Comparison and optimization of hiPSC forebrain cortical differentiation protocols

Several protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual...

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Bibliographic Details
Published in:PloS one 2014-08, Vol.9 (8), p.e105807-e105807
Main Authors: Muratore, Christina R, Srikanth, Priya, Callahan, Dana G, Young-Pearse, Tracy L
Format: Article
Language:English
Subjects:
Amino acids
Astrocytes
Astrocytes - cytology
Biology and Life Sciences
Brain
Cell culture
Cell Differentiation
Cell morphology
Cells, Cultured
Coculture Techniques
Cytology
Differentiation (biology)
Embryoid Bodies - cytology
Embryos
Enzymes
Expansion
Flow cytometry
Forebrain
Gene expression
Growth factors
Hospitals
Humans
Induced Pluripotent Stem Cells - cytology
Induced Pluripotent Stem Cells - physiology
Inhibitory postsynaptic potentials
Insulin
Medical schools
Methods
Monolayers
Monomolecular films
Morphology
Neural stem cells
Neurons
Neurons - cytology
Neurons - metabolism
Optimization
Pluripotency
Progenitor cells
Prosencephalon
Purity
Smad protein
Stem cells
Substrates
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Summary:Several protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual SMAD inhibition differentiation protocols, manual vs. AggreWell aggregate formation, plating substrates, neural progenitor cell (NPC) isolation methods, NPC maintenance and expansion, and astrocyte co-culture. The embryoid aggregate protocol, using a Matrigel substrate, consistently generates a high yield and purity of neurons. NPC isolation by manual selection, enzymatic rosette selection, or FACS all are efficient, but exhibit some differences in resulting cell populations. Expansion of NPCs as neural aggregates yields higher cell purity than expansion in a monolayer. Finally, co-culture of iPSC-derived neurons with astrocytes increases neuronal maturity by day 40. This study directly compares commonly employed methods for neuronal differentiation of iPSCs, and can be used as a resource for choosing between various differentiation protocols.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0105807