title: Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.
abstract: Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.
authors.affiliation: National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California.
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Stover
authors.forename: David J
authors.initials: DJ
authors.lastname: Brick
authors.forename: Hubert E
authors.initials: HE
authors.lastname: Nethercott
authors.forename: Maria G
authors.initials: MG
authors.lastname: Banuelos
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.forename: Diane K
authors.initials: DK
authors.lastname: O'Dowd
authors.forename: Philip H
authors.initials: PH
authors.lastname: Schwartz
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D058953:Neural Stem Cells / Q000166:cytology*; D009474:Neurons / Q000166:cytology / Q000502:physiology / Q000637:transplantation; D018408:Patch-Clamp Techniques
keywords: cGMP; cellular models of disease; cellular therapy; differentiation; drug discovery; glia; iPSCs; methods; neural stem cells; neurons; nomenclature
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