Introduction The notion that phenotypic fate is indissolubly bound to the embryonic germ layer derivation of the cell has been challenged by several observations on different types of pluripotent stem cells. A number of authors have shown that several adult tissues harbour rare populations of stem cells with mesenchymal characteristics. When grown in vitro under specific conditions, or after genetic modification, these cells populations show an extensive capacity for self-renewal and differentiate into cell types not necessarily restricted to the mesodermic lineage (Blau et al., 2001; Prockop, 2003; Rafii and Lyden, 2003; Verfaillie, 2002). Similar results have been obtained after syngenic or xenogenic transplantations of such cells in a number of animal species, including primates, and in most cases it was possible to demonstrate integration of the transplanted donor cells in different host tissues (Krause et al., 2001; Liechty et al., 2000). In parallel, a number of studies have reported cases of chimerism after bone marrow infusions in humans (Gussoni et al., 2002; Mezey et al., 2003; Quaini et al., 2002; Weimann et al., 2003b), suggesting that the behaviour of the cells observed in animal models could to some extent be reproduced in man. Cells with neural characteristics appear to have been generated in vitro from adult stem cells of putative mesodermal origin and isolated from a variety of connective tissues, including bone marrow, umbilical cord blood, dermis and adipose tissue (Hermann et al., 2004; Jin et al., 2003; Kang et al., 2004; Padovan et al., 2003; Sanchez-Ramos et al., 2000; Sanchez-Ramos, 2002; Wislet-Gendebien et al., 2005; Wislet- Gendebien et al., 2003). However, attempts to assert transdifferentiation of adult bone-marrow-derived cells into neural lineages in vivo have produced discordant results. Some authors have reported the integration and differentiation of such cells in the brain (Munoz-Elias et al., 2004), whereas others have found that the few cells that were capable of engrafting into the nervous tissue fused with endogenous cells 3925 The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells (hMSCs) by simple in vitro treatments is appealing both conceptually and practically. However, whether phenotypic modulations observed after chemical manipulation of such stem cells truly represent a genuine trans-lineage differentiation remains to be established. We have re-evaluated the effects of a frequently reported biochemical approach, based on treatment with butylated hydroxyanisole and dimethylsulphoxide, to bring about such phenotypic conversion by monitoring the morphological changes induced by the treatment in real time, by analysing the expression of phenotype-specific protein markers and by assessing the modulation of transcriptome. Video time- lapse microscopy showed that conversion of mesenchymal stem cells to a neuron-like morphology could be reproduced in normal primary fibroblasts as well as mimicked by addition of drugs eliciting cytoskeletal collapse and disruption of focal adhesion contacts. Analysis of markers revealed that mesenchymal stem cells constitutively expressed multi-lineage traits, including several pertaining to the neural one. However, the applied ‘neural induction’ protocol neither significantly modulated the expression of such markers, nor induced de novo translation of other neural-specific proteins. Similarly, global expression profiling of over 21,000 genes demonstrated that gene transcription was poorly affected. Most strikingly, we found that the set of genes whose expression was altered by the inductive treatment did not match those sets of genes differentially expressed when comparing untreated mesenchymal stem cells and immature neural tissues. Conversely, by comparing these gene expression profiles with that obtained from comparisons between the same cells and an unrelated non- neural organ, such as liver, we found that the adopted neural induction protocol was no more effective in redirecting human mesenchymal stem cells toward a neural phenotype than toward an endodermal hepatic pathway. Key words: Bone-marrow-derived stem cells, Neurogenesis, Transdifferentiation Summary Neurogenic potential of human mesenchymal stem cells revisited: analysis by immunostaining, time- lapse video and microarray Nicoletta Bertani 1,‡ , Paolo Malatesta 1, * ,‡,§ , Giorgia Volpi 1 , Paolo Sonego 2 and Roberto Perris 1,2 1 Department of Evolutionary and Functional Biology, University of Parma, Viale delle Scienze 11/a, 43100 Parma, Italy 2 Division for Experimental Oncology 2, The National Cancer Institute, CRO-IRCCS, Via Pedemontana Occidentale 1, Aviano 33081, Italy *Present address: National Institute for Cancer Research (IST), 16132 Genoa, Italy ‡ These authors contributed equally to this work § Author for correspondence (e-mail: paolo.malatesta@istge.it) Accepted 25 May 2005 Journal of Cell Science 118, 3925-3936 Published by The Company of Biologists 2005 doi:10.1242/jcs.02511 Research Article Journal฀of฀Cell฀Science