Modulated Generation of Neuronal Cells from Bone Marrow by Expansion and Mobilization of Circulating Stem Cells with in Vivo Cytokine Treatment S. Corti,* F. Locatelli,* S. Strazzer,† S. Salani,† R. Del Bo,* D. Soligo,‡ P. Bossolasco,§ N. Bresolin,* , † G. Scarlato,* and G. P. Comi* *Centro Dino Ferrari, Dipartimento di Scienze Neurologiche, Universita ` degli Studi di Milano, and ‡Centro Trapianti di Midollo, I.R.C.C.S. Ospedale Maggiore Policlinico, 20122 Milan, Italy; †I.R.C.C.S. Eugenio Medea, Bosisio Parini, Lecco, Italy; and §Fondazione Matarelli, Milan, Italy Received March 5, 2002; accepted July 9, 2002 The aim of the present study is to determine whether the expansion and mobilization of circulating bone marrow (BM) stem cells by in vivo treatment with granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF) increase the amount of BM- derived neuronal cells in mouse brain. The presence of BM-derived cells in the brain was traced by trans- planting into lethally irradiated adults and newborns adult BM from transgenic mice that ubiquitously ex- pressed enhanced green fluorescent protein (GFP). GFP and Y-chromosome donor-derived cells were present in several brain areas of all treated mice (cor- tical and subcortical areas, cerebellum, olfactory bulb). The presence of GFP cells expressing nuclear neural specific antigen (NeuN), neurofilament, and -III tubulin in cortical forebrain and olfactory bulb (OB) was higher in G-CSF–SCF treated groups (P < 0.05, analysis of variance, Fisher post hoc). We ob- served that overall the amount of double positive cells was higher in animals treated at birth than in adults and in OB than in forebrain areas (P < 0.05). Temporal cortical areas of cytokine-treated adult animals re- vealed a mean threefold increase in the number of GFP cells expressing the nuclear neural specific an- tigen (211  86 GFPNeuN/mm 3 in G-CSF  SCF treated mice and 66  33 GFPNeuN/mm 3 in control animals). GFP cells coexpressing neuronal markers contain only one nucleus and have a DNA index (a measure of DNA ploidy) identical to that of surround- ing neurons, thus excluding donor cell fusion with endogenous cells as a relevant phenomenon under these experimental conditions. Our results indicate that G-CSF and SCF administration modulates the availability of GFP cells in the brain and enhances their capacity to acquire neuronal characteristics. Cy- tokine stimulation of autologous stem cells might be seen as a new strategy for neuronal repair in neuro- degenerative diseases. © 2002 Elsevier Science (USA) Key Words: neuronal neogenesis; stem cells; bone marrow transplantation; GFP. INTRODUCTION Stem cell transplantation is a potential strategy for the treatment of neurodegenerative diseases and cen- tral nervous system injury. Recently, stem cells capa- ble of differentiating in tissues other than those where they reside have been found in adult tissues. These cells can be used as a possible transplantable cell source (1). Bone marrow (BM) contains not only blood precursors, but also cells that can differentiate in vivo and in vitro in mesenchymal tissues (bone, adipocyte, cartilage, cardiac, and skeletal muscle) and neural tis- sues as well (6, 14). It has been demonstrated that human bone marrow stromal cells and umbilical cord blood can be induced in vitro to exhibit a neuronal phenotype (17, 22). Intravascular transplantation of genetically marked adult mouse BM into irradiated normal adult hosts demonstrated that BM cells migrate into the brain and differentiate in cells expressing several neuronal anti- gens (3). Generation of neuronal cells was also ob- served when adult mouse BM were transplanted into newborn mice incapable of developing cells of the my- eloid and lymphoid lineages, which could thus be transplanted without being irradiated (9). Although the percentage of neuronal cells derived from BM is low, accounting for 0.2– 0.3% of the total neurons (as estimated in the olfactory bulb of adult mice), it still provides relevant evidence of the BM contribution to the neuronal cell population (3). The investigation of factors that can promote this process could be useful toward the development of a clinically applicable ther- apy. Two main determinants seem to influence the incorporation of BM stem cells into tissues: (1) tissue Experimental Neurology 177, 443– 452 (2002) doi:10.1006/exnr.2002.8004 443 0014-4886/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.