title: Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens.
abstract: Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Diogo
authors.initials: D
authors.lastname: Ribeiro
authors.affiliation: Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: Rocio
authors.initials: R
authors.lastname: Laguna Goya
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Geeta
authors.initials: G
authors.lastname: Ravindran
authors.affiliation: Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: Romina
authors.initials: R
authors.lastname: Vuono
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Clare L
authors.initials: CL
authors.lastname: Parish
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Claire
authors.initials: C
authors.lastname: Foldi
authors.affiliation: Laboratory of Molecular Neurosurgery and Neurological Clinic, Department of Stereotactical Neurosurgery, University Freiburg Medical Center, Breisacher Str. 64, 79106 Freiburg i. B., Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Piroth
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Shanzheng
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, BMC A11, 22184 Lund, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Parmar
authors.affiliation: Laboratory of Molecular Neurosurgery and Neurological Clinic, Department of Stereotactical Neurosurgery, University Freiburg Medical Center, Breisacher Str. 64, 79106 Freiburg i. B., Germany.
authors.forename: Guido
authors.initials: G
authors.lastname: Nikkhah
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Jens
authors.initials: J
authors.lastname: Hjerling-Leffler
authors.affiliation: Laboratory of Neurogenesis and Cell Therapy, Wallenberg Neuroscience Center and Lund Stem Cell Center, University Hospital, SE-221 84 Lund, Sweden.
authors.forename: Olle
authors.initials: O
authors.lastname: Lindvall
authors.affiliation: Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Barker
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden. Electronic address: ernest.arenas@ki.se.
authors.forename: Ernest
authors.initials: E
authors.lastname: Arenas
mesh_terms: D002452:Cell Count; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology* / Q000502:physiology*; D058953:Neural Stem Cells / Q000166:cytology / Q000502:physiology*; D055495:Neurogenesis / Q000502:physiology*; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000008:administration & dosage; D051153:Wnt Proteins / Q000008:administration & dosage; D000071818:Wnt-5a Protein
chemical_list: D011518:Proto-Oncogene Proteins; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D004298:Dopamine