiate, and myelinate axons in postnatal stages (Pfeiffer et al., 1993; Miller, 1996). These bipotential precursor cells or oligodendrocyte precursor cells (OPCs) give rise to as- trocytes (in cultures containing 10% FCS) or to oligo- dendrocytes (in serum-free cultures) (Bo ¨gler et al., 1990; Barres et al., 1992; Kondo and Raff, 2000) when cultured in vitro. Neurotrophin-3 (NT-3) regulates the proliferation and differentiation of oligodendrocytes in culture (Barres et al., 1994; Cohen et al., 1996; Hein- rich et al., 1999). Platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) promote division and prevent the differentiation of OPCs (Noble et al., 1988; Bo ¨gler et al., 1990). There have been many reports of experimental ther- apeutic interventions in rats aimed at promoting the regeneration of demyelinated corticospinal tracts in vivo. The development of appropriate animal models and these successful cellular therapeutic interventions will be crucial for the treatment of all kinds of central nervous system disorders in human clinical practice. There has also been previous reports of the stimulation of myelinogenesis in culture by microglia (Hamilton and Rome, 1994) or by the interleukin-6 receptor-inter- leukin-6 fusion protein (Haggiag et al., 2001). The present experiments were designed to determine whether oligodendrocyte precursor cells transfected Grant sponsor: Direccion General de Investigacion, Ministerio de Ciencia y Tecnologia, Spain; Grant number: PM99-0102. *Correspondence to: Nazario Rubio, Instituto Cajal, CSIC, Dr. Arce Avenue 37, 28002 Madrid, Spain. E-mail: nazario@cajal.csic.es Received 7 November 2003; Accepted 23 December 2003 DOI 10.1002/glia.20035 Published online 24 March 2004 in Wiley InterScience (www.interscience. wiley.com). GLIA 47:78 – 87 (2004) © 2004 Wiley-Liss, Inc.