An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors

• DOI: 10.1371/journal.pone.0135170
• Publication Date: 2015-08-05T18:11:10Z
• Authors: Amranul Haque; Nihad Adnan; Ali Motazedian; Farhima Akter; Sharif Hossain; Koichi Kutsuzawa; Kakon Nag; Eiry Kobatake; Toshihiro Akaike
• Keywords: Pluripotent Stem Cells; 0301 basic medicine; Cell Survival; Science; Recombinant Fusion Proteins; Blotting, Western; Induced Pluripotent Stem Cells; Cell Culture Techniques; 610; Gene Expression; Time-Lapse Imaging; Cell Line; Mice; 03 medical and health sciences; Neural Stem Cells; Neurites; Animals; Embryonic Stem Cells; Microscopy, Confocal; Reverse Transcriptase Polymerase Chain Reaction; Q; R; 500; Reproducibility of Results; Cell Differentiation; Cadherins; Immunoglobulin Fc Fragments; Medicine; Research Article

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.