DNA Methylation Landscape of ReNcell Common Neural Progenitor Cell Lines Reveals Distinct Lineage Bias

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Martina Gyimesi et al, have conducted an important study "DNA methylation landscape of ReNcell common neural progenitor cell lines reveals distinct lineage bias" which describes the epigenetic lineage differentiation between the two ReNcell cell models, widely studied in neurogenesis and neurodegenerative disease studies.

The authors have conducted a systemic epigenetic analysis and have found DNA methylation as a determinant of lineage-specific signaling in humans. The manuscript requires some more clarification for better publication.

1. The rationale for examining the ReNcell CX and ReNcell VM should be more clearly emphasized in the Introduction section, which would the strenghten the manuscript.
2.  More background details of these cells need to be incorporated in the Material section, like procurement details, isolation, source and their culturing conditions, to make the manuscript better.
3. Functional assays need to be done for the verification of the epigenetic modifications between the cells. 
4. Recent references should be added to the manuscript.

I congratulate the authors for their valuable study, and giving the readers the opportunity to select the appropriate cells for particular experiments.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, authors looked at the inherent DNA methylation variabilities in two types of human NPC models - ReNcell VM (RVM) and ReNcell CX (RCX). This study showed that immortalized RCX cells from human cortex are more homogenous and showed hypomethylation in some neuronal genes suggesting that RCX has bias towards neuronal differentiation. On the other hand, RVM cells are more heterogenous population collected from ventral mesencephalon and has hypomethylation in glial genes like GFAP. Furthermore, beta values of Notch signaling genes and qPCR suggest the presence of different combinations of Notch receptors and ligands in RCX and RVM populations that could lead to the inherent bias towards different lineages. The introduction and discussion is well written and easy to follow. Although the study answers interesting questions, authors have to address the following major and minor points:

 

1. In the graphical abstract, authors mentioned “Foetal cerebral cortex” for both ReNcell CX and ReNcell VM.
2. Authors overall came up with conclusions based on DNA methylation and qPCR. This could be an issue majorly with the Notch signaling related experiments. As there are good antibodies available for Notch signaling components, it would be better to show specific Notch receptor and ligand stainings in both RCX and RVM populations.
3. Can authors give explanation why they specifically choose to knock down SDC4. For example, why not SDC2?
4. In Figure 6, I don’t see what blue vs red plots refer to. Can the authors mention which sets of plots belong to RCX vs RVM cells.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made appropriate changes as per the comments. I recommend this manuscript for acceptance in this journal.

Reviewer 2 Report

Comments and Suggestions for Authors

No more comments.