Region-specific generation of functional neurons from naive embryonic stem cells in adult brain Tibor Harkany,* ,1 Michael Anda ¨ng,* ,1 Hylke Jan Kingma,* Tama ´s J. Go ¨rcs, Carl D. Holmgren,à Yuri Zilberterà and Patrik Ernfors* *Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics/Center of Excellence in Developmental Biology and àDepartment of Neuroscience, Karolinska Institutet, Stockholm, Sweden  C & O Vogt Institute for Brain Research, University of Du ¨sseldorf, Du ¨sseldorf, Germany Abstract Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into func- tional neurons when transplanted in the central nervous sys- tem. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipi- ent tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue charac- teristics in formulating cell replacement strategies for neuro- degenerative disorders. Keywords: differentiation, engraftment, grafting, mouse. J. Neurochem. (2004) 88, 1229–1239. In the central nervous system (CNS), grafts of in vitro differentiated neural stem cells (NSC) established from the embryonic, early postnatal and adult CNS, and embryonic stem (ES) cell-derived progenitors of multiple developmental stages generated in vitro survive for long periods, undergo phenotypic differentiation, and acquire neurochemical and electrophysiological properties similar to those of host neurons, with a positive impact on disease behaviour and depleted brain functions (McDonald et al. 1999; Temple 2001; Bjo ¨rklund et al. 2002; Kim et al. 2002). The ability of transplanted embryonic and adult NSC to generate neurons in contrast to glia; or different classes of neurons varies greatly among distinct regions of the CNS. NSC differentiate primarily into glia when transplanted into non-neurogenic brain regions such as the cerebellum, striatum and spinal cord (Gage et al. 1995; Fricker et al. 1999; Chow et al. 2000; Shihabuddin et al. 2000), and hippocampus-derived progenitors become olfactory neurons when placed in the rostral migratory stream (Suhonen et al. 1996). Transplan- tation of cells firmly committed to a neuronal lineage and expressing immature or mature neuronal markers have a greater propensity for neuronal differentiation in non-neuro- genic brain regions, in particular the spinal cord (Shin et al. 2000). Similar results have been obtained by transplanting ES cell-derived embryoid bodies and ES cell-derived in vitro-differentiated neural cells (McDonald et al. 1999; Liu et al. 2000; Andressen et al. 2001; Zhang et al. 2001; Bjo ¨rklund et al. 2002; Kim et al. 2002). Received May 22, 2003; revised manuscript received September 28, 2003; accepted October 31, 2003. Address correspondence and reprint requests to Dr Patrik Ernfors, Laboratory of Molecular Neurobiology, Department of Medical Bio- chemistry and Biophysics, Scheeles va ¨g 1:A1, Karolinska Institutet, S- 171 77 Stockholm, Sweden. E-mail: Patrik.Ernfors@mbb.ki.se 1 These authors contributed equally to the present study. Abbreviations used: ALP, alkaline phosphatase; bFGF, basic fibroblast growth factor; Cy, carbocyanine; DBX, doublecortin; DMEM, Dul- becco’s modified Eagle’s medium; ES, embryonic stem; FCS, fetal calf serum; GFP, green fluorescent protein; LIF, leukaemia inhibitory factor; mES, mouse ES; PB, sodium phosphate buffer (0.1 M, pH 7.4); PFA, paraformaldehyde; ir, immunoreactive; NSC, neural stem cells; TH, tyrosine hydroxylase; Tuj1, b-111-tubulin. Journal of Neurochemistry , 2004, 88, 1229–1239 doi:10.1046/j.1471-4159.2003.02243.x Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 88, 1229–1239 1229