Comparison and Optimization of hiPSC Forebrain Cortical Differentiation Protocols |
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| Authors: | Christina R. Muratore Priya Srikanth Dana G. Callahan Tracy L. Young-Pearse |
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| Affiliation: | 1. Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.; 2. Program in Neuroscience, Graduate School of Arts and Sciences, Harvard University, Cambridge, Massachusetts, United States of America.; 3. Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, United States of America.; University Of Melbourne, Australia, |
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| Abstract: | 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. |
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