Large-scale generation of highly enriched neural stem-cell-derived oligodendroglial cultures: maturation-dependent differences in insulin-like growth factor-mediated signal transduction.
Multipotent neural stem cells (NSCs) are competent for commitment to the oligodendrocyte (OL) lineage both in vitro and in vivo. We exploited this property to develop a rat neurospheres (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 generation 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 differences 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 kinase 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.
Broughton
Sarah K
SK
Department of Pediatrics, Oregon Health & Science University, Portland, OR 97239-3098, USA.
Chen
Hanqin
H
Riddle
Art
A
Kuhn
Scott E
SE
Nagalla
Srinivasa
S
Roberts
Charles T
CT
Back
Stephen A
SA
D000818:Animals; D000831:Animals, Newborn; D016380:Brain Tissue Transplantation / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000378:metabolism / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000187:drug effects / Q000378:metabolism; D053613:Janus Kinase 1 / Q000187:drug effects / Q000378:metabolism; D039902:Multipotent Stem Cells / Q000187:drug effects / Q000378:metabolism*; D009836:Oligodendroglia / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013002:Somatomedins / Q000378:metabolism* / Q000494:pharmacology; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism / Q000494:pharmacology
D020934:Ciliary Neurotrophic Factor; D017077:Culture Media, Conditioned; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D013002:Somatomedins; D014284:Triiodothyronine; D007334:Insulin-Like Growth Factor I; D053613:Janus Kinase 1; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases