[CANCER RESEARCH 63, 8877– 8889, December 15, 2003] Cellular and Genetic Characterization of Human Adult Bone Marrow-Derived Neural Stem-Like Cells: A Potential Antiglioma Cellular Vector Jeongwu Lee, 1 Abdel G. Elkahloun, 2 Steven A. Messina, 1 Nicolay Ferrari, 1 Dan Xi, 1 Carolyn L. Smith, 3 Ronald Cooper, Jr., 2 Paul S. Albert, 4 and Howard A. Fine 1 1 Neuro-Oncology Branch, National Cancer Institute, National Institute of Neurological Disorders and Stroke, NIH; 2 Cancer Genetics Branch, National Human Genome Research Institute-NIH, Microarray Unit, National Institute of Neurological Disorders and Stroke, NIH; 3 National Institute of Neurological Disorders and Stroke Light Imaging Facility, National Institute of Neurological Disorders and Stroke; and 4 Biometric Research Branch, DCTD, National Cancer Institute, Bethesda, Maryland ABSTRACT We describe the in vitro isolation and expansion of cells capable of forming neurosphere-like aggregates from human adult bone marrow. Cells within these passaged spheroids can differentiate into astrocytes, specific neuronal subtypes, and oligodendrocytes and have gene expres- sion profiles similar to human fetal brain-derived neural stem cells. Ge- netically modified neural-competent bone marrow-derived cells efficiently migrate toward distant sites of brain injury and tumor in vivo, where they differentiate and express therapeutic transgenes when transplanted into the brains of mice. These studies suggest that adult bone marrow may serve as a large reservoir for autologous neural stem-like cells for future therapeutic strategies. INTRODUCTION Malignant gliomas represent an important cause of cancer-related mortality for which standard treatments are suboptimal (1). Although gliomas do not generally metastasize, their propensity to deeply infiltrate adjacent cerebral cortex precludes definitive surgical resec- tion or other local therapies. Intrinsic resistance of glioma cells to radiation and chemotherapy limits traditional cancer therapies, whereas newer approaches such as gene therapy are confounded by the inefficiencies of viral vectors to transduce the deeply infiltrating glioma cells. An autologous cellular vector that could migrate through brain parenchyma and deliver a therapeutic transgene to the site of infiltrating tumor would theoretically represent an optimal gene de- livery strategy for brain tumors. Neural stem cell (NSC) lines have been demonstrated to be effective for delivering transgenes to brain tumors based on their unique migratory properties within the central nervous system [CNS (2– 4)]; however, the ability to safely obtain sufficient numbers of autologous NSCs represents a significant and potentially prohibitive technical challenge. The demonstration that freshly isolated bone marrow cells injected into the brains of animals can express neuronal and glial markers in mice suggests that tissue- specific stem cells possess broad developmental potential and raises the possibility of a readily available source of autologous NSC-like cells (5–15). Here we demonstrate the in vitro isolation, expansion, and therapeutic potential of a neural-competent population of cells from adult human bone marrow. MATERIALS AND METHODS Cell Culture. RBCs from human adult (donors, 20 – 40 years old) bone marrow cells purchased from AllCells (Berkely, CA) were depleted by density gradient, and the remaining cells were plated in uncoated tissue culture plastic dishes. After 10 days of culture in 15% ES-qualified serum (Life Technologies, Inc., Gaithersburg, MD) containing media with basic fibroblast growth factor [bFGF (50 ng/ml)]/fibroblast growth factor (FGF) 8 (100 ng/ml)/sonic hedge- hog (500 ng/ml)/leukemia inhibitory factor (10 ng/ml; from R&D Systems, Minneapolis, MN), nestin-positive cell aggregates were formed. Cells were then trypsinized and replated in serum-free N2/B27 neurobasal media (Life Technologies, Inc.) with bFGF/FGF8/sonic hedgehog. Floating aggregates were separated and cultured for 10 days. Brain-derived neural stem cells (BDNSCs) were obtained from Clonetics (Walkersville, MD). Conditioned media from BDNSC culture were filtered through 0.22 m Stericup (Milli- pore, Bedford, MA) and used in the culture of BDNSCs and marrow-derived neural-competent cells [MDNCCs (25% conditioned media +75% fresh me- dia)]. To induce differentiation in vitro, cell aggregates were plated on fi- bronectin-coated plates in B27 media containing 10% fetal bovine serum. In vivo conditioning consisted of the injection of cell aggregate-dissociated, nestin-positive cells into the lateral ventricle of neonatal severe combined immunodeficient mouse. Transplanted human cells were examined by confocal microscopy (Zeiss LSM 510; Zeiss). For ex vivo characterization, mice trans- planted with human cells were killed; brains were dissociated by digestion with trypsin, collagenase, and papain; and then the harvested cells were cultured in the presence of bFGF and differentiated by the removal of bFGF (16). Immunohistochemistry. Immunohistochemistry was carried out using standard protocols. The following primary antibodies were used at the follow- ing dilutions: (a) TuJ1 rabbit polyclonal antibody, 1:2000; green fluorescence protein (GFP) monoclonal antibody, 1:200 (both from Babco, Richmond, CA); (b) TuJ1 mouse monoclonal antibody, 1:1000; MAP2 mouse monoclonal antibody, 1:2000; glial fibrillary acidic protein (GFAP) mouse monoclonal antibody, 1:400; 2',3'-cyclic nucleotide 3'-phosphodiesterase monoclonal an- tibody, 1: 1000; and -aminobutyrate rabbit polyclonal antibody, 1:1000 (all from Sigma, St. Louis, MO); (c) A2B5 monoclonal antibody, 1:100 (BioTrend); (d) nestin polyclonal antibody, 1:1000; NeuN mouse monoclonal antibody, 1:100; GFAP rabbit polyclonal antibody, 1:400; human ribonucleo- protein monoclonal antibody, 1:50; HuMac mouse monoclonal antibody, 1:50; P1H12 monoclonal antibody, 1:100; and neurofilament M rabbit polyclonal antibody, 1:200 (all from Chemicon); (e) Ki67 rabbit polyclonal antibody (Novocastra); (f) GFP rabbit polyclonal antibody, 1:200 (Molecular Probes, Eugene, OR); (g) F4/80 rat monoclonal antibody, 1:50; S100 rabbit poly- clonal antibody, 1:100 (Abcam); (h) Glut1 rabbit polyclonal antibody, 1:1000 (Calbiochem); (i) CD45 mouse monoclonal antibody, 1:100 (PharMingen), (j) O4 monoclonal antibody, 1:40 (Developmental Studies Hybridoma Bank); (k) aquaporin4 rabbit polyclonal antibody, 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA); (l) tyrosine hydroxylase rabbit monoclonal antibody, 1:400 (Pel-Freez, Rogers, AK); and (m) bromodeoxyuridine rat monoclonal anti- body, 1:100 (Accurate, Westbury, NY). For detection of primary antibodies, fluorescence-labeled secondary antibodies (Molecular Probes) were used ac- cording to the manufacturer’s recommendations. Telomeric Repeat Amplification Protocol Assay. Telomeric repeat am- plification protocol assay was carried out using Telomerase PCR kit (Roche) following the manufacturer’s recommendations. Microarray. Human cDNA microarray chips consisting of 13,826 se- quence-verified cDNA clones were printed onto glass slides as described online. 5 Gene names are according to build 138 of the Unigene human sequence collection. 6 Total RNA from all samples was extracted using Trizol Reagent (Life Technologies, Inc.), and further purified using the RNeasy kit (Qiagen). One ug of total RNA was subjected to linear RNA amplification using the modified Eberwine protocol. 7 Microarrays were hybridized and Received 3/27/03; revised 8/27/03; accepted 10/8/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Howard A. Fine, Room 225, The Bloch Building (Building 82), 9030 Old Georgetown Road, Bethesda, Maryland 20892. Phone: (301) 402-6298; Fax: (301) 480-2246; E-mail: hfine@mail.nih.gov. 5 http://www.nhgri.nih.gov/UACORE/. 6 http://www.ncbi.nlm.nih.gov/UniGene/build.html. 7 http://cmgm.stanford.edu/pbrown/protocols/ampprotocol_2.txt. 8877 Research. on May 4, 2021. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from