Modeling Human Neural Functionality In Vitro: Three-Dimensional Culture for Dopaminergic Differentiation

Autor: Paula M. Alves, André E.P. Bastos, Stefania Piersanti, Ana P. Teixeira, Giampietro Schiavo, Lucy M. Collinson, Daniel Simão, Catarina Brito, Margarida Serra, Sigrid C. Schwarz, Isabella Saggio, Catarina Pinto, Christopher J. Peddie, Eric J. Kremer, Pedro A. Lima, Valerio Licursi, Sara Salinas, Anne Weston
Přispěvatelé: Instituto de Biologia Experimental e Tecnológica (IBET), Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Department of Biology and Biotechnology 'Charles Darwin', Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Lincoln's Inn Fields Laboratories, Cancer Research UK, Departamento de Química e Bioquímica, Faculdade de Ciencias da Universidade de Lisboa, NOVA Medical School - Faculdade de Ciências Médicas (NMS), Universität Leipzig [Leipzig], Pathogénèse et contrôle des infections chroniques (PCCI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre Hospitalier Universitaire de Montpellier (CHU Montpellier ), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Istituto di Biologia e Patologia Molecolari - CNR [Roma, Italy], Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Sobell Department of Motor Neuroscience and Movement Disorders [London WC, UK] (Institute of Neurology), University College of London [London] (UCL), he authors gratefully acknowledge Dr. Johannes Schwarz for the supply of hmNPCs within the scope of the EU project BrainCAV (FP7-222992), Pedro Almada and Dr. Emilio Gualda for support on confocal microscopy, Dr. Ana Amaral for support on hmNPC 3D cultures and, Dr. Rodolfo Negri and Dr. Enrico Tagliafico for chip bioinformatics. IBET Analytical Services Unit, Portugal, is acknowledged for HPLC analysis and Institute of Ophthalmology, UCL, United Kingdom, is acknowledged for collection of SBFSEM data. The NMR spectrometers are part of The National NMR Facility, supported by Fundação para a Ciência e a Tecnologia (RECI/BBB-BQB/0230/2012). This work was supported by BrainCAV (FP7-222992) and Brainvectors (FP7-286071), funded by the EU (PTDC/EBB-BIO/112786/2009 and PTDC/EBB-BIO/119243/2010), funded by Fundação para a Ciência e Tecnologia, Portugal, and Cancer Research, United Kingdom. D.S. was recipient of a PhD fellowship from FCT, Portugal (SFRH/BD/78308/2011, FCT)., European Project: 222992,EC:FP7:HEALTH,FP7-HEALTH-2007-B,BRAINCAV(2008), European Project: 286071,EC:FP7:PEOPLE,FP7-PEOPLE-2011-IAPP,BRAINVECTORS(2012), European Project: 112786,FCT::,PTDC/2009,PTDC/EBB-BIO/112786/2009(2011), European Project: 119243,FCT::,PTDC/2010,PTDC/EBB-BIO/119243/2010(2012), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), Universität Leipzig, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), CNR Istituto di Biologia e Patologia Molecolari [Roma] (CNR | IBPM), National Research Council of Italy | Consiglio Nazionale delle Ricerche (CNR)
Rok vydání: 2015
Předmět:
MESH: Neural Stem Cells/cytology
Dopamine
MESH: Printing
Three-Dimensional
Biomedical Engineering
Synaptogenesis
Bioengineering
MESH: Dopamine
Biology
Biochemistry
MESH: Tissue Engineering/methods
Biomaterials
Neural Stem Cells
Biomimetics
Neurosphere
Humans
Induced pluripotent stem cell
MESH: Neural Stem Cells/physiology
Neural cell
Cells
Cultured
MESH: Biomimetics/methods
MESH: Humans
Tissue Engineering
Dopaminergic Neurons
MESH: Dopaminergic Neurons/cytology
MESH: Cell Differentiation/physiology
Dopaminergic
Glutamate receptor
Cell Differentiation
3d cellular models
dopaminergic differentiation
cellular modeling
neuronal progenitors
Neural stem cell
MESH: Batch Cell Culture Techniques/methods
Batch Cell Culture Techniques
Printing
Three-Dimensional
Stem cell
MESH: Dopaminergic Neurons/physiology
Neuroscience
[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
MESH: Cells
Cultured
Zdroj: Tissue Engineering: Parts A, B, and C
Tissue Engineering: Parts A, B, and C, Mary Ann Liebert, 2015, 21 (3-4), pp.654-668. ⟨10.1089/ten.TEA.2014.0079⟩
Tissue Engineering: Parts A, B, and C, 2015, 21 (3-4), pp.654-668. ⟨10.1089/ten.TEA.2014.0079⟩
ISSN: 1937-335X
1937-3341
DOI: 10.1089/ten.tea.2014.0079
Popis: International audience; Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca(2+) transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons (Vm=-70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.
Databáze: OpenAIRE
Externí odkaz: