Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules Maria Grazia Aluigi c , Rosella Coradeghini a , Chiara Guida a, * , Chiara Scanarotti a , Anna Maria Bassi b , Carla Falugi c , PierLuigi Santi a , Edoardo Raposio a a University of Genova, Department of Surgery, Morphology Disciplines and Integrated Methodologies (DICMI), Laboratory of Tissue Engineering, Via De Toni 14, 16132 Genova, Italy b University of Genova, Department of Experimental Medicine (DIMES), 16132 Genova, Italy c University of Genova, Department of Biology (DIBIO), 16132 Genova, Italy Received 21 July 2008; revised 30 October 2008; accepted 25 February 2009 Abstract A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer’s disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre- adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes. Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Pre-adipocytes; Adult stem cells; Neural differentiation; Cholinergic molecules; Choline acetyltransferase 1. Introduction Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. Pre- adipocytes (PA), like the mesodermic derivative cells (such as bone marrow, mesenchymal and endothelial cells) retain the high plasticity to convert from one tissue into cells with multiple potentialities (Wosnitza et al., 2007). PA differentiate into various cell types including the osteo- genic, chondrogenic, adipogenic, myogenic and endothelial lineages (Zuk et al., 2001, 2002; Rodriguez et al., 2005; Strem et al., 2005; Wosnitza et al., 2007; Schaffler and Buchler, 2007). Recently Huang et al. (2007), Ashjian et al. (2003), Safford et al. (2002) and Raposio et al. (2007) reported on the possi- bility of obtaining populations of neurogenic-like cells, expressing neural and/or glial markers. Raposio et al. (2007) exposed pre-adipocytes to a neurogenic medium, mimicking the natural conditions for neural differentiation, by adding insulin, indomethacin and isobutylmethylxanthine to the culture medium (Ashjian et al., 2003). Insulin can promote the maturation of differentiating neocortical cells in rat brain (Wan et al., 1997; Plitzko et al., 2001). Indomethacin, an inhibitor of cyclooxygenase, can promote the survival of neural cells after ischemic injury to the central nervous system (Grilli et al., 1996; Asanuma et al., 2001). Isobutylmethylxanthine, an inhibitor of phosphodiesterase, can stimulate the increase of * Corresponding author. Tel.: þ39 0103537311; fax: þ39 0103537803. E-mail address: chiara.guida@unige.it (C. Guida). 1065-6995/$ - see front matter Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2009.02.014 Cell Biology International 33 (2009) 594e601 www.elsevier.com/locate/cellbi