Poulsen Espersen posted an update 4 days, 16 hours ago
One limitation of this study is that the timedependent changes of differentiation markers such as SOX and TBR were not examined alongside APOE.However, timedependent changes of various markers of differentiation would add further validity to our observations and unequivocally clarify whether APOE expression is indeed correlated with the differentiation state of the cells.Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study.Highresolution microscopy techniques would have been more ideal to identify the accurate loci of APOE expression and overcome the challenges of imaging densely packed cells at the earliest stages of neural induction. Further investigations with improved imaging capacity will therefore allow us to characterise APOE during the earlier stages of neural induction and hint at potential mechanisms underlying its role in neurodevelopment.To address this knowledge gap, more data from both in vitro and in vivo samples derived from various species should be generated and compared against each other.We hope that our focused study has laid a strong foundation to such collaborative investigations that may be conducted in the future.Combining our observations and previous evidence reported in the literature, we speculate that APOE has an important role in stem cell maintenance and propose that further investigations should be carried out to validate our findings including methods that were not employed in this study.Moreover, it would be interesting to examine the exact underlying mechanisms such as whether APOE is an upstream or downstream factor of stem cell maintenance, and whether APOE genotype and APOE lossoffunction would produce similar phenotypes.International license. Psychopharmacology. Notably, D is also used as the baseline for the qPCR data.The authors describe an increase in intracellular localisation of APOE following NSC differentiation providing higher magnification images may reveal changes in APOE distribution more clearly.Fig C: APOE appears to be more widely expressed at D for all three NSC lineages. The authors would like to thank the reviewer for the comment on the quantification of ICC images.We now include a quantification of the images in the updated manuscript.The authors would like to thank the reviewer for mentioning this important aspect of the ICC experiment reported in our manuscript.While the authors confirm that the ICC experiments were conducted for APOE on D cells, the data were not included in the manuscript due to the following reasons.According to the differentiation protocol, the cells were maintained at high density approaching near confluence from D to D.We observed that this inadvertently diminishes the quality of immunocytochemistry images for D cells, since clear boundaries of nuclei could not be easily identified with epifluorescence microscopy and further complicated the downstream quantification process.The possibility of dissociating D cells and plating them on to a different surface for better image quality and quantification was considered briefly.However, such additional handling was not done to the cells so that any potential source of artefacts that could mask the true state of D cells can be ruled out in our experiments.While the use of epifluorescence microscopy in our study can be seen as a clear limitation, APOE reasch Doxycycline hyclate immunostaining patterns of D cells was not qualitatively different from that of D cells in our observations.