ABSTRACT
BACKGROUND: It has been widely accepted that both bone marrow-derived mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have the capacity to differentiate into neural lineages. Some scholars believe that in addition to HSCs and MSCs, bone marrow (BM) also harbors a highly mobile population of CXCR4+ tissue committed stem cells (TCSCs), including skeletal muscles, heart, liver, and neural tissue. OBJECTIVE: To make sure that neural tissue-committed stem cells (NTCSCs) reside in the bone marrow, and to establish a purification and culture method for bone marrow-derived NTCSCs.DESIGN: Opening animal study.SETTING: Department of Anatomy, the Second Military Medical University of Chinese PLA. MATERIALS: Adult Sprague-Dawley (SD) rats (pathogen-free) were provided by the Animal Center of the Second Military Medical University of Chinese PLA. Dulbecco's modified eagle's medium (DMEM)/F12, B27, N2 and epidermal growth factor (EGF, Invitrogen Company), basic fibroblast growth factor (bFGF, CytoLab Ltd), rabbit anti-rat Nestin,CXCR4, β-Tublin Ⅲ, glial fibrillary acidic protein (GFAP, Santa Cruz Company), mouse anti-rat microtubule associated protein 2ab (MAP2ab) (Clone11-5B), cyclic nucleotide 3'phosphohydrolase (CNPase, Clone AP20, NeoMarkers Company), fluorescent(fluorescein isothiocyanate, Cy3) marker reagents (Wuhan Boster Bioengineering Co., Ltd), nuclear fluorescent dyes 4,6-diamidino-2-phenylindole(DAPI)(Sigma), immunohistochemistry reagents (Vector Laboratories Company) , and NycoPrepTM separation liquid (1.077A, Axis-Shield Company) were used in this study.METHODS: This study was performed in the Department of Anatomy, the Second Military Medical University of Chinese PLA from January 2004 to December 2006. Bone marrow was harvested from bilateral femurs and tibias of 2-3 weeks SD rats. Mononuclear cell layer was isolated by NycoPrepTM separation liquid and suspended in DMEM/F12(1:1)serum-free medium supplemented with 2% B27,1% N2, 20 μg/L bFGF, 20μg/L EGF, 1×105 U/L penicillin and 100 mg/L streptomycin. NTCSCs were isolated and propagated by suspensive growing from adherent cells in bone marrow in DMEM/F12 free-serum medium. MAIN OUTCOME MEASURES: NTCSCs were identified by immunocytochemistry for CXCR4, a marker of TCSCs and nestin, a marker of neural stem cells, and neural lineages marker protein after differentiation of cellular spheres. RESULTS: The NTCSCs spheres expressed nestin, a neural stem cell marker as well as CXCR4, a marker of TCSCs. The NTCSCs' spheres were naturally differentiated in DMEM medium with 15% fetal bovine serum. The differentiated cells expressed β-Tublin Ⅲ, MAP2ab, CNPase and GFAP, markers of neural lineages. CONCLUSION: NTCSCs reside in bone marrow and naturally differentiate into neural lineages in vitro.