Efficient Generation of Expandable Dorsal Forebrain Neural Rosette Stem Cell Lines

Reviewer #1 (Public review):

Summary:

The authors aimed to develop a fully scalable, feeder-free protocol for deriving dorsal forebrain neural rosette stem cells (NRSCs) from human pluripotent stem cells, eliminating the need for manual rosette isolation. Using dynamic suspension culture combined with single-SMAD inhibition (RepSox), they sought to generate FOXG1⁺/OTX2⁺ NRSCs within ten days and expand them through at least twelve passages while retaining regional identity. They also aimed to demonstrate the cells' capacity to differentiate into functional neurons, astrocytes, and oligodendrocytes under defined conditions.

Strengths:

A key strength is the elimination of labour-intensive manual rosette picking, which significantly reduces operator variability and enhances throughput. The authors provide diverse validation in the form of flow cytometry showing >95% OTX2⁺ over passages 2-12, immunocytochemistry, single-cell RNA-seq, and functional MEA recordings, confirming both regional fidelity and neuronal activity. They also demonstrate glial differentiation and reproducibility across two hESC lines.

The results convincingly demonstrate that the RepSox/suspension approach yields high-purity dorsal forebrain neural progenitor cells (NRSCs) that maintain marker expression and multipotency through passage 12 and differentiate into electrophysiologically active neurons and mature glia. Thus, the authors have achieved their primary objectives.

This protocol addresses a significant bottleneck in neural stem cell production by providing a reproducible, high-throughput alternative that is well-suited to drug screening, disease modelling, and potential cell therapy manufacturing. Standardised, scalable NRSC banks will accelerate neurodevelopmental and neurodegenerative disorder studies, enable automated bioreactor workflows, and encourage the sharing of resources across academia and industry.

Weaknesses:

Weaknesses include a lack of direct comparison to conventional manual-selection protocols, and the need to improve the statistical rigor of all quantitative assays by applying appropriate hypothesis tests (e.g., t-tests or ANOVA with multiple-comparison correction) rather than reporting mean {plus minus} SD alone.

Additional Context:

Beyond the core technical advance, it's important to situate this work within the broader landscape of neural stem cell research and its downstream applications. Traditionally, dorsal forebrain NSCs have been generated via manual rosette picking after dual-SMAD inhibition (Chambers et al., 2009), a process that is labor-intensive, low-throughput, and prone to operator-dependent variability. By eliminating that step, this protocol directly addresses a key barrier to standardizing NSC production under GMP-compatible conditions - critical for both large-scale drug screening and eventual clinical use. Stable, regionally specified forebrain NSCs are especially valuable for modeling early neurodevelopmental disorders (e.g., autism spectrum disorders, microcephaly) and late-onset pathologies (e.g., Alzheimer's disease) in vitro, where precise cortical patterning is essential to recapitulate disease phenotypes. Moreover, establishing long-term epigenetic fidelity (e.g., via future ATAC-seq or histone-mark profiling) will further reassure users that transcriptional consistency reflects preserved regulatory networks, not just transient marker expression. Finally, demonstrating robust cryopreservation viability (>80%) makes these cells a readily shareable resource for the community, accelerating cross-lab reproducibility and comparative studies of patient-derived iPSC lines. This context underscores how scalable, high-purity forebrain NSCs can transform both basic neuroscience research and translational pipelines.

Reviewer #2 (Public review):

In the present manuscript, Dannulat Frazier et al. provide a novel and advanced protocol for obtaining almost pure populations of neural rosette stem cells (NRSCs) expressing the general markers NES and SOX2. These NSCs are expandable and exhibit dorsal forebrain properties and markers that are maintained throughout passages in culture (at least until passage 12). The authors also demonstrate the multipotency of these NSCs by their ability to differentiate into functional neurons, and precursors of astrocytes and oligodendrocytes.

This method does not require the usual step of manual rosette selection and allows a greater homogeneity of the NSCs obtained and the standardization of the protocol, which will allow greater advances in the applications of these NSCs in research and as models of disease or compound testing. The manuscript is of great interest for the research area, since it describes a new methodology that can facilitate the research and therapeutic application of NSCs.

The manuscript is well-written; the results are clear, robust, and well-explained. The conclusions reached in this paper are well-supported by the data, but some aspects could be better clarified.

(1) The results presented in the present manuscript of the NSCS are performed up to passage 12; it would be interesting to know up to which passages these cells can be expanded, maintaining their initial properties. Have the authors analyzed passages beyond 12?

(2) In Figure 2A, where different markers are shown in NSCs at different passages, it seems that at passage 12, there is a decrease in TJP1+ zones in relation to earlier passages, which could indicate a reduction in the potential to generate rosettes. Have the authors done any quantification along these lines? Could this be the case, or is it just an effect of the image chosen?

(3) In Figure 3A, it is very striking and intriguing that the decrease in the expression of the PAX6 gene in passage 8 in relation to passage 2, which does not correspond to what is observed at the protein level. Have the authors verified this result using another technique, such as for example RT-q-PCR?

(4) In Figure 5B, the labeling for GFAP, appears rather nuclear, despite being a cytoskeleton protein. How can the authors explain this?

Author response:

Reviewer #1 (Public review):

Thank you for your thoughtful and constructive feedback on our manuscript. We greatly appreciate your insights regarding our work, as they are invaluable in refining our research.

We are very happy to hear that you recognize the strengths of our method, particularly the elimination of manual rosette picking, which significantly enhances throughput and reduces variability. We are also pleased that our validation efforts—through flow cytometry, immunocytochemistry, single-cell RNA-sequencing, and functional MEA recordings—effectively demonstrate both the identity and functionality of our derived dorsal forebrain neural rosette stem cells (NRSCs).

Regarding the identified weaknesses, we agree that a direct comparison with conventional manual-selection protocols, specifically those utilizing dual-SMAD inhibition, would be a significant improvement. To address this, we have initiated additional experiments that will directly compare our single-SMAD inhibition approach (RepSox) with dual-SMAD inhibition (SB/LDN), aiming for a comprehensive evaluation of both protocols.

In terms of statistical rigor, we appreciate your suggestion on improving our quantitative assays. All data were collected from at least three independent experiments and presented as mean ±standard deviation unless otherwise specified. Due to the qualitative nature of the data, no formal statistical tests were performed for most of the experiments and the mean and standard deviation were calculated for some quantitative measurements obtained, providing a descriptive summary of the data. When possible, we will incorporate appropriate statistical tests, to present our data in a more robust manner, rather than merely reporting mean ± SD.

Finally, we recognize the importance of situating our work within the broader landscape of neural stem cell research. We aim to elucidate the potential downstream applications for our protocol, which we believe will significantly impact neurodevelopmental and neurodegenerative disorder studies.

Thank you again for your valuable suggestions. We look forward to refining our manuscript and enhancing the contribution of our research to the field.

Reviewer #2 (Public review):

Thank you for your thoughtful and constructive feedback on our manuscript. We appreciate your recognition of the novelty and potential impact of our protocol for obtaining neural rosette stem cells (NRSCs). Your comments are invaluable in improving our work.

We are pleased that you found our methodology to be a significant advancement in the field, particularly the elimination of the manual rosette selection step, which hopefully will enhance homogeneity and standardization. We agree that this development has implications for research, disease modelling, and compound testing.

Regarding your specific points:

Passage expansion: Thank you for your insightful suggestion regarding the analysis beyond passage 12. We have continued passaging our NRSC line for more than 12 passages while maintaining the rosette structure. Although we do not yet have comprehensive and detailed analyses at these later passages, we will include some data and relevant information on our findings in the revised manuscript.

TJP1+ zones: We appreciate your observation regarding the decreased TJP1+ zones at passage 12. We have not consistently detected a reduction in the number of rosettes or TJP1+ lumens across our cultures between passages. While some variability has been noted, we occasionally observe minor reductions at specific time points, followed by a recovery of rosettes in subsequent passages. This suggests that monitoring the number of rosettes is indeed a useful indicator of cell culture health. Cultures should be discarded if rosettes are completely lost. We will take a closer look at this aspect and report the findings in the revised manuscript.

PAX6 Gene expression verification: Thank you for highlighting the discrepancy between PAX6 gene expression levels and protein levels. Unfortunately, we have not yet validated these results using an alternative technique. One potential explanation for this discrepancy may be the phenomenon of negative autoregulation, where increased levels of PAX6 protein can inhibit its own mRNA expression (Manuel et al., 2007). Moreover, Hsieh and Yang (2009) observed that during neurogenesis, PAX6 protein levels may not correlate linearly with mRNA levels, particularly in variable cellular environments. Additionally, post-transcriptional regulatory mechanisms, such as translation initiation mediated by Internal Ribosome Entry Sites (IRES), have been documented in various contexts involving PAX6, suggesting that mRNA levels may not fully represent functional protein levels in developing tissues (Li et al., 2023). We will go deeper into this discussion in the revised manuscript.

GFAP Labeling: We appreciate your comments regarding the nuclear labeling of GFAP. In our astrocyte cultures, we have indeed observed GFAP localization in both the nucleus and the cytoplasm (Figure 5B). We will investigate this phenomenon further and provide a clearer explanation, supported by relevant literature, in the revised version. Although GFAP is primarily categorized as an intermediate filament protein localized in the cytoplasm, evidence suggests its nuclear localization may indicate additional regulatory roles during astrocyte development, activation, and pathology. This finding highlights the potential complexity of GFAP's role during fetal development and cellular stress, suggesting a broader functional scope that may extend into the nuclear space.

Once again, thank you for your insightful feedback and for recognizing the potential of our research. We are committed to addressing your comments and enhancing the quality of our manuscript.

Manuel, M. et al. (2007) ‘Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization’, Development, 134(3), pp. 545–555. Available at: https://doi.org/10.1242/dev.02764.

Hsieh, Y.-W. and Yang, X.-J. (2009) ‘Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors’, Neural Development, 4(1), p. 32. Available at: https://doi.org/10.1186/1749-8104-4-32.

Li, Q. et al. (2023) ‘Translation of paired box 6 (PAX6) mRNA is IRES-mediated and inhibited by cymarin in breast cancer cells’, Genes & Genetic Systems, 98(4), pp. 161–169. Available at: https://doi.org/10.1266/ggs.23-00039.