Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells

Neural progenitor cells (NPCs) fromhuman induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culturemethodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior tomonolayer (2D) neural induction. Our aimwas to compare the efficiency of 2D inductionwith 3Dinduction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two inductionmethods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background. (c) 2017 The Authors. Published by Elsevier B. V. This is an open access article under the CC BY license.