Abstract
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 S100s-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.
Highlights
We report a robust two-step neural induction protocol for the generation of human induced Pluripotent Stem Cells (hiPSCs)-derived Neural Precursor Cell (NPC), closing the gap between previously published monolayer protocols and commonly used FGF/Epidermal Growth Factor (EGF)-containing media conditions
The use of cellular reprogramming of human somatic cells into self-renewable human induced Pluripotent Stem Cells [1] represents a major breakthrough in biomedical research. hiPSCs can be differentiated into stably proliferating Neural Stem Cells (NSCs) and provide a heretofore unattainable, virtually unlimited access to patient-specific neural cells in a sustainable and standardized manner
This study aims to bridge this gap by applying the rapid monolayer induction protocol to hiPSCs with a subsequent shift to well-established defined media conditions, thereby generating a monolayer-derived Neural Precursor Cell (NPC) population, which depends on basic Fibroblast Growth Factor (bFGF) and EGF
Summary
The use of cellular reprogramming of human somatic cells into self-renewable human induced Pluripotent Stem Cells (hiPSCs) [1] represents a major breakthrough in biomedical research. hiPSCs can be differentiated into stably proliferating Neural Stem Cells (NSCs) and provide a heretofore unattainable, virtually unlimited access to patient-specific neural cells in a sustainable and standardized manner. HiPSCs can be differentiated into stably proliferating Neural Stem Cells (NSCs) and provide a heretofore unattainable, virtually unlimited access to patient-specific neural cells in a sustainable and standardized manner. This study aims to bridge this gap by applying the rapid monolayer induction protocol to hiPSCs with a subsequent shift to well-established defined media conditions, thereby generating a monolayer-derived Neural Precursor Cell (NPC) population, which depends on bFGF and EGF. We show that such rapidly and efficiently generated NPCs can be expanded, cryopreserved and employed for standardized differentiation into glial and neuronal lineages. Our protocol provides a robust basis to rapidly generate patient-specific neural cells for pharmacological studies, cell replacement therapies and tissue engineering
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