title: Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment.
abstract: 3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up.
authors: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors: Cláudia C
authors: CC
authors: Miranda
authors: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors: Tiago G
authors: TG
authors: Fernandes
authors: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors: Jorge F
authors: JF
authors: Pascoal
authors: Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany.|LIFE & BRAIN GmbH, Bonn, Germany.
authors: Simone
authors: S
authors: Haupt
authors: Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany.|LIFE & BRAIN GmbH, Bonn, Germany.
authors: Oliver
authors: O
authors: Brüstle
authors: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors: Joaquim M S
authors: JM
authors: Cabral
authors: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. margarida.diogo@tecnico.ulisboa.pt.
authors: Maria Margarida
authors: MM
authors: Diogo
mesh_terms: D002449:Cell Aggregation / Q000235:genetics*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000235:genetics*; D049109:Cell Proliferation / Q000235:genetics; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology; D006801:Humans; D058953:Neural Stem Cells / Q000166:cytology*; D055495:Neurogenesis / Q000235:genetics; D009474:Neurons / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000166:cytology*
chemical_list: D003470:Culture Media
keywords: Defined conditions; Human pluripotent stem cells; Neural commitment; Neural precursor cells; Suspension aggregates