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Abstrait
Rapid Monolayer Neural Induction of induced Pluripotent Stem Cells Yields Stably Proliferating Neural Stem Cells
Katharina Günther, Antje Appelt-Menzel, Chee Keong Kwok, Heike Walles, Marco Metzger and Frank Edenhofer
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 GFAP- and 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.