In vitro hypoxic preconditioning of embryonic stem cells as a strategy of promoting cell survival and functional benefits after transplantation into the ischemic rat brain Michelle Hedrick Theus a,b , Ling Wei a , Lin Cui a , Kevin Francis a,b , Xinyang Hu a , Christine Keogh a , Shan Ping Yu a,b, ⁎ a Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA b Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA Received 13 June 2007; revised 24 November 2007; accepted 16 December 2007 Available online 5 January 2008 Abstract Hypoxic preconditioning (HP) and stem cell transplantation have been extensively studied as individual therapies for ischemic stroke. The present investigation is an initial effort to combine these methods to achieve increased therapeutic effects after brain ischemia. Sublethal in vitro hypoxia pretreatment significantly enhanced the tolerance of neurally-differentiating embryonic stem (ES) cells and primary bone marrow mesenchymal stem cells (BMSC) to apoptotic cell death (40–50% reduction in cell death and caspase-3 activation). The HP protective effects on cultured cells lasted for at least 6 days. HP increased secretion of erythropoietin (EPO) and upregulated expression of bcl-2, hypoxia-inducible factor (HIF-1α), erythropoietin receptor (EPOR), neurofilament (NF), and synaptophysin in ES cell-derived neural progenitor cells (ES-NPCs). The HP cytoprotective effect was diminished by blocking EPOR, while pretreatment of ES-NPCs with recombinant human EPO mimicked the HP effect. HP-primed ES-NPCs survived better 3 days after transplantation into the ischemic brain (30–40% reduction in cell death and caspase-3 activation). Finally, transplanted HP-primed ES-NPCs exhibited extensive neuronal differentiation in the ischemic brain, accelerated and enhanced recovery of sensorimotor function when compared to transplantation of non-HP-treated ES-NPCs. The cell-priming strategy aimed to promote transplanted cell survival and their tissue repair capability provides a simple yet effective way of optimizing cell transplantation therapy. © 2007 Elsevier Inc. All rights reserved. Keywords: Hypoxia; Embryonic stem cells; Bone marrow mesenchymal stem cells; Ischemic stroke; Transplantation; Preconditioning; Erythropoietin; Apoptosis; Functional recovery Introduction Stem cell transplantation has emerged as a potential restorative therapy for CNS disorders such as ischemic stroke and spinal cord injury. Embryonic stem (ES) cells are capable of proliferating and differentiating into neural progenitor cells with the use of induction protocols, such as those involving retinoic acid (RA), ultimately leading to the development of functionally mature neurons and glial cells (Bain et al., 1995, 1996; Gottlieb and Huettner, 1999; McDonald et al., 1999; Wei et al., 2005). Using stem cells including ES cells as grafts has provided hope for tissue repair and functional restoration after CNS injury (Holm and Isacson, 1999; Bjorklund and Lindvall, 2000; Roy et al., 2006; Sonntag et al., 2007). This may be achieved through the integration of differentiating stem cells into neural networks within host tissues and/or by contributing trophic support to protect ‘at risk’ cells and promote endogenous neurogenesis and angiogenesis in and around the damaged brain areas (Polezhaev and Alexandrova, 1984; Kordower et al., 1996; Borlongan et al., 1998). To this end, infusion of growth factors has been shown to promote endogenous progenitor proliferation in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons (Nakatomi et al., 2002). Although Available online at www.sciencedirect.com Experimental Neurology 210 (2008) 656 – 670 www.elsevier.com/locate/yexnr ⁎ Corresponding author. Department of Pharmaceutical and Biomedical Sciences, 280 Calhoun Street, Medical University of South Carolina, Charleston, SC 29425, USA. Fax: +1 843 792 1712. E-mail address: yusp@musc.edu (S.P. Yu). 0014-4886/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2007.12.020