Interplay of Leukemia Inhibitory Factor and Retinoic Acid on Neural Differentiation of Mouse Embryonic Stem Cells Raquel Martı ´n-Iba ´n ˜ ez, Noelia Urba ´n, Sole `ne Sergent-Tanguy, Jose ´ Ramo ´ n Pineda, Nu ´ ria Garrido-Clua, Jordi Alberch, and Josep M. Canals * Departament de Biologia Cellular i Anatomia Patolo ` gica, Facultat de Medicina, IDIBAPS, Universitat de Barcelona, Spain Embryonic stem (ES) cells have great potential for cell replacement in neurodegenerative disorders. Implantation of these cells into the brain, however, requires their prior differentiation. We examined the interplay between leuke- mia inhibitory factor (LIF) and retinoic acid (RA) on neural differentiation of mouse ES (mES) cells. Mouse embryonic stem cells were allowed to form cell aggregates, the so- called embryoid bodies (EBs), in the absence or presence of LIF. In the absence of LIF, mES cells downregulated the expression of the undifferentiated mES cell marker Oct-3/ 4, and increased mRNA levels of two neural precursor markers, Sox-1 and Nestin, as well as the neuronal marker b-tubulin III. This neuronal differentiation was enhanced by treating EBs with RA. Moreover, RA irreversibly increased the number of postmitotic neurons in culture, as shown by the reduction of proliferating mES cells and the increase in b-tubulin III-positive cells 6 days after RA removal, which in turn affected mES cell viability. The addition of LIF during EBs formation, however, blocked completely this neuronal differentiation. Our findings also showed that pre-differentiation of mES cells in vitro avoided the terato- carcinoma formation observed when proliferating mES cells were grafted into the brain. In addition, mES cells pre-differentiated with RA in culture showed a reduction in proliferation and the presence of neural phenotypes after grafting. In conclusion, the present results indicate that RA enhances neuronal differentiation of mES cells in the absence of LIF, although it compromises cell viability and transplantation. V V C 2007 Wiley-Liss, Inc. Key words: neurodegenerative diseases; cell replace- ment; GABA; transplantation Embryonic stem (ES) cells, derived from the inner cell mass of the blastocyst, can be maintained in culture indefinitely in an undifferentiated state, and are capable of forming many specialized cell types, including those of the three primary embryonic germ layers, in response to proper instructional cues (Martin, 1981; Evans and Potten, 1991). Embryonic stem cells self-renewal can be main- tained in vitro via the growth factors provided by feeder cells (Smith, 2001) or by the addition to the culture me- dium of exogenous factors such as leukemia inhibitory fac- tor (LIF). Leukemia inhibitory factor and its close relatives are the only pro-pluripotency factors associated with mouse ES (mES) cells. Leukemia inhibitory factor enables mES propagation as a relative homogeneous population of undifferentiated stem cells (Burdon et al., 2002). Thus, re- moval of LIF induces mES cell differentiation, as shown by the reduction in Oct-3/4 expression levels (Faherty et al., 2005). Oct-3/4 is a POU domain homeobox tran- scription factor that is expressed during gametogenesis and in early embryonic cells, where it has been shown to be important for the maintenance of pluripotency (Nichols et al., 1998; Feldman et al., 2006). One strategy for mES cell differentiation involves the induction of cell aggregation-forming embryoid bodies (EBs) in suspension cultures. Different inducing factors and conditions during EBs formation have been assayed for neural differentiation in the presence (Tighe and Gudas, 2004; He et al., 2006) or absence of LIF (Bain et al., 1995; Okabe et al., 1996; Okada et al., 2004). Reti- noic acid induces differentiation of stem cells into several cell types resembling specific embryonic lineages (Simeone et al., 1990; Stornaiuolo et al., 1990; Chen and Gudas, 1996; Dinsmore et al., 1996; Rohwedel et al., 1999; Smith et al., 2001; Okada et al., 2004). Exposure of EBs to high concentrations of RA strongly drives neural differentia- tion, whereas low concentrations induce cardiomyocyte Sole `ne Sergent-Tanguy is currently at INSERM U643, CHU Ho ˆtel Dieu, 30 bvb Jean Monnet, 44093 Nantes, cedex 01, France. *Correspondence to: Josep M Canals, Departament de Biologia Cellular i Anatomia Patolo ` gica, Facultat de Medicina, IDIBAPS, Universitat de Bar- celona, C/Casanova, 143, E-08036 Barcelona, Spain. E-mail: jmcanals@ ub.edu Contract grant sponsor: Ministerio de Educacio ´n y Ciencia (Spain); Con- tract grant number: SAF2005-01335, SAF2005-00147; Contract grant sponsor: Ministerio de Sanidad y Consumo (Spain); Contract grant number: G03/167, G03/210, PI040659; Contract grant sponsor: Fundacio ´ La Caixa (Spain). Received 14 September 2006; Revised 5 December 2006; Accepted 6 December 2006 Published online 8 March 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21228 Journal of Neuroscience Research 85:2686–2701 (2007) ' 2007 Wiley-Liss, Inc.