title: Induction of midbrain dopaminergic neurons from primate embryonic stem cells by coculture with sertoli cells.
abstract: The aim of this study was to produce dopaminergic neurons from primate embryonic stem (ES) cells following coculture with mouse Sertoli cells. After 3 weeks of induction, immunostaining revealed that 90% +/- 9% of the colonies contained tyrosine hydroxylase-positive (TH(+)) neurons, and 60% +/- 7% of the tubulin beta III-positive (Tuj III(+)) neurons were TH(+). Reverse transcription-polymerase chain reaction analyses showed that Sertoli-induced neurons expressed midbrain dopaminergic neuron markers, including TH, dopamine transporter, aromatic amino acid decarboxylase (AADC), receptors such as TrkB and TrkC, and transcription factors NurrI and Lmx1b. Neurons that had been differentiated on Sertoli cells were positive for Pax2, En1, and AADC, midbrain-related markers, and negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. These Sertoli cell-induced dopaminergic cells can release dopamine when depolarized by high K(+). Sertoli cell-conditioned medium contained glial cell line-derived neurotrophic factor (GDNF) and supported neuronal differentiation. After pretreatment with anti-GDNF antibody, the percentage of Tuj III(+) colonies was reduced to 14%. Thus, GDNF contributed significantly to inducing primate ES cells into dopaminergic neurons. When transplanted into a 6-hydroxydopamine-treated Parkinson's disease model, primate-derived dopaminergic neurons integrated into the mouse striatum. Two weeks after transplantation, surviving TH(+) cells were present. These TH(+) cells survived for 2 months. Therefore, the induction method of coculture ES cells with Sertoli cells provides an unlimited source of primate cells for the study of pathogenesis and transplantation in Parkinson's disease.
authors.affiliation: Department of Anatomy and Organ Technology, Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan. yuefm@sch.md.shinshu-u.ac.jp
authors.forename: Fengming
authors.initials: F
authors.lastname: Yue
authors.forename: Li
authors.initials: L
authors.lastname: Cui
authors.forename: Kohei
authors.initials: K
authors.lastname: Johkura
authors.forename: Naoko
authors.initials: N
authors.lastname: Ogiwara
authors.forename: Katsunori
authors.initials: K
authors.lastname: Sasaki
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002454:Cell Differentiation; D018920:Coculture Techniques; D004298:Dopamine / Q000378:metabolism*; D004627:Embryonic Induction; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism / Q000494:pharmacology; D008252:Macaca fascicularis / Q000196:embryology; D008297:Male; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology*; D051379:Mice; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D011323:Primates / Q000196:embryology; D012708:Sertoli Cells / Q000378:metabolism* / Q000502:physiology; D013234:Stem Cells / Q000378:metabolism*; D013997:Time Factors; D014183:Transplantation, Heterologous
chemical_list: D051100:Glial Cell Line-Derived Neurotrophic Factor; D004298:Dopamine