Neurogenic differentiation of murine and human adipose-derived stromal cells Kristine M. Safford, a Kevin C. Hicok, b Shawn D. Safford, a Yuan-Di C. Halvorsen, b William O. Wilkison, b Jeffrey M. Gimble, b and Henry E. Rice a, * a Department of Surgery, Division of Pediatric Surgery, Box 3815, Duke University Medical Center, Durham, NC 27710, USA b Artecel Sciences, Inc., Durham, NC, USA Received 1 May 2002 Abstract The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1–3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Fol- lowing neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal dif- ferentiation of stromal cells from adipose tissue has broad biological and clinical implications. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Adipose-derived adult stem cell; Mesenchymal stem cell; Neurogenic differentiation Neural tissue has long been regarded as incapable of regeneration and the identification of cell populations capable of neuronal differentiation has generated intense interest [1,2]. Stem cells from embryonic tissue as well as adult brain are capable of undergoing expansion and neuronal differentiation in vitro and in vivo [3–6]. However, the inaccessibility of these stem cells limits their clinical utility and has led to the search for alter- native cells that are capable of neuronal differentiation. Pluripotent mesenchymal stem and progenitor cells have been detected in multiple tissues, including bone marrow and umbilical cord blood [7–11]. Under appropriate conditions, bone marrow stromal cells se- lectively differentiate into mesenchymal lineages [8,9,12– 14]. Recent studies have shown that bone marrow stromal cells can be induced to neuronal differentiation in vitro and in vivo [15–19]. Adipose tissue has been identified as an alternative source of pluripotent stromal cells [20–22]. These cells have been termed adipose-derived adult stem (ADAS) cells, as they are self-renewing and can be induced to various mesenchymal tissues, including chondrocytes, adipocytes, osteoblasts, and myocytes [21,23,24]. To date, ADAS cells have not been demonstrated to be capable of differentiation towards non-mesenchymal lineages. We now report that murine and human ADAS cells can be induced to undergo morphologic and phenotypic changes consistent with neuronal differentiation. Adipose tissue may represent an alternative source of cells capable of neuronal differentiation, potentially enhancing their use- fulness in the treatment of neurological disease. Materials and methods Cellharvestandculture. For isolation of murine ADAS (muADAS) cells, we used BalbC mice (Charles River Laboratories). Animals were housed under standard conditions and the IACUC of Duke University Biochemical and Biophysical Research Communications 294 (2002) 371–379 www.academicpress.com BBRC * Corresponding author. Fax: +1-919-681-8353. E-mail address: rice0017@mc.duke.edu (H.E. Rice). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)00469-2