EFFICIENT EXPANSION AND GENE TRANSDUCTION OF MOUSE NEURAL STEM/PROGENITOR CELLS ON RECOMBINANT FIBRONECTIN G. RAPPA, a1,2 D. KUNKE, b1 J. HOLTER, b D. B. DIEP, b J. MEYER, c C. BAUM, c O. FODSTAD, a2 S. KRAUSS b AND A. LORICO a2 * a Department of Tumor Biology, Norwegian Radium Hospital, Monte- bello, Oslo 0310, Norway b Department of Cellular and Genetic Therapy, The National Hospital, Oslo 0027, Norway c Department of Hematology/Oncology, Hannover Medical School, Hannover 30625, Germany Abstract—Neural stem/progenitor cells (NSCs) are com- monly grown as floating neurospheres in medium contain- ing basic fibroblast growth factor and epidermal growth factor. Under these conditions, about 1% of the cells retain multipotentiality. We developed a protocol based on cul- ture of NSCs in adherence on recombinant fibronectin (rFN) to transduce up to 90% NSCs at a multiplicity of infection of 2 with no need for viral concentration or pro- duction of serum-free retroviral supernatants. NSCs grew faster on rFN than as neurospheres on tissue culture plas- tic and did not lose their stem cell nature or multipotenti- ality. Furthermore, retroviral-mediated transgene expres- sion was sustained with time in culture and upon differen- tiation into neurons and astrocytes. These experimental conditions may be utilized to study the function of various genes in NSCs, and to manipulate NSCs for gene and cell therapy of several neurological diseases. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: stem cells, neural cells, retrovirus, gene therapy. Neural stem/progenitor cells (NSCs) carry potential for the replacement of neurons lost to injury or disease (Riess et al., 2002), and for cell and gene therapy of neurodegen- erative syndromes (Le Belle and Svendsen, 2002; Pluchino et al., 2003). They can be engineered to produce therapeutic molecules against brain cancer cells (Aboody et al., 2000; Benedetti et al., 2000). To expand the poten- tial of NSCs by introducing specific gene products, efficient gene transfer is a prerequisite. Several methods are used to introduce genetic infor- mation into NSCs. Protocols based on cationic lipids (Falk et al., 2002) generally yield low efficiency and stability of the transgene. Viral vectors including adenovirus (Falk et al., 2002), recombinant adeno-associated virus (Hughes et al., 2002; Wu et al., 2002), feline immunodeficiency virus (Hughes et al., 2002) and retrovirus (Owens et al., 2002) are more efficient and therefore more generally used. However, use of viral vectors may trigger differentiation of NSCs (Hughes et al., 2002). Recombinant fibronectin (rFN) has been shown to fa- cilitate retroviral infection by co-localization of hematopoi- etic cells and viral particles on the same rFN molecule (Hanenberg et al., 1996). In this work, we investigated whether it was possible to grow and transduce NSCs on rFN without loss of their stem cell characteristics. Our results show that culture of NSCs in adherence on rFN allows almost complete retroviral gene transfer without intrinsic alterations of the differentiation potential of the transduced cells. EXPERIMENTAL PROCEDURES Preparation and culture of NSCs and skin-derived stem/progenitor cells (SSCs) NSCs were prepared from neonatal forebrains of C57BL/6 mice (P0 stage) as previously described (Machon et al., 2002). Skin- derived stem/progenitor cells (SSCs) were prepared from the same mice according to the protocol of Toma et al. (2001). NSCs and SSCs were cultured in serum-free Neurobasal-A medium containing B27 (Gibco-BRL, Rockville, MD, USA), 2 mM L-glu- tamine, 10 ng/ml basic fibroblast growth factor, and 20 ng/ml epidermal growth factor (both from R&D Systems, Minneapolis, MN, USA). Low passage (passages 2–5) NSCs and SSCs were used throughout all the experiments. Untreated tissue culture 24-well plates were coated with 10 g/cm2 rFN (Retronectin; Takara Shuzo Co., Shiga, Japan), according to the manufacturer’s instructions. For culture on rFN, neurospheres growing on tissue culture plastic (TCP) were incubated in the presence of 0.05% trypsin with 0.02% EDTA for 7 min at 37 °C. The single cell suspensions were then pelleted by centrifugation at 400g for 3 min, resuspended in complete medium, filtered through a 70-m filter to remove eventual remaining cell clumps, and added to rFN-coated plates. The cells were then incubated at 37 °C in an atmosphere of 5% CO 2 in air. 1 These two authors contributed equally to the present work. 2 Present address: Cancer Research Institute, University of South Alabama, Mobile, AL 36688, USA. *Corresponding author. Tel: +1-251-461-1636; fax: +1-251-460-6994. E-mail address: alorico@usouthal.edu (A. Lorico). Abbreviations: EGFP, enhanced green fluorescence protein; FMEV, Friend mink cell focus forming/murine embryonic stem cell virus; F-RNA, RNA from cells growing as floating cultures; -GCSh, catalytic (heavy) subunit of -glutamyl cysteine synthetase; GFAP, glial fibrillary acidic protein; IRES, internal ribosomal entry site; MAP, microtubule- associated protein; MOI, multiplicity of infection; NSC, neural stem/ progenitor cell; NSCs/rFN, untransduced neural stem/progenitor cells as monolayers on recombinant fibronectin; NSCs/TCP, untransduced neural stem/progenitor cells as floating neurospheres on tissue culture plastic; rFN, recombinant fibronectin; RF-RNA, RNA from cells grow- ing 6 days on recombinant fibronectin, then 1 week as floating culture; SSC, skin-derived stem/progenitor cell; TCP, tissue culture plastic; T-RNA, RNA from transduced cells. Neuroscience 124 (2004) 823– 830 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.11.030 823