Generation of cerebellar neuron precursors from embryonic stem cells.
Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.
Su
Hong-Lin
HL
Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan.
Muguruma
Keiko
K
Matsuo-Takasaki
Mami
M
Kengaku
Mineko
M
Watanabe
Kiichi
K
Sasai
Yoshiki
Y
D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation; D017690:Cell Transplantation; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology*; D051524:Fibroblast Growth Factor 8 / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism*; D011689:Purkinje Cells / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism; D060508:Wnt3 Protein; D060509:Wnt3A Protein
C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D016895:Culture Media, Serum-Free; C496457:Fgf8 protein, mouse; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D049452:Green Fluorescent Proteins; D051524:Fibroblast Growth Factor 8