Ontogeny-recapitulating generation and tissue integration of ES cell-derived Purkinje cells.
Purkinje cells are the sole output neurons of the cerebellar cortex and their dysfunction causes severe ataxia. We found that Purkinje cells could be robustly generated from mouse embryonic stem (ES) cells by recapitulating the self-inductive signaling microenvironments of the isthmic organizer. The cell-surface marker Neph3 enabled us to carry out timed prospective selection of Purkinje cell progenitors, which generated morphologically characteristic neurons with highly arborized dendrites that expressed mature Purkinje cell-specific markers such as the glutamate receptor subunit GluRδ2. Similar to mature Purkinje cells, these neurons also showed characteristic spontaneous and repeated action potentials and their postsynaptic excitatory potentials were generated exclusively through nonNMDA glutamate receptors. Fetal transplantation of precursors isolated by fluorescence-activated cell sorting showed orthotopic integration of the grafted neurons into the Purkinje cell layer with their axons extending to the deep cerebellar nuclei and dendrites receiving climbing and parallel fibers. This selective preparation of bona fide Purkinje cells should aid future investigation of this important neuron.
Muguruma
Keiko
K
Organogenesis and Neurogenesis Group, RIKEN Center for Developmental Biology, Kobe, Japan.
Nishiyama
Ayaka
A
Ono
Yuichi
Y
Miyawaki
Hiroyuki
H
Mizuhara
Eri
E
Hori
Seiji
S
Kakizuka
Akira
A
Obata
Kunihiko
K
Yanagawa
Yuchio
Y
Hirano
Tomoo
T
Sasai
Yoshiki
Y
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000166:cytology / Q000196:embryology; D004622:Embryo, Mammalian; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D005968:Glutamate Decarboxylase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D011247:Pregnancy; D011689:Purkinje Cells / Q000502:physiology*; D013234:Stem Cells / Q000502:physiology*
D007136:Immunoglobulins; D008565:Membrane Proteins; C546601:Neph3 protein, mouse; D009419:Nerve Tissue Proteins; D049452:Green Fluorescent Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1