title: Three-dimensional culture of single embryonic stem-derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling.
abstract: In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% βIII-tubulin+cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES-NSPCs expressed matriptase and secreted MMP-2/9, with MMP-2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES-NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three-dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES-NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin-based hydrogels for ES-NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.|Faculdade de Engenharia, Universidade do Porto, Portugal.
authors.forename: Ana R
authors.identifier: 0000-0002-9578-8237
authors.initials: AR
authors.lastname: Bento
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Quelhas
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.|Departamento de Patologia e Oncologia da Faculdade de Medicina da Universidade do Porto de Medicina da Universidade do Porto, Portugal.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Oliveira
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.|Faculdade de Engenharia, Universidade do Porto, Portugal.|Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Pêgo
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.|Faculdade de Engenharia, Universidade do Porto, Portugal.
authors.forename: Isabel F
authors.identifier: 0000-0003-2921-3627
authors.initials: IF
authors.lastname: Amaral
mesh_terms: D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D005337:Fibrin / Q000494:pharmacology*; D005782:Gels; D006801:Humans; D020100:Hydrogels / Q000494:pharmacology*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D009415:Nerve Net / Q000187:drug effects / Q000502:physiology*; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010641:Phenotype; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D005782:Gels; D020100:Hydrogels; D005337:Fibrin; D012697:Serine Endopeptidases; C485595:matriptase; D020782:Matrix Metalloproteinases
keywords: extracellular matrix; fibrin hydrogel; fibronectin; laminin; matrix metalloproteinase activity; neural stem cells; neuronal maturation; three-dimensional culture