Rapid Monolayer Neural Induction of induced Pluripotent Stem Cells Yields Stably Proliferating Neural Stem Cells

Objective: The induction of neural stem cells (NSCs) from human induced pluripotent stem cells (hiPSCs) developed into an important strategy to derive patient-specific neuronal and glial cells. Several neural differentiation protocols have been developed mainly involving laborious experimentation such as embryoid body (EB) formation or manual neural rosette isolation. The aim of this study is to develop a rapid neural induction protocol, which combines a previously published monolayer approach with common cultivation methods. Methods and results: hiPSCs were differentiated into primitive NSCs (pNSC) using a rapid monolayer differentiation protocol within 7 days. pNSCs were expanded up to 5 passages and showed a downregulation of the pluripotency gene POU5F1 and expressed NSC markers such as SOX1, SOX2, Nestin and PAX6. In a second step we adapted pNSCs to a widely used FGF/EGF-dependent NSC state by culturing in media supplemented with FGF, EGF and Wnt agonist CHIR99021. Under these conditions, cells underwent a rapid and prominent morphological change to rosette-like structures. These cells remained proliferative for more than 30 passages and maintained the expression profile of neural marker genes. Moreover, they could be efficiently differentiated into neurons as well as GFAPand S100ß-positive astrocytes. Conclusion: We report a robust two-step neural induction protocol for the generation of hiPSC-derived NPCs, closing the gap between previously published monolayer protocols and commonly used FGF/EGF-containing media conditions. Our protocol will serve as a fast and efficient neural induction strategy to derive patient-specific neural cells for biomedical applications such as disease modeling and cell replacement therapy. Rapid Monolayer Neural Induction of induced Pluripotent Stem Cells Yields Stably Proliferating Neural Stem Cells Katharina Gunther1#, Antje Appelt-Menzel2,3#, Chee Keong Kwok1, Heike Walles2,3, Marco Metzger2,3 and Frank Edenhofer1,4* 1Institute of Anatomy and Cell Biology, Stem Cell and Regenerative Medicine Group, 97070 Wurzburg, Germany 2University Hospital Würzburg, Chair Tissue Engineering and Regenerative Medicine, 97070 Wurzburg, Germany 3Translational Center Würzburg “Regenerative Therapies for Oncology and Musculoskeletal Diseases”, Branch of Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, 97070 Würzburg, Germany 4Institute of Molecular Biology, Department Genomics, Stem Cell Biology and Regenerative Medicine, Leopold-Franzens-University Innsbruck, 6020 Innsbruck, Austria #These two authors have contributed equally to this work and are sharing the first authorship *Corresponding author: Frank Edenhofer, Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany; and Department Genomics, Stem Cell Biology and Regenerative Medicine, Institute of Molecular Biology, Leopold-Franzens-University Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria, Tel:+49 931 31-88113; +43 507 51411; Fax: +49 931 31-82087; E-mail: [email protected]; [email protected] Received June 03, 2016; Accepted June 14, 2016; Published June 20, 2016 Citation: Gunther K, Appelt-Menzel A, Kwok CK, Walles H, Metzger M, et al. (2016) Rapid Monolayer Neural Induction of induced Pluripotent Stem Cells Yields Stably Proliferating Neural Stem Cells. J Stem Cell Res Ther 6: 341. doi: 10.4172/21577633.1000341 Copyright: © 2016 Gunther K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.