Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties Gabriela F. Barnabe´ 1., Telma T. Schwindt1., Maria E. Calcagnotto1, Fabiana L. Motta1, Gilberto Jr. Martinez2, Allan C. de Oliveira3, Leda M. N. Keim5, Vaˆ nia D’Almeida3,4, Rosa´ lia Mendez-Otero5, Luiz E. Mello1* 1 Departamento de Fisiologia, Universidade Federal de Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Sa˜o Paulo, Brazil, 2 Departamento de Biof´ısica, Universidade Federal de Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Sa˜o Paulo, Brazil, 3 Departamento de Pediatria, Universidade Federal de Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Sa˜o Paulo, Brazil, 4 Departamento de Biocieˆncias, Universidade Federal de Sa˜o Paulo (UNIFESP), Santos, Sa˜o Paulo, Brazil, 5 Instituto de Biof´ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Rio de Janeiro, Brazil Abstract Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (b-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, b-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch- clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro. Citation: Barnabe´ GF, Schwindt TT, Calcagnotto ME, Motta FL, Martinez G Jr, et al. (2009) Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties. PLoS ONE 4(4): e5222. doi:10.1371/journal.pone.0005222 Editor: Mauricio Rojas, Emory University, United States of America Received August 25, 2008; Accepted March 12, 2009; Published April 16, 2009 Copyright: 2009 Filoso Barnabe´ et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by FAPESP and CNPq. Barnabe´ G.F., Schwindt T.T., are and Calcagnotto M.E. and Motta F.L. were FAPESP fellows. D’Almeida V. and Mello L.E. are recipients of CNPq fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lemello@unifesp.br . These authors contributed equally to this work. Introduction Mesenchymal stem cells (MSC) from bone marrow stroma have been broadly studied for use in the therapy of neurological disorders. The great interest in studying this multipotent cell population largely derives from the advantages it offers. For instance, MSC can be easily harvested for autologous transplan- tation and do not involve ethical issues. Many studies have demonstrated that MSC can give rise to neural cells, both in vitro [1–14] and in vivo [15–20]. In vitro experiments seeking the ideal conditions to obtain neural cells from MSC were initially conducted by two main research groups: Sanchez-Ramos et al. [6] and Woodbury et al. [9]. The former tried to differentiate MSC using molecules specifically involved in neural development (growth factors, neurotrophins, cytokines and retinoic acid) [6], while the latter proposed that neural induction could be performed simply by the addition of specific chemical compounds to the culture medium (b-mercaptoethanol [BME], dimethyl sulfoxide [DMSO] and butylated hydroxyanizole [BHA]) [9]. The chemical induction protocol used herein has two intriguing aspects: I) while the neuronal differentiation of embryonic stem cells and neural progenitor cells take days to happen [21], only a few hours are necessary for chemically-induced MSC to acquire morphological features and express specific markers of mature neurons (e.g., NeuN, MAP-2, NSE); and II) the percentage of differentiated neuronal cells exceeds 70%, which is extremely high compared to neuronal differentiation using growth factors [22]. Such results contradict the molecular mechanisms currently accepted for neural development because the development of mature neurons is a very slow process characterized by the induction and regulation of specific gene sets, as well as by distinct PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5222