Large-scale generation of highly enriched neural stem-cell-derived oligodendroglial cultures: maturation-dependent differences in insulin-like growth factor-mediated signal transduction Sarah K. Broughton,* ,1 Hanqin Chen,* ,1 Art Riddle,* Scott E. Kuhn,* Srinivasa Nagalla,* Charles T. Roberts, Jr* ,2 and Stephen A. Back* ,   ,2 Departments of *Pediatrics and  Neurology, Oregon Health & Science University, Portland, Oregon, USA Abstract Multipotent neural stem cells (NSCs) are competent for com- mitment to the oligodendrocyte (OL) lineage both in vitro and in vivo. We exploited this property to develop a rat neuro- spheres (NS)/oligospheres (OS)-based culture system to generate large numbers of highly enriched late OL progenitors (preOLs) and mature OLs (MatOLs). CNS neuroblastoma cell line B104-derived conditioned medium promoted the genera- tion of nearly pure populations of preOLs from dissociated OS. The subsequent culture of preOLs with ciliary neurotrophic factor (CNTF) and 3,3¢,5¢-triiodo-L-thyronine (T 3 ) generated nearly pure populations of MatOLs. OL lineage specificity was confirmed by immunocytochemistry, quantitative RT-PCR and gene expression profiling, which demonstrated large differ- ences between preOLs and MatOLs. The insulin-like growth factors (IGFs) are potent neuro-protective agents required for OL survival. We used this system to systematically define maturation-dependent changes in IGF signaling during the course of OL differentiation. The IGF-I and insulin receptors, insulin receptor substrate-1 (IRS-1) and IRS-2, protein kin- ase B (PKB)/Akt and Janus kinase (JNK) were expressed at higher levels in NS and preOLs compared with OS and MatOLs. Erk expression increased markedly from NS to OS, decreased only partially upon commitment to preOLs, and, in MatOLs, returned to a low level similar to NS. IGF activation of the generally proliferative Erk pathway was gradually acquired during NSC differentiation, whereas IGF activation of the generally pro-survival, anti-apoptotic PI3K/PKB pathway was consistently robust at each developmental stage. Keywords: Akt/protein kinase B, Erk, myelin, neural stem cell, neurosphere, oligodendrocyte. J. Neurochem. (2007) 100, 628–638. The late oligodendrocyte (OL) progenitor (pre-OL) is a mitotically active, post-migratory cell that resides at a critical control point in OL lineage progression (Pfeiffer et al. 1981, 1993; Back and Volpe 1999). PreOLs are the source of all mature Ols (MatOLs), and their differentiation normally coincides with the initiation of CNS myelinogenesis (Hardy and Friedrich 1996; Back et al. 2002b). Additional potential roles in CNS regeneration and repair are supported by the recognition that OL progenitors (preOLs) can commit to additional fates in both gliogenesis and neurogenesis (Kondo and Raff 2000; Belachew et al. 2003). Despite growing evidence for a central role for preOLs in the pathogenesis of CNS white matter disorders, there is limited information regarding the unique molecular features that distinguish preOLs from MatOLs. PreOLs display a markedly greater susceptibility to death triggered by oxida- tive stress than do more mature OL lineage stages (Back et al. 1998, 2002a; Fragoso et al. 2004; Lin et al. 2004). The Received April 12, 2006; revised manuscript received May 10, 2006; accepted June 30, 2006. Address correspondence and reprint requests to Stephen A. Back MD PhD, Department of Pediatrics, NRC 5, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239– 3098, USA. E-mail: backs@ohsu.edu 1 These authors contributed equally to this work. 2 Joint senior authors. Abbreviations used: B104-CM, B104-derived conditioned medium; BDM, basal-defined medium; bFGF, basic fibroblast growth factor; CNTF, ciliary neurotrophic factor; C T , cycle threshold; EGF, epidermal growth factor; GFAP, glial fibrillary acidic protein; GluR2, type-2 glu- tamate receptor; IGF, insulin-like growth factor; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; KA2, kainate receptor; MAS,MICROARRAY SUITE; MAP2, microtubule-associated protein 2; MatOL, mature OL; MBP, myelin basic protein; NM, NS medium; NS, neurospheres; NSC, neural stem cell; OL, oligodendrocyte; OS, oligospheres; PI3K, phos- phatidylinositol 3-kinase; PKB, protein kinase B; preOL, OL progenitor; SDS, sodium dodecyl sulfate; T 3 , 3,3¢,5¢-triiodo-L-thyronine; TBS-T, Tris-buffere saline with Tween 20. Journal of Neurochemistry , 2007, 100, 628–638 doi:10.1111/j.1471-4159.2006.04171.x 628 Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 628–638 Ó 2006 The Authors