In Vitro Effects of Methylprednisolone over Oligodendroglial Cells: Foresight to Future Cell Therapies

Round 1

Reviewer 1 Report

In the present manuscript entitled “In vitro effects of methylprednisolone over oligodendroglial cells: foresight to future cells therapies”, Gomez-Pinedo et al. showed the effect of corticosteroids on the ability of oligodendroglioma cells to proliferate and differentiate into oligodendrocytes. They used the HOG cell line derived from a human oligodendroglioma and found that methylprednisolone reduces the cell viability, proliferation rate, and differentiation in dose-dependent manner. Although the results are of potential interest, the data quality is not sufficient to make proper conclusions and several key questions remain. Overall, the present data are not sufficient to support the conclusions of the effect of methylprednisolone on oligodendroglioma.

 

1) The quality of cell images is poor and the results are questionable as the morphological patterns of oligodendrocytes are unusual (especially in Figure 4B). Some cells stained with an anti-MBP antibody appear to be undifferentiated. Other markers for OPC versus mature oligodendrocytes are needed.

 

2) Comparing DAPI staining in Figs. 3B and 4B, there is variation in cell density (e.g. 3 days MP 5mM is significantly different between two figures). These might affect the proliferation and differentiation of cells. In addition, for comparison, it is better to add the images of day 0.

 

3) Furthermore, the concentrations of 30 mM and 50 mM of methylprednisolone are too high, because most cells appear dead as shown by DAPI staining. How does it compare with clinical blood concentrations?

 

4) Finally, there is a contradiction between immunoblotting data and immunofluorescence data using an anti-MBP antibody. For example, in Fig. 4A, the expression of MBP decreased as the days proceeded in sham group, while the intensity of MBP is increased as the days passed. I’m concerned about the accuracy of these assays. Also, other mature oligodendrocytes markers are also needed for immunoblotting.

 

5) How to calculate the proliferative activity (Table 2) and oligodendroglial maturation? Please mention them elsewhere.

 

6) N values should be clearer in Figure legends.

Minor editing of English language required.

Author Response

Reviewer(s)' Comments to Author:

Reviewer 1

In the present manuscript entitled “In vitro effects of methylprednisolone over oligodendroglial cells: foresight to future cells therapies”, Gomez-Pinedo et al. showed the effect of corticosteroids on the ability of oligodendroglioma cells to proliferate and differentiate into oligodendrocytes. They used the HOG cell line derived from a human oligodendroglioma and found that methylprednisolone reduces the cell viability, proliferation rate, and differentiation in dose-dependent manner. Although the results are of potential interest, the data quality is not sufficient to make proper conclusions and several key questions remain. Overall, the present data are not sufficient to support the conclusions of the effect of methylprednisolone on oligodendroglioma.

Dear reviewer, thanks for your comments, then we answer your comments

1) The quality of cell images is poor and the results are questionable as the morphological patterns of oligodendrocytes are unusual (especially in Figure 4B). Some cells stained with an anti-MBP antibody appear to be undifferentiated. Other markers for OPC versus mature oligodendrocytes are needed.

Response: Thanks for the comments

The images were acquired in the confocal microscope, following a sequential acquisition protocol at a speed of 4px/msec and a resolution of 1600px/1600px. Since they are panoramic images, they can be a handicap for better observation. In our methodology we use a basal culture medium, without factors or hormones that can promote morphological changes, for example cells with arborized or cellular projections. With conventional oligodendroglia differentiation media, they drastically induce their maturation in 10 days(Marton et al., 2019), so our medium is considered as a maintenance medium, used previously in others papers(Gomez-Pinedo et al., 2021). Therefore, the labeling of the MBP in our experience and that of other colleagues is correct for this cell line and culture medium used. In addition, we have made other markers, such as CNPase or PLP, observing similar results to those found. We have proceeded to modify the images so that they are more consistent with the data described. 

2) Comparing DAPI staining in Figs. 3B and 4B, there is variation in cell density (e.g. 3 days MP 5mM is significantly different between two figures). These might affect the proliferation and differentiation of cells. In addition, for comparison, it is better to add the images of day 0.

 

Response: Thanks for the comments

Thanks for the observation. We have modified the images to make them more consistent and we have added day 0 to have a better comparison of the findings described.

 

3) Furthermore, the concentrations of 30 mM and 50 mM of methylprednisolone are too high, because most cells appear dead as shown by DAPI staining. How does it compare with clinical blood concentrations?

 

Thanks for your comments.  This observation was also made by another reviewer,

With the support of the clinical pharmacology service of our hospital, the calculations were carried out following the maximum recommended dosage for patients with acute flare-ups with multiple sclerosis, 1gr of methylprednisolone, considered the limit therapeutic dose used, being the dose of 50 mM of methylprednisolone in culture, 30 mM would be the corresponding to 600mg, 5 mM would be the corresponding at 100mg and 0,5 mM will be the equivalent of 10mg, approximately.

 

 

4) Finally, there is a contradiction between immunoblotting data and immunofluorescence data using an anti-MBP antibody. For example, in Fig. 4A, the expression of MBP decreased as the days proceeded in sham group, while the intensity of MBP is increased as the days passed. I’m concerned about the accuracy of these assays. Also, other mature oligodendrocytes markers are also needed for immunoblotting.

 

Thanks for your comments,

In reference to the previous points and based on this comment, we have decided to make at least another two replicates, in order to have at least 4 replicates of each cell culture. We have modified the images to be more consistent.

 

5) How to calculate the proliferative activity (Table 2) and oligodendroglial maturation? Please mention them elsewhere.

Response: Thanks for the comments,

In the point: 2.2 Analysis of HOG cell proliferation and maturation in the final part we have attached the software used for the analysis of fluorescence intensity, we have used the image analysis program IMAGE-J (Schneider, Rasband, & Eliceiri, 2012).

6) N values should be clearer in Figure legends.

Response: Thanks for your comments,

We attach the N values of each procedure at the bottom of the figure

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, the authors investigated effects of methylprednisolone on the capacity for oligodendroglioma cell proliferation and differentiation in vitro. Major results are negative effects on both cell proliferation and differentiation and the authors demonstrated that corticosteroids should not be included in cell transplantation protocols for the purpose of remyelination.

 

This study contains potentially important information for considering protocols of cell transplantations, but there are several concerns associated with the study.

 

1. In figure 2, cell viability is shown in a graph and demonstrating decreased viability. This result should be confirmed in other methods, such as immunostaining for c-cas3 expression and the TUNEL assay. Or by showing pictures stained with methylene blue as described in method.

 

2. In figure 3, the data between graph and pictures looks inconsistent. for example, 10days picture for SHAM have much less numbers of Ki67+ cells than in 5 days. The authors should prepare pictures consistent with the graph.

 

3. In figure4, show a quantification of the ratio of MBP+ cells. According to the western blotting, it looks like MBP expression decreases from day 3 to 10. However, it seems that the figureB shows opposite.

 

4. What is the expression level of steroid receptors like in HOG cells? Is it possible that these cells are much more sensitive to steroid administration than other subtypes of OPC because of the origin of HOG cells? 

 

5. it is necessary to show the translocation of receptors by the administration of methylprednisolone.

 

6. MBP expression decreased by the administration of methylprednisolone. Do the remaining cells express other marker proteins, such as PDGFRalpha, CNPase, CC1? or Did they lose characteristics as OPCs?

 

7. The authors had better show the validity of the concentration used in this study. 

Author Response

Reviewer(s)' Comments to Author:

Reviewer 2

In this manuscript, the authors investigated effects of methylprednisolone on the capacity for oligodendroglioma cell proliferation and differentiation in vitro. Major results are negative effects on both cell proliferation and differentiation and the authors demonstrated that corticosteroids should not be included in cell transplantation protocols for the purpose of remyelination.

This study contains potentially important information for considering protocols of cell transplantations, but there are several concerns associated with the study.

 Response: Dear reviewer, thanks for your comments, then we answer your comments

 

1. In figure 2, cell viability is shown in a graph and demonstrating decreased viability. This result should be confirmed in other methods, such as immunostaining for c-cas3 expression and the TUNEL assay. Or by showing pictures stained with methylene blue as described in method.

Response: Thank you for your comments, it is very accurate, so we have performed an immunostaining for ssDNA (Millipore MAB3299), provides a cellular marker specific for apoptotic death that is independent of internucleosomal DNA fragmentation and is useful for the detection of different stages of apoptosis in various cell types.

We attach the graph with the data obtained:

 

 

These data will be integrated into the manuscript.

 

 

2. In figure 3, the data between graph and pictures looks inconsistent. for example, 10days picture for SHAM have much less numbers of Ki67+ cells than in 5 days. The authors should prepare pictures consistent with the graph.

 Response: Thank you for your comments. We correct the data to make them consistent.

 

3. In figure4, show a quantification of the ratio of MBP+ cells. According to the western blotting, it looks like MBP expression decreases from day 3 to 10. However, it seems that the figure B shows opposite.

Response: Thank you for your comments. We correct the data to make them consistent.

 

4. What is the expression level of steroid receptors like in HOG cells? Is it possible that these cells are much more sensitive to steroid administration than other subtypes of OPC because of the origin of HOG cells? 
5. it is necessary to show the translocation of receptors by the administration of methylprednisolone.

Response:

Thanks for your comments          
We greatly appreciate your efforts in reviewing the article, which we understand to be relevant in future cell therapy designs with the therapeutic objective of remyelination and repair, due the extensive use of corticoids in the stem cell therapy protocols.

The approach used in our manuscript is focused on seeing the effect of corticosteroids (methyl prednisolone) on cells of oligodendroglial lineage, of great interest for our lines of research in cell therapy, in demyelinating pathologies, where treatment with methyl prednisone is a pharmacological agent of first choice. Therefore, we consider that with a series of clear and simple experiments it has allowed us to reach the conclusions in our manuscript. In reference to the writing of the results, we have corrected the writing style and we have corrected the format of the references.

In relation to the mechanisms involved, several works indicate that glucocorticoids impair proliferation and differentiation.

Kino et al. have described that exogenous administration of corticosterone decreases proliferation and survival of hippocampal progenitor cells in several animal species (Brummelte & Galea, 2010; Cameron & Gould, 1994; Kino et al., 2010; Lau et al., 2007; Schoenfeld & Gould, 2013). Injection of glucocorticoids in rats also induces apoptosis both in neural progenitor cells and in immature granular cell neurons of their dentate gyri (Kino, 2015; Yu et al., 2010). Thus, excess amounts of glucocorticoids act as negative regulators for the function/activity of hippocampal progenitor cells in vivo, and may mediate in some part the effects of stressful stimuli on these cells. This mechanism may be mediated by modulating the transcriptional activity of cyclin-dependent kinase 5 (CDK5), involved in the morphogenesis and functions of the central nervous system. Further, high doses of glucocorticoids inhibit production of brain-derived neurotrophic factor, affect the myelination in the CNS (Xiao et al., 2010).

We clearly understand your concern about the analysis of how methylprednisolone can affect the translocation of receptors, it is an interesting point, but we consider presenting it in a new, more extensive and detailed study, logistical problems with the commercial company prevent us from preparing the experiments at this time, since the delivery times of the antibodies that we have requested are scheduled to arrive on June 20^th, which would delay the response of this manuscript.

We consider that although this point is important, our message continues to be maintained.

 

6. MBP expression decreased by the administration of methylprednisolone. Do the remaining cells express other marker proteins, such as PDGFRalpha, CNPase, CC1? or Did they lose characteristics as OPCs?

Response: Thank you for your comment,

In preliminary experiments we observed that the cells preserve the labeling of oligodendroglial progenitors surch PDGF-R alpha and a low expression of CNPase or PLP, so our data are consistent.

 

Example of image shows that the specific markers of oligodendroglial progenitors are maintained after corticosteroid administration (10 days), but with a different pattern.

 

7. The authors had better show the validity of the concentration used in this study. 

Thanks for your comments.  This observation was also made by another reviewer,

With the support of the clinical pharmacology service of our hospital, the calculations were carried out following the maximum recommended dosage for patients with acute flare-ups with multiple sclerosis, 1gr of methylprednisolone, considered the limit therapeutic dose used, being the dose of 50 mM of methylprednisolone in culture, 30 mM would be the corresponding to 600mg, 5 mM would be the corresponding at 100mg and 0,5 mM will be the equivalent of 10mg, approximately.

 

 

 

Kind regards,

 

 

 

 

PhD ULISES ALFONSO GOMEZ PINEDO

Senior Research

Head of the LAB Neurobiology

 

 

 

Prof JORGE MATIAS-GUIU

Scientific Director

 

 

 

 

REFERENCE

 

Brummelte, S., & Galea, L. A. (2010). Chronic high corticosterone reduces neurogenesis in the dentate gyrus of adult male and female rats. Neuroscience, 168(3), 680-690. doi:10.1016/j.neuroscience.2010.04.023

Cameron, H. A., & Gould, E. (1994). Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neuroscience, 61(2), 203-209. doi:10.1016/0306-4522(94)90224-0

Kino, T. (2015). Stress, glucocorticoid hormones, and hippocampal neural progenitor cells: implications to mood disorders. Front Physiol, 6, 230. doi:10.3389/fphys.2015.00230

Kino, T., Jaffe, H., Amin, N. D., Chakrabarti, M., Zheng, Y. L., Chrousos, G. P., & Pant, H. C. (2010). Cyclin-dependent kinase 5 modulates the transcriptional activity of the mineralocorticoid receptor and regulates expression of brain-derived neurotrophic factor. Mol Endocrinol, 24(5), 941-952. doi:10.1210/me.2009-0395

Lau, W. M., Qiu, G., Helmeste, D. M., Lee, T. M., Tang, S. W., So, K. F., & Tang, S. W. (2007). Corticosteroid decreases subventricular zone cell proliferation, which could be reversed by paroxetine. Restor Neurol Neurosci, 25(1), 17-23. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/17473392

Schoenfeld, T. J., & Gould, E. (2013). Differential effects of stress and glucocorticoids on adult neurogenesis. Curr Top Behav Neurosci, 15, 139-164. doi:10.1007/7854_2012_233

Xiao, J., Wong, A. W., Willingham, M. M., van den Buuse, M., Kilpatrick, T. J., & Murray, S. S. (2010). Brain-derived neurotrophic factor promotes central nervous system myelination via a direct effect upon oligodendrocytes. Neurosignals, 18(3), 186-202. doi:10.1159/000323170

Yu, S., Patchev, A. V., Wu, Y., Lu, J., Holsboer, F., Zhang, J. Z., . . . Almeida, O. F. (2010). Depletion of the neural precursor cell pool by glucocorticoids. Ann Neurol, 67(1), 21-30. doi:10.1002/ana.21812

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

This manuscript is a resubmission of a previous version. The authors have made significant efforts to address the concerns of the reviewer. In particular, I found the revised figures (Fig.3 and 5) to be much improved. As a result, the paper is much clearer and presents the new insights of the effects of methylprednisolone on oligodendrocyte proliferation and differentiation. Thus, I recommend publication without re-review.

Author Response

Thanks for your comments and suggestions.

Reviewer 2 Report

I understand the authors answered most of my concerns.

I have a couple of more suggestions.

i) include pictures for ssDNA staining in figure4

ii) show PDGFRa,  CNPase, or PLP staining data as shown in the letter

iii) include explanations about the concentrations as written in the letter

The manuscript should be rearranged including these suggestions.

 

Author Response

Thank you for your comments and valuable suggestions that have improved the quality of our manuscript. Following your comments we have attached:

i) include pictures for ssDNA staining in figure 4:

Done, we have added the representative images of the analysis carried out

ii) show PDGFRa,  CNPase, or PLP staining data as shown in the letter: 

The images that were presented in the previous answer were attached in supplementary material, attaching a paragraph after figure 5, where we cited the new material.

iii) include explanations about the concentrations as written in the letter 

In the methods we have annexed the explanation about the doses used

Thanks ...gracias