title: Engineering of midbrain organoids containing long-lived dopaminergic neurons.
abstract: The possibility to generate dopaminergic (DA) neurons from pluripotent stem cells represents an unlimited source of material for tissue engineering and cell therapy for neurodegenerative disease. We set up a protocol based on the generation of size-calibrated neurospheres for a rapid production (3 weeks) of a high amount of DA neurons (>60%) oriented toward a midbrain-like phenotype, characterized by the expression of FOXA2, LMX1A, tyrosine hydroxylase (TH), NURR1, and EN1. By using γ-secretase inhibitors and varying culture time of neurospheres, we controlled maturation and cellular composition of a three-dimensional (3D) engineered nervous tissue (ENT). ENT contained neurons and glial cells expressing various markers of maturity, such as synaptophysin, neuronal nuclei-specific protein (NeuN), and glial fibrillary acidic protein (GFAP), and were electrophysiologically active. We found that 3-week-old neurospheres were optimal to generate 3D tissue containing DA neurons with typical A9 morphology. ENT generated from 4-week-old neurospheres launched glial cell type since astrocytes and myelin could be detected massively at the expense of TH-immunoreactive neurons. All γ-secretase inhibitors were not equivalent; compound E was more efficient than DAPT in generating DA neurons. This DA tissue provides a tool for drug screening, and toxicology. It should also become a useful biomaterial for studies on Parkinson's disease.
authors.affiliation: 1 Department of Pathology and Immunology, University Medical Center, University of Geneva , Geneva, Switzerland .
authors.forename: Vannary
authors.initials: V
authors.lastname: Tieng
authors.forename: Luc
authors.initials: L
authors.lastname: Stoppini
authors.forename: Sabrina
authors.initials: S
authors.lastname: Villy
authors.forename: Marc
authors.initials: M
authors.lastname: Fathi
authors.forename: Michel
authors.initials: M
authors.lastname: Dubois-Dauphin
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Krause
mesh_terms: D002478:Cells, Cultured; D059290:Dopaminergic Neurons / Q000502:physiology*; D053595:Embryonic Stem Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells; D008636:Mesencephalon / Q000166:cytology*; D009940:Organoids / Q000166:cytology*; D051880:Receptors, Notch / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology; D023822:Tissue Engineering
chemical_list: D051880:Receptors, Notch