JOURNAL OF CELLULAR PHYSIOLOGY 204:51–62 (2005) Nestin-Expressing Neural Stem Cells Identified in the Scar Following Myocardial Infarction JESSICA DRAPEAU, 1,2 VIVIANE EL-HELOU, 1,2 ROBERT CLEMENT, 1 SAMAR BEL-HADJ, 1,2 HUGUES GOSSELIN, 1 LOUIS-ERIC TRUDEAU, 3 LOUIS VILLENEUVE, 1 AND ANGELINO CALDERONE 1,2 * 1 Department of Physiology, University of Montreal, Montreal, Quebec, Canada 2 Montreal Heart Institute, Montreal, Quebec, Canada 3 Department of Pharmacology, University of Montreal, Montreal, Quebec, Canada Nerve fiber innervation of the scar following myocardial damage may have occurred either via the growth of pre-existing fibers and/or the mobilization of neural stem cells. The present study examined whether neural stem cells were recruited to the infarct region of the rat heart following coronary artery ligation. The neural stem cell marker nestin was detected in the infarct region of 1-week post-myocardial infarct (MI) male rats and cultured scar-derived neural-like cells. By contrast, nestin staining was undetected in either scar myofibroblasts or cardiac myocytes residing in the non-infarcted left ventricle. Reactive astrocytes were isolated from the infarct region and characterized by the co-expression of nestin, glial fibrillary acidic protein, and vimentin. Specific staining of oligodendrocytes and neurons was also detected in the infarct region and cultured scar-derived neural-like cells. Furthermore, neurofilament-M positive fibers were identified in the scar and tyrosine hydroxylase immunoreactivity was observed in peripherin-positive neurons. Neurite formation was induced in PC12 cells treated with the conditioned-media of primary passage scar-derived cells, highlighting the synthesis and secretion of neurotrophic factors. Nerve growth factor (NGF) and brain-derived neurotrophic factor were detected in myofibroblasts and neural cells, and both cell types expressed the NGF receptors trkA and p75. These data highlight the novel observation that neural stem cells were recruited to the infarct region of the damaged rat heart and may contribute in part to nerve fiber growth and subsequent innervation of the scar. J. Cell. Physiol. 204: 51–62, 2005. ß 2004 Wiley-Liss, Inc. Scar formation of the damaged heart following coronary artery ligation was defined as reparative fibrosis and constituted an essential physiological event that prevented cardiac rupture (Sun and Weber, 2000). The myofibroblast, characterized by the concomitant expression of a-smooth muscle actin and vimentin was the principal cell implicated in reparative fibrosis (Gabbiani, 1998). In response to local growth factors and inflammatory cytokines, myofibroblast prolifera- tion was associated with the increased synthesis and deposition of extracellular matrix proteins that led to the formation of a collagen scar (Gabbiani, 1998). Concomitant with scar formation was the observation that Schwann cells and axons were detected along the periphery and within the damaged region of the heart induced by either coronary artery occlusion or trans- diaphragmatic freeze-thaw injury (Vracko et al., 1990). The authors concluded that nerve fiber regeneration occurred in the newly formed scar of the damaged heart. A similar paradigm was described during cutaneous scar formation and healing as sensory nerve innervation and/or sprouting had occurred and was facilitated in part by the local release of nerve growth factor (NGF) by invading myofibroblasts (Matsuda et al., 1998; Liu et al., 1999; Hasan et al., 2000). Nerve fiber regeneration and subsequent innervation of the scar, regardless the tissue may have occurred either via the growth of pre-existing fibers and/or the recruitment of neural stem cells. Indirect evidence to support the latter premise was recently reported as multipotent stem cells were identified in the dermis and differentiated in culture to neural cells (Toma et al., 2001). The neural stem cell marker nestin was co-expressed with the neuron- specific markers bIII-tubulin and neurofilament-M (Lendahl et al., 1990; Toma et al., 2001). Moreover, nestin-positive neural stem cells in the dermis further differentiated to either an astrocyte or oligodendrocyte (Toma et al., 2001). In the CNS, the mobilization of resident stem cells represented a putative response to tissue damage, and the scar microenvironment provided the appropriate stimuli to induce nestin-expressing neural stem cell differentiation to neurons and/or astrocytes (Johansson et al., 1999; Rice et al., 2003). Recent studies support the premise that the infarct region of the damaged heart was capable of recruiting stem cells. The injection of either Sca-1 positive cardiac progenitor cells or c-Kit positive bone marrow cells in the peri-infarct region of the heart were subsequently identified in the scar (Orlic et al., 2001; Oh et al., 2003). Based on these latter observations and the postulated role of neural stem cell recruitment to damaged tissue, the present study examined whether scar formation post-myocardial infarct (MI) mobilized nestin-expres- sing neural stem cells. Second, the differentiation of neural stem cells to either a neuron, astrocyte, and/or oligodendrocyte phenotype was examined. Lastly, as ß 2004 WILEY-LISS, INC. Contract grant sponsor: Heart and Stroke Foundation of Canada; Contract grant sponsor: Quebec, Canadian Institutes of Health Research; Contract grant sponsor: ‘‘La Fondation de l’Institut de Cardiologie de Montre´al.’’ *Correspondence to: Angelino Calderone, Research Center, Institut de Cardiologie de Montre´al, 5000 rue Be´langer Est, Montre´al, Que´bec, Canada H1T 1C8. E-mail: angelo.calderone@umontreal.ca Received 10 August 2004; Accepted 13 October 2004 DOI: 10.1002/jcp.20264