Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells.
Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6-8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na(+) or Ca(2+) spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro.
Tao
Osamu
O
Department of Physiology, Keio University School of Medicine, Tokyo, Japan.
Shimazaki
Takuya
T
Okada
Yohei
Y
Naka
Hayato
H
Kohda
Kazuhisa
K
Yuzaki
Michisuke
M
Mizusawa
Hidehiro
H
Okano
Hideyuki
H
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002531:Cerebellum / Q000166:cytology / Q000502:physiology; D018920:Coculture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D019706:Excitatory Postsynaptic Potentials; D053444:Inhibitory Postsynaptic Potentials; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D055495:Neurogenesis / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000502:physiology; D011689:Purkinje Cells / Q000166:cytology / Q000502:physiology*; D012964:Sodium / Q000378:metabolism; D013569:Synapses / Q000502:physiology
D016895:Culture Media, Serum-Free; D012964:Sodium; D002118:Calcium