title: Automated large-scale culture and medium-throughput chemical screen for modulators of proliferation and viability of human induced pluripotent stem cell-derived neuroepithelial-like stem cells.
abstract: The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.
authors.affiliation: Stem Cell Sciences UK Ltd, Cambridge, UK. donna.mclaren@lonza.com
authors.forename: Donna
authors.initials: D
authors.lastname: McLaren
authors.forename: Thorsten
authors.initials: T
authors.lastname: Gorba
authors.forename: Anita
authors.initials: A
authors.lastname: Marguerie de Rotrou
authors.forename: Gopalan
authors.initials: G
authors.lastname: Pillai
authors.forename: Clare
authors.initials: C
authors.lastname: Chappell
authors.forename: Alison
authors.initials: A
authors.lastname: Stacey
authors.forename: Sarah
authors.initials: S
authors.lastname: Lingard
authors.forename: Anna
authors.initials: A
authors.lastname: Falk
authors.forename: Austin
authors.initials: A
authors.lastname: Smith
authors.forename: Philipp
authors.initials: P
authors.lastname: Koch
authors.forename: Oliver
authors.initials: O
authors.lastname: Brüstle
authors.forename: Richard
authors.initials: R
authors.lastname: Vickers
authors.forename: Jon
authors.initials: J
authors.lastname: Tinsley
authors.forename: David
authors.initials: D
authors.lastname: Flanders
authors.forename: Paul
authors.initials: P
authors.lastname: Bello
authors.forename: Stewart
authors.initials: S
authors.lastname: Craig
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005500:Follow-Up Studies; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects*; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects*
chemical_list: D016895:Culture Media, Serum-Free; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2