Poulsen Espersen posted an update 4 days, 13 hours ago
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. The authors would like to thank the reviewer for this comment.In the updated manuscript, these images are shown in a separate figure. The PDF version should enable sufficient magnification to view the composite panels and clearly demonstrate more intracellular localisation on D cells.However, no data is available on the expression pattern of APOE in human neural stem cells.They report a dramatic reduction in APOE mRNA levels during differentiation, as well as a change in the cellular distribution of APOE protein.However, while the QPCR data is convincing and very robust, the immunocytochemistry studies should be further analysedimproved in order to draw any strong conclusions.The images presented are not of very good quality, and if judging by them, APOE expression rather seems to increase globally during differentiation, with few cells expressing high levels at D and most cells expressing moderate levels at D.If this is not possible, the reasch Midostaurin conclusions should be toned down and further experiments suggested in the discussion for example, protein quantification by WB and cellular fractionation and quantification of protein in the media to assess intracellular protein localization and secretion, respectively.It would be very informative to see ifhow these two markers change over the course of the differentiation protocol.Either in the introduction or the discussion, it could be noted that astrocytes express very high levels of APOE in the brain.If possible, provide higher magnificationhigher quality images of APOE stainings, including also the other time points during differentiation.Day would be particularly important to include, since it displays the highest levels of expression by QPCR.The authors would like to thank the reviewer for this comment.In the updated manuscript, these images are shown in a separate figure. The PDF version should enable sufficient magnification to view the composite panels and clearly demonstrate more intracellular localisation on D cells.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.