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Neuroprotection by cord blood neural progenitors involves antioxidants, neurotrophic and angiogenic factors ☆ Hadar Arien-Zakay a , Shimon Lecht a , Marian M. Bercu a , Rinat Tabakman a , Ron Kohen b , Hanan Galski c , Arnon Nagler c , Philip Lazarovici a, ⁎ a Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel b Department of Pharmacy, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, 91120, Israel c Laboratory of Molecular Immunobiology, Division of Hematology and Bone Marrow Transplantation and Cord Blood Bank, Chaim Sheba Medical Center, Tel-Hashomer, Israel a b s t r a c t a r t i c l e i n f o Article history: Received 5 October 2008 Revised 6 November 2008 Accepted 14 November 2008 Available online 25 November 2008 Keywords: Neuroprotection Oxygen–glucose deprivation Free radicals NGF VEGF FGF-2 PC12 cells Cord blood progenitors Human umbilical cord blood (HUCB) is a valuable source for cell therapy since it confers neuroprotection in str oke ani mal mod els . Howeve r, the res ponsib le sub- popula tio ns rema in to be est abli she d and the mechanisms involved are unkno wn. To explo re HUCB neuroprot ective properti es in a PC12 cell-bas ed ischemic neuron al model, we used an HUCB mononuclea r-en riched populatio n of collagen-ad herent cells, which can be differentia ted in vitro into a neuronal pheno type (HUCBNP). Upon co-cult ure with insulted- PC12 cells, HUCBNP conferred ∼30% neuroprotection, as evaluated by decreased lactate dehydrogenase and caspase-3 activities. HUCBNP decreas ed by 95% the level of free radica ls in the insulted-PC12 cells, in cor rel ati on wit h the appe ara nce of ant iox ida nts, as mea sur ed by cha nge s in the oxi dat ion –reduction potential of the medium using cyclic-voltammetry. An increased level of nerve growth factor (NGF), vascular endoth elial growth factor and basic fibrobla st growth facto r in the co-culture medium was tempo rally correlated with a -medium neuropr otect ion effect, which was parti ally abolishe d by heat denaturat ion. HUCBNP-induced neuroprotection was correlated with changes in gene expression of these neurotrophic factors, while blocke d by K252a, an antagonist of the TrkA/NGF recept or. These findings indicate that HUCBNP-induced neuroprotection involves antioxidant(s) and neurotrophic factors, which, by paracrine and/ or autocrine interactions between the insulted-PC12 and the HUCBNP cells, conferred neuroprotection. © 2008 Elsevier Inc. All rights reserved. Introduction Research on neuronal progenitors is driven by their potential use for cellular therapy of neurodegenerative disorders (Raedt and Boon, 2005) and damage to the central ne rvous system following stro ke and trauma (Chopp and Li, 2006). Sinc e the ava ilab ility of huma n neur onal stem cells derived from early embryos is extremely limited, progeni- tors of other origins, such as bone marrow (Sanchez-Ramos, 2002) or human umbilical cord blood (HUCB) (Buzańska et al., 2006), ar e bein g considered. HUCB contains multiple populations of pluripotent stem cells and can be considered an alternative to embryonic stem cells. Inter esti ngly , sub- popul ation s of HUCB cell s, either hema topoi etic (Chen et al., 2005) or mesenchymal-like (Low et al., 2008) cells upon treatment with specific growth factors are able to differentiate into neuron-like cells in culture with functional voltage- and ligand-gated chan nels (Sun et al ., 2005), and thus ame nab le to treatment of neurologic diseases (Harris et al., 2007; Low et al., 2008; Newman et al. , 2004). Sinc e HUCB cell s hav e been prove d ther apeutica lly benefici al in ani malmodel s of str oke (Chen et al ., 2001) and trau matic bra in inj ury( Luet al ., 200 2) alb eitwithout evi den ce of neuron al tis sue replacement, the promise of using HUCB-derived stem cells in the treatment of brain disorders relies on a “bystander ” function through secretion of growth factors and cytokines inducing neuroprotection and immuno-modulation ( Martino and Pluchino, 2006). Using neuronal conditioning medium and nerve growth factor (NGF) , we recently establis hed a novel appro ach for the isolation and in vitro differ entiation of a populatio n of collage n-adher ent, nestin- positive progeni tors from HUCB, operationall y defined as neurona l- like progenitor s (HUCBNP ) (Arien-Zakay et al., 2007). We hypothe- size that these cell s, in the undiff erentiated or neuronal-like differ entiat ed phenotype may be useful for neuronal therapy . Therefore, our aims in the present study were to evaluate in vitro the neuroprotective potential of HUCBNP in an ischemic PC12 cell model and to assess some of the cellular mechanisms involved in the neuroprotective effect. Using a special ischemic device ( Tabakman et al., 2002) we developed an in vitro model of ischemia based on combined oxygen and glucose deprivation (OGD) insult, followed by reoxygenation (OGD/reoxygenation), thus mimicking the pathophy- siol ogic alconditions of brain isc hemia (Setaetal., 2002; Taba kman et Experimental Neurology 216 (2009) 83–94 ☆ This study is part of a PhD thesis to be submitted to The Hebrew University of Jerusalem by H.A.Z. ⁎ Corresponding author. Fax: +972 2 6757490. E-mail address: [email protected] (P. Lazarovici). 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reser ved. doi:10.1016/j.expneurol.2008.11.006 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
