title: Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells.
abstract: Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.
authors.affiliation: Washington University School of Medicine, Center for the Study of Nervous System Injury, St. Louis, MO 63110, USA.
authors.forename: Chul-Sang
authors.initials: CS
authors.lastname: Lee
authors.forename: Lee Y
authors.initials: LY
authors.lastname: Tee
authors.forename: Susan
authors.initials: S
authors.lastname: Dusenbery
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Takata
authors.forename: Judith P
authors.initials: JP
authors.lastname: Golden
authors.forename: Brian A
authors.initials: BA
authors.lastname: Pierchala
authors.forename: David I
authors.initials: DI
authors.lastname: Gottlieb
authors.forename: Eugene M
authors.initials: EM
authors.lastname: Johnson
authors.forename: Dennis W
authors.initials: DW
authors.lastname: Choi
authors.forename: B Joy
authors.initials: BJ
authors.lastname: Snider
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008024:Ligands; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D009414:Nerve Growth Factors / Q000378:metabolism / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*
chemical_list: D018690:Excitatory Amino Acid Agonists; C495040:Gdnf protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008024:Ligands; D009414:Nerve Growth Factors; D017475:Receptors, Nerve Growth Factor; D016202:N-Methylaspartate