Efficient Generation of Expandable Dorsal Forebrain Neural Rosette Stem Cell Lines

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Abstract Neural stem cells (NSCs) represent an interesting option for developing in vitro disease models and drug screening assays due to their differentiation capacity into neurons and glial cells. Additionally, NSCs are under investigation in on-going clinical trials for treatment of various human neurological disorders. NSCs can be isolated from the central nervous system or derived in vitro from human pluripotent stem cells (hPSCs). However, the current methods for generating NSCs typically include a phase of neural rosette formation and subsequent manual isolation of these tiny structures. As this is a laborious process characterized by operator-dependent variability and scalability challenges, there is a pressing need to develop optimized and scalable protocols to obtain pure NSC populations. In this study, we present a new method for generating highly pure and expandable dorsal forebrain FOXG1 + OTX2 + TLE4 + SOX5 + neural rosette stem cell (NRSC) lines without the necessity for manual isolation of rosette structures. Our findings demonstrate the reproducibility of this protocol through the characterization of different NRSC lines over multiple passages, highlighting the robustness of the process. These NRSCs can be expanded for at least 12 passages without compromising their rosette-formation capacity or their initial dorsal forebrain identity. Furthermore, we show the differentiation capacity of these NRSCs to generate pure populations of TUBB3 + neurons, and under specific conditions, their ability to differentiate into early glial progenitor cells including GFAP + astrocytes and O4 + oligodendrocytes. Collectively, these results show the capabilities of our protocol to generate an expandable NRSC population suitable for in vitro disease modeling and drug screening, while also suggesting a viable strategy for scalable NRSC production for clinical application.

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