title: Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells.
abstract: Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. tfernandes@tecnico.ulisboa.pt.
authors.forename: Tiago G
authors.initials: TG
authors.lastname: Fernandes
authors.affiliation: Instituto de Medicina Molecular, Universidade de Lisboa and Hospital de Dona Estefânia, CHLC, Lisbon, Portugal.
authors.forename: Sofia T
authors.initials: ST
authors.lastname: Duarte
authors.affiliation: Erasmus Medical Center iPS facility, Erasmus Medical Center, University Medical Center, Rotterdam, The Netherland.
authors.forename: Mehrnaz
authors.initials: M
authors.lastname: Ghazvini
authors.affiliation: Instituto de Medicina Molecular, Universidade de Lisboa and Hospital de Dona Estefânia, CHLC, Lisbon, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Gaspar
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Diana C
authors.initials: DC
authors.lastname: Santos
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Ana R
authors.initials: AR
authors.lastname: Porteira
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Gonçalo M C
authors.initials: GM
authors.lastname: Rodrigues
authors.affiliation: Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Haupt
authors.affiliation: Instituto de Medicina Molecular, Universidade de Lisboa and Hospital de Dona Estefânia, CHLC, Lisbon, Portugal.|Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Diogo M
authors.initials: DM
authors.lastname: Rombo
authors.affiliation: Department of Neurology, Hospital Sant Joan de Déu (HSJD), Barcelona and CIBER-ER (Biomedical Network Research Centre on Rare Diseases, Instituto de Salud Carlos III), Madrid, Spain.
authors.forename: Judith
authors.initials: J
authors.lastname: Armstrong
authors.affiliation: Instituto de Medicina Molecular, Universidade de Lisboa and Hospital de Dona Estefânia, CHLC, Lisbon, Portugal.|Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Sebastião
authors.affiliation: Department of Developmental Biology, Erasmus Medical Center, University Medical Center, Rotterdam, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Gribnau
authors.affiliation: Department of Neurology, Hospital Sant Joan de Déu (HSJD), Barcelona and CIBER-ER (Biomedical Network Research Centre on Rare Diseases, Instituto de Salud Carlos III), Madrid, Spain.
authors.forename: Àngels
authors.initials: À
authors.lastname: Garcia-Cazorla
authors.affiliation: Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Brüstle
authors.affiliation: Instituto de Medicina Molecular, Universidade de Lisboa and Hospital de Dona Estefânia, CHLC, Lisbon, Portugal.
authors.forename: Domingos
authors.initials: D
authors.lastname: Henrique
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Joaquim M S
authors.initials: JM
authors.lastname: Cabral
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Maria Margarida
authors.initials: MM
authors.lastname: Diogo
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation / Q000235:genetics; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D051783:Methyl-CpG-Binding Protein 2 / Q000096:biosynthesis / Q000235:genetics; D058953:Neural Stem Cells / Q000166:cytology; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology; D015518:Rett Syndrome / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D051897:Smad Proteins, Inhibitory / Q000235:genetics
chemical_list: D003470:Culture Media; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D051897:Smad Proteins, Inhibitory
keywords: Defined culture conditions; Dual-SMAD inhibition; Induced pluripotent stem cells; Neurodevelopment modeling; Rett syndrome