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Further investigations using threedimensional imaging techniques such as confocal microscopy will enable better imaging and reasch Astragaloside A quantification of densely packed cells on D.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. It would be particularly interesting to examine the timecourse of APOE expression changes from the stem cell stage to the mature neuronalglial stage.We hope that the data reported in our manuscript can serve as a foundation to such experiments to be conducted in the future.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.To validate this observation, however, additional experiments with a more direct quantitative approach should be conducted.Since APOE has been shown to exist in both secreted and intracellular forms, it will be interesting to see which form of APOE is produced at each differentiation stage.It is possible that more APOE is secreted in undifferentiated cells compared to differentiated cells, which may not be fully captured using immunocytochemistry techniques performed on fixed cells.Therefore, further investigations on secreted and intracellular APOE using quantitative approaches will be able to clarify whether cells indeed produce different forms and levels of APOE depending on its differentiation state.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.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.It would be very informative to see ifhow these two markers change over the course of the differentiation protocol.If not possible by immuno, a QPCR for these genes would also be enough to show the trends of expression during differentiation.The authors would like to thank the reviewer for pointing out the expression changes of SOX and TBR.While we have not examined the timedependent changes of these markers in this study, the authors can confirm that SOX and TBR expression was consistently observed at D and D by other experienced members of the lab using the differentiation protocols reported in this study.These data were generated by qPCR, ICC, and microarray experiments that collectively show the expression of SOX and TBR similar to the ICC data reported in our manuscript.While we are confident with the SOX and TBR expression pattern in our study, we agree with the reviewer that APOE expression should be examined alongside the differentiation markers.

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