Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells.
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.
Lee
Chul-Sang
CS
Washington University School of Medicine, Center for the Study of Nervous System Injury, St. Louis, MO 63110, USA.
Tee
Lee Y
LY
Dusenbery
Susan
S
Takata
Toshihiro
T
Golden
Judith P
JP
Pierchala
Brian A
BA
Gottlieb
David I
DI
Johnson
Eugene M
EM
Choi
Dennis W
DW
Snider
B Joy
BJ
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*
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